C H A P T E R

1
PREPARATION OF TISSUES FOR STUDY
Fixation 1
1
Histology & Its
Methods of Study
AUTORADIOGRAPHY 9
CELL & TISSUE CULTURE 10
Embedding & Sectioning 3
ENZYME HISTOCHEMISTRY 10
Staining 3
LIGHT MICROSCOPY 4 VISUALIZING SPECIFIC MOLECULES 10
Bright-Field Microscopy 4 Immunohistochemistry 11
Fluorescence Microscopy 5 Hybridization Techniques 12
Phase-Contrast Microscopy 5 INTERPRETATION OF STRUCTURES IN TISSUE
Confocal Microscopy 5 SECTIONS 14
Polarizing Microscopy 7 SUMMARY OF KEY POINTS 15
ELECTRON MICROSCOPY 8 ASSESS YOUR KNOWLEDGE 16
Transmission Electron Microscopy 8
Scanning Electron Microscopy 9

H istology is the study of the tissues of the body and

how these tissues are arranged to constitute organs.
This subject involves all aspects of tissue biology, with
the focus on how cells’ structure and arrangement optimize
functions specific to each organ.
a better knowledge of tissue biology. Familiarity with the tools
and methods of any branch of science is essential for a proper
understanding of the subject. This chapter reviews common
methods used to study cells and tissues, focusing on micro-
scopic approaches.
Tissues have two interacting components: cells and extra-
cellular matrix (ECM). The ECM consists of many kinds of
macromolecules, most of which form complex structures,
such as collagen fibrils. The ECM supports the cells and con-
››PREPARATION OF TISSUES
tains the fluid transporting nutrients to the cells, and carry- FOR STUDY
ing away their wastes and secretory products. Cells produce The most common procedure used in histologic research is
the ECM locally and are in turn strongly influenced by matrix the preparation of tissue slices or “sections” that can be exam-
molecules. Many matrix components bind to specific cell ined visually with transmitted light. Because most tissues and
surface receptors that span the cell membranes and connect organs are too thick for light to pass through, thin translu-
to structural components inside the cells, forming a contin- cent sections are cut from them and placed on glass slides for
uum in which cells and the ECM function together in a well- microscopic examination of the internal structures.
coordinated manner. The ideal microscopic preparation is preserved so that the
During development, cells and their associated matrix tissue on the slide has the same structural features it had in the
become functionally specialized and give rise to fundamen- body. However, this is often not feasible because the prepara-
tal types of tissues with characteristic structural features. tion process can remove cellular lipid, with slight distortions
Organs are formed by an orderly combination of these tissues, of cell structure. The basic steps used in tissue preparation for
and their precise arrangement allows the functioning of each light microscopy are shown in Figure 1–1.
organ and of the organism as a whole.
The small size of cells and matrix components makes his-
tology dependent on the use of microscopes and molecular Fixation
methods of study. Advances in biochemistry, molecular biol- To preserve tissue structure and prevent degradation by
ogy, physiology, immunology, and pathology are essential for enzymes released from the cells or microorganisms, pieces of

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 2 CHAPTER 1  ■  Histology & Its Methods of Study

FIGURE 1–1  Sectioning fixed and embedded tissue.

52°- 60°C

(a) Fixation Dehydration Clearing Infiltration Embedding

Drive wheel

Block holder
Paraffin block

Tissue
Steel knife

Most tissues studied histologically are prepared as shown, with Similar steps are used in preparing tissue for transmission elec-
this sequence of steps (a): tron microscopy (TEM), except special fixatives and dehydrating
solutions are used with smaller tissue samples and embedding
■■ Fixation: Small pieces of tissue are placed in solutions of
involves epoxy resins which become harder than paraffin to allow
chemicals that cross-link proteins and inactivate degradative
very thin sectioning.
enzymes, which preserve cell and tissue structure.
■■ Dehydration: The tissue is transferred through a series of (b) A microtome is used for sectioning paraffin-embedded tissues
increasingly concentrated alcohol solutions, ending in 100%, for light microscopy. The trimmed tissue specimen is mounted
which removes all water. in the paraffin block holder, and each turn of the drive wheel by
■■ Clearing: Alcohol is removed in organic solvents in which the histologist advances the holder a controlled distance, gener-
both alcohol and paraffin are miscible. ally from 1 to 10 μm. After each forward move, the tissue block
■■ Infiltration: The tissue is then placed in melted paraffin until it passes over the steel knife edge and a section is cut at a thickness
becomes completely infiltrated with this substance. equal to the distance the block advanced. The paraffin sections
■■ Embedding: The paraffin-infiltrated tissue is placed in a small are placed on glass slides and allowed to adhere, deparaffinized,
mold with melted paraffin and allowed to harden. and stained for light microscope study. For TEM, sections less than
■■ Trimming: The resulting paraffin block is trimmed to expose 1 μm thick are prepared from resin-embedded cells using an ultra-
the tissue for sectioning (slicing) on a microtome. microtome with a glass or diamond knife.

organs are placed as soon as possible after removal from the microscopy, react with the amine groups (NH2) of proteins,
body in solutions of stabilizing or cross-linking compounds preventing their degradation by common proteases. Glutaral-
called fixatives. Because a fixative must fully diffuse through dehyde also cross-links adjacent proteins, reinforcing cell and
the tissues to preserve all cells, tissues are usually cut into ECM structures.
small fragments before fixation to facilitate penetration. To Electron microscopy provides much greater magni-
improve cell preservation in large organs, fixatives are often fication and resolution of very small cellular structures,
introduced via blood vessels, with vascular perfusion allowing and fixation must be done very carefully to preserve addi-
fixation rapidly throughout the tissues. tional “ultrastructural” detail. Typically in such studies,
One widely used fixative for light microscopy is forma- glutaraldehyde-treated tissue is then immersed in buffered
lin, a buffered isotonic solution of 37% formaldehyde. Both osmium tetroxide, which preserves (and stains) cellular lip-
this compound and glutaraldehyde, a fixative used for electron ids as well as proteins.

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 Preparation of Tissues for Study 3

Embedding & Sectioning Staining

C H A P T E R
To permit thin sectioning, fixed tissues are infiltrated and Most cells and extracellular material are completely color-
embedded in a material that imparts a firm consistency. less, and to be studied microscopically tissue sections must
Embedding materials include paraffin, used routinely for light be stained (dyed). Methods of staining have been devised that
microscopy, and plastic resins, which are adapted for both make various tissue components not only conspicuous but also
light and electron microscopy. distinguishable from one another. Dyes stain material more or
Before infiltration with such media, the fixed tissue must less selectively, often behaving like acidic or basic compounds
undergo dehydration by having its water extracted gradually and forming electrostatic (salt) linkages with ionizable radicals

1
by transfers through a series of increasing ethanol solutions, of macromolecules in tissues. Cell components, such as nucleic

Histology & Its Methods of Study  ■  Preparation of Tissues for Study

ending in 100% ethanol. The ethanol is then replaced by an acids with a net negative charge (anionic), have an affinity for
organic solvent miscible with both alcohol and the embedding basic dyes and are termed basophilic; cationic components,
medium, a step referred to as clearing because infiltration with such as proteins with many ionized amino groups, stain more
the reagents used here gives the tissue a translucent appearance. readily with acidic dyes and are termed acidophilic.
The fully cleared tissue is then placed in melted paraffin Examples of basic dyes include toluidine blue, alcian blue,
in an oven at 52°-60°C, which evaporates the clearing solvent and methylene blue. Hematoxylin behaves like a basic dye,
and promotes infiltration of the tissue with paraffin, and then staining basophilic tissue components. The main tissue com-
embedded by allowing it to harden in a small container of ponents that ionize and react with basic dyes do so because of
paraffin at room temperature. Tissues to be embedded with acids in their composition (DNA, RNA, and glycosaminogly-
plastic resin are also dehydrated in ethanol and then infiltrated cans). Acid dyes (eg, eosin, orange G, and acid fuchsin) stain
with plastic solvents that harden when cross-linking polymer- the acidophilic components of tissues such as mitochondria,
izers are added. Plastic embedding avoids the higher tempera- secretory granules, and collagen.
tures needed with paraffin, which helps avoid tissue distortion. Of all staining methods, the simple combination of
The hardened block with tissue and surrounding embed- hematoxylin and eosin (H&E) is used most commonly.
ding medium is trimmed and placed for sectioning in an Hematoxylin stains DNA in the cell nucleus, RNA-rich por-
instrument called a microtome (see Figure 1–1). Paraffin sec- tions of the cytoplasm, and the matrix of cartilage, produc-
tions are typically cut at 3-10 μm thickness for light microscopy, ing a dark blue or purple color. In contrast, eosin stains other
but electron microscopy requires sections less than 1 μm thick. cytoplasmic structures and collagen pink (Figure 1–2a). Here
One micrometer (1 μm) equals 1/1000 of a millimeter (mm) eosin is considered a counterstain, which is usually a single
or 10−6 m. Other spatial units commonly used in microscopy dye applied separately to distinguish additional features of a
are the nanometer (1 nm = 0.001 μm = 10−6 mm = 10−9 m) and tissue. More complex procedures, such as trichrome stains (eg,
angstrom (1 Å = 0.1 nm or 10−4 μm). The sections are placed on Masson’s trichrome), allow greater distinctions among various
glass slides and stained for light microscopy or on metal grids extracellular tissue components.
for electron-microscopic staining and examination. The periodic acid–Schiff (PAS) reaction utilizes the
hexose rings of polysaccharides and other carbohydrate-rich
› ›› MEDICAL APPLICATION tissue structures and stains such macromolecules distinctly
purple or magenta. Figure 1–2b shows an example of cells with
Biopsies are tissue samples removed during surgery or routine carbohydrate-rich areas well-stained by the PAS reaction. The
medical procedures. In the operating room, biopsies are fixed DNA of cell nuclei can be specifically stained using a modifica-
in vials of formalin for processing and microscopic analysis in tion of the PAS procedure called the Feulgen reaction.
a pathology laboratory. If results of such analyses are required Basophilic or PAS-positive material can be further identi-
before the medical procedure is completed, for example to fied by enzyme digestion, pretreatment of a tissue section with
know whether a growth is malignant before the patient is an enzyme that specifically digests one substrate. For example,
closed, a much more rapid processing method is used. The pretreatment with ribonuclease will greatly reduce cytoplas-
biopsy is rapidly frozen in liquid nitrogen, preserving cell mic basophilia with little overall effect on the nucleus, indicat-
structures and at the same time making the tissue hard and ing the importance of RNA for the cytoplasmic staining.
ready for sectioning. A microtome called a cryostat in a cabi- Lipid-rich structures of cells are revealed by avoiding the
net at subfreezing temperature is used to section the block processing steps that remove lipids, such as treatment with
with tissue, and the frozen sections are placed on slides for heat and organic solvents, and staining with lipid-soluble
rapid staining and microscopic examination by a pathologist. dyes such as Sudan black, which can be useful in diagnosis
Freezing of tissues is also effective in histochemical stud- of metabolic diseases that involve intracellular accumulations
ies of very sensitive enzymes or small molecules because of cholesterol, phospholipids, or glycolipids. Less common
freezing, unlike fixation, does not inactivate most enzymes. methods of staining can employ metal impregnation tech-
Finally, because clearing solvents often dissolve cell lipids in niques, typically using solutions of silver salts to visual certain
fixed tissues, frozen sections are also useful when structures ECM fibers and specific cellular elements in nervous tissue.
containing lipids are to be studied histologically. The Appendix lists important staining procedures used for
most of the light micrographs in this book.

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 4 CHAPTER 1  ■  Histology & Its Methods of Study

FIGURE 1–2  Hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining.

G G
G
L
L

a b

Micrographs of epithelium lining the small intestine, (a) stained lumen, where projecting microvilli have a prominent layer of
with H&E, and (b) stained with the PAS reaction for glycoproteins. glycoproteins at the lumen (L) and in the mucin-rich secretory
With H&E, basophilic cell nuclei are stained purple, while cyto- granules of goblet cells. Cell surface glycoproteins and mucin are
plasm stains pink. Cell regions with abundant oligosaccharides PAS-positive because of their high content of oligosaccharides
on glycoproteins, such as the ends of the cells at the lumen (L) and polysaccharides, respectively. The PAS-stained tissue was
or the scattered mucus-secreting goblet cells (G), are poorly counterstained with hematoxylin to show the cell nuclei.
stained. With PAS, however, cell staining is most intense at the (a. X400; b. X300)

Slide preparation, from tissue fixation to observation (or ocular lens) further magnifying this image and projecting
with a light microscope, may take from 12 hours to 2½ days, it onto the viewer’s retina or a charge-coupled device (CCD)
depending on the size of the tissue, the embedding medium, highly sensitive to low light levels with a camera and monitor.
and the method of staining. The final step before microscopic The total magnification is obtained by multiplying the magni-
observation is mounting a protective glass coverslip on the fying power of the objective and ocular lenses.
slide with clear adhesive. The critical factor in obtaining a crisp, detailed image
with a light microscope is its resolving power, defined as the
smallest distance between two structures at which they can be
››LIGHT MICROSCOPY seen as separate objects. The maximal resolving power of the
light microscope is approximately 0.2 μm, which can permit
Conventional bright-field microscopy and more specialized
clear images magnified 1000-1500 times. Objects smaller or
applications like fluorescence, phase-contrast, confocal, and
thinner than 0.2 μm (such as a single ribosome or cytoplasmic
polarizing microscopy are all based on the interaction of light
microfilament) cannot be distinguished with this instrument.
with tissue components and are used to reveal and study tissue
Likewise, two structures such as mitochondria will be seen as
features.
only one object if they are separated by less than 0.2 μm. The
microscope’s resolving power determines the quality of the
Bright-Field Microscopy image, its clarity and richness of detail, and depends mainly on
With the bright-field microscope, stained tissue is examined the quality of its objective lens. Magnification is of value only
with ordinary light passing through the preparation. As shown when accompanied by high resolution. Objective lenses pro-
in Figure 1–3, the microscope includes an optical system and viding higher magnification are designed to also have higher
mechanisms to move and focus the specimen. The optical resolving power. The eyepiece lens only enlarges the image
components are the condenser focusing light on the object obtained by the objective and does not improve resolution.
to be studied; the objective lens enlarging and projecting the Virtual microscopy, typically used for study of bright-
image of the object toward the observer; and the eyepiece field microscopic preparations, involves the conversion of a

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 Light Microscopy 5

FIGURE 1–3  Components and light path of a

Fluorescence Microscopy

C H A P T E R
bright-field microscope. When certain cellular substances are irradiated by light of a
proper wavelength, they emit light with a longer wavelength—
Eyepiece
Interpupillar
adjustment
a phenomenon called fluorescence. In fluorescence
Binocular
tubes Head microscopy, tissue sections are usually irradiated with ultra-
violet (UV) light and the emission is in the visible portion of
the spectrum. The fluorescent substances appear bright on
Stand
a dark background. For fluorescent microscopy, the instru-

1
Measuring
ment has a source of UV or other light and filters that select

Histology & Its Methods of Study  ■  Light Microscopy

graticule
Beamsplitter
rays of different wavelengths emitted by the substances to be
Revolving
nosepiece visualized.
Specimen Objective Fluorescent compounds with affinity for specific cell
holder macromolecules may be used as fluorescent stains. Acridine
Mechanical
stage orange, which binds both DNA and RNA, is an example.
On/off
switch When observed in the fluorescence microscope, these nucleic
Condenser Illumination
intensity
acids emit slightly different fluorescence, allowing them to be
Field lens
control localized separately in cells (Figure 1–4a). Other compounds,
Field such as DAPI and Hoechst, stain specifically bind DNA and
diaphragm
Collector
are used to stain cell nuclei, emitting a characteristic blue fluo-
lens rescence under UV. Another important application of fluores-
cence microscopy is achieved by coupling compounds such as
X-Y
Base translation fluorescein to molecules that will specifically bind to certain
Tungsten mechanism
cellular components and thus allow the identification of these
halogen
lamp structures under the microscope (Figure 1–4b). Antibodies
labeled with fluorescent compounds are extremely important
Photograph of a bright-field light microscope showing its in immunohistologic staining. (See the section Visualizing
mechanical components and the pathway of light from the Specific Molecules.)
substage lamp to the eye of the observer. The optical system
has three sets of lenses:
Phase-Contrast Microscopy
■■ The condenser collects and focuses a cone of light that
illuminates the tissue slide on the stage. Unstained cells and tissue sections, which are usually trans-
■■ Objective lenses enlarge and project the illuminated parent and colorless, can be studied with these modified
image of the object toward the eyepiece. Interchangeable light microscopes. Cellular detail is normally difficult to see
objectives with different magnifications routinely used in
in unstained tissues because all parts of the specimen have
histology include X4 for observing a large area (field) of the
tissue at low magnification; X10 for medium magnification roughly similar optical densities. Phase-contrast micros-
of a smaller field; and X40 for high magnification of more copy, however, uses a lens system that produces visible images
detailed areas. from transparent objects and, importantly, can be used with
■■ The two eyepieces or oculars magnify this image another living, cultured cells (Figure 1–5).
X10 and project it to the viewer, yielding a total magnifica-
Phase-contrast microscopy is based on the principle that
tion of X40, X100, or X400.
light changes its speed when passing through cellular and
 (Used with permission from Nikon Instruments.) extracellular structures with different refractive indices. These
changes are used by the phase-contrast system to cause the
structures to appear lighter or darker in relation to each other.
stained tissue preparation to high-resolution digital images Because they allow the examination of cells without fixation or
and permits study of tissues using a computer or other digi- staining, phase-contrast microscopes are prominent tools in
tal device, without an actual stained slide or a microscope. In all cell culture laboratories. A modification of phase-contrast
this technique, regions of a glass-mounted specimen are cap- microscopy is differential interference contrast micros-
tured digitally in a grid-like pattern at multiple magnifications copy with Nomarski optics, which produces an image of liv-
using a specialized slide-scanning microscope and saved as ing cells with a more apparent three-dimensional (3D) aspect
thousands of consecutive image files. Software then converts (Figure 1–5c).
this dataset for storage on a server using a format that allows
access, visualization, and navigation of the original slide with
common web browsers or other devices. With advantages in Confocal Microscopy
cost and ease of use, virtual microscopy is rapidly replacing With a regular bright-field microscope, the beam of light
light microscopes and collections of glass slides in histology is relatively large and fills the specimen. Stray (excess) light
laboratories for students. reduces contrast within the image and compromises the

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 6 CHAPTER 1  ■  Histology & Its Methods of Study

FIGURE 1–4  Appearance of cells with fluorescent microscopy.

N R

a b

Components of cells are often stained with compounds visible by filaments show nuclei with blue fluorescence and actin filaments
fluorescence microscopy. stained green. Important information such as the greater density
(a) Acridine orange binds nucleic acids and causes DNA in cell of microfilaments at the cell periphery is readily apparent. (Both
nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to X500)
appear orange in these cells of a kidney tubule. (Figure 1–4b, used with permission from Drs Claire E. Walczak
and Rania Rizk, Indiana University School of Medicine,
(b) Cultured cells stained with DAPI (4′,6-diamino-2-phenylindole) Bloomington.)
that binds DNA and with fluorescein phalloidin that binds actin

FIGURE 1–5  Unstained cells’ appearance in three types of light microscopy.

a b c

Living neural crest cells growing in culture appear differently in-phase light differently and produce an image of these features
with various techniques of light microscopy. Here the same field in all the cells.
of unstained cells, including two differentiating pigment cells, is (c) Differential interference contrast microscopy: Cellular details
shown using three different methods (all X200): are highlighted in a different manner using Nomarski optics.
(a) Bright-field microscopy: Without fixation and staining, only Phase-contrast microscopy, with or without differential interfer-
the two pigment cells can be seen. ence, is widely used to observe live cells grown in tissue culture.
(b) Phase-contrast microscopy: Cell boundaries, nuclei, and (Used with permission from Dr Sherry Rogers, Department of Cell
cytoplasmic structures with different refractive indices affect Biology and Physiology, University of New Mexico, Albuquerque, NM.)

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 Light Microscopy 7

such optical sections at a series of focal planes through the

FIGURE 1–6  Principle of confocal microscopy.
specimen allows them to be digitally reconstructed into a

C H A P T E R
3D image.

Laser Polarizing Microscopy

Polarizing microscopy allows the recognition of stained or
unstained structures made of highly organized subunits.
When normal light passes through a polarizing filter, it exits

1
Scanner vibrating in only one direction. If a second filter is placed in

Histology & Its Methods of Study  ■  Light Microscopy

the microscope above the first one, with its main axis per-
Detector
pendicular to the first filter, no light passes through. If, how-
ever, tissue structures containing oriented macromolecules
are located between the two polarizing filters, their repeti-
tive structure rotates the axis of the light emerging from the
polarizer and they appear as bright structures against a dark
background (Figure 1–7). The ability to rotate the direction
Plate with of vibration of polarized light is called birefringence and is
pinhole
Beam
splitter FIGURE 1–7  Tissue appearance with bright-field
and polarizing microscopy.

Lens

Other
Focal plane out-of-focus
Specimen planes

Although a very small spot of light originating from one plane

of the section crosses the pinhole and reaches the detector,
rays originating from other planes are blocked by the blind.
Thus, only one very thin plane of the specimen is focused at a
time. The diagram shows the practical arrangement of a confo- a
cal microscope. Light from a laser source hits the specimen and
is reflected. A beam splitter directs the reflected light to a pin-
hole and a detector. Light from components of the specimen
that are above or below the focused plane is blocked by the
blind. The laser scans the specimen so that a larger area of the
specimen can be observed.

resolving power of the objective lens. Confocal microscopy

(Figure 1–6) avoids these problems and achieves high reso-
lution and sharp focus by using (1) a small point of high-
intensity light, often from a laser and (2) a plate with a
pinhole aperture in front of the image detector. The point b
light source, the focal point of the lens, and the detector’s
pinpoint aperture are all optically conjugated or aligned to
Polarizing light microscopy produces an image only of material
each other in the focal plane (confocal), and unfocused light having repetitive, periodic macromolecular structure; features
does not pass through the pinhole. This greatly improves without such structure are not seen. Pieces of thin, unsec-
resolution of the object in focus and allows the localization tioned mesentery were stained with red picrosirius, orcein, and
of specimen components with much greater precision than hematoxylin, placed on slides and observed by bright-field (a)
with the bright-field microscope. and polarizing (b) microscopy.
Confocal microscopes include a computer-driven mirror (a) With bright-field microscopy, collagen fibers appear red,
with thin elastic fibers and cell nuclei darker. (X40)
system (the beam splitter) to move the point of illumination
across the specimen automatically and rapidly. Digital images (b) With polarizing microscopy, only the collagen fibers are
visible and these exhibit intense yellow or orange birefrin-
captured at many individual spots in a very thin plane of focus gence. (a: X40; b: X100)
are used to produce an “optical section” of that plane. Creating

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 8 CHAPTER 1  ■  Histology & Its Methods of Study

a feature of crystalline substances or substances containing The wavelength in an electron beam is much shorter than that
highly oriented molecules, such as cellulose, collagen, micro- of light, allowing a 1000-fold increase in resolution.
tubules, and actin filaments.
The utility of all light microscopic methods is greatly Transmission Electron Microscopy
extended through the use of digital cameras. Many features The transmission electron microscope (TEM) is an imag-
of digitized histologic images can be analyzed quantitatively ing system that permits resolution around 3 nm. This high
using appropriate software. Such images can also be enhanced resolution allows isolated particles magnified as much as
to allow objects not directly visible through the eyepieces to be 400,000 times to be viewed in detail. Very thin (40-90 nm),
examined on a monitor. resin-embedded tissue sections are typically studied by TEM
at magnifications up to approximately 120,000 times.
››ELECTRON MICROSCOPY Figure 1–8a indicates the components of a TEM and the
basic principles of its operation: a beam of electrons focused
Transmission and scanning electron microscopes are based on using electromagnetic “lenses” passes through the tissue sec-
the interaction of tissue components with beams of electrons. tion to produce an image with black, white, and intermediate

FIGURE 1–8  Electron microscopes.

Electron gun Electron gun

Cathode Cathode

3 mm
Anode Anode

Copper grid
Condensor lens with three sections Lens

Specimen Column
Objective lens holder Lens
Column Scanner
Intermediate lens Electron detector

TEM image Lens SEM image

Projector lens

Image on viewing
screen Specimen
Electron detector
with CCD camera

(a) Transmission electron microscope (b) Scanning electron microscope

Electron microscopes are large instruments generally housed in a In a TEM image areas of the specimen through which electrons
specialized EM facility. passed appear bright (electron lucent), while denser areas or
(a) Schematic view of the major components of a transmission elec- those that bind heavy metal ions during specimen preparation
tron microscope (TEM), which is configured rather like an upside- absorb or deflect electrons and appear darker (electron dense).
down light microscope. With the microscope column in a vacuum, a Such images are therefore always black, white, and shades of gray.
metallic (usually tungsten) filament (cathode) at the top emits elec- (b) The scanning electron microscope (SEM) has many similarities
trons that travel to an anode with an accelerating voltage between to a TEM. However, here the focused electron beam does not pass
60 and 120 kV. Electrons passing through a hole in the anode form through the specimen, but rather is moved sequentially (scanned)
a beam that is focused electromagnetically by circular electric from point to point across its surface similar to the way an electron
coils in a manner analogous to the effect of optical lenses on light. beam is scanned across a television tube or screen. For SEM speci-
The first lens is a condenser focusing the beam on the section. mens are coated with metal atoms with which the electron beam
Some electrons interact with atoms in the section, being absorbed interacts, producing reflected electrons and newly emitted secondary
or scattered to different extents, while others are simply transmit- electrons. All of these are captured by a detector and transmitted to
ted through the specimen with no interaction. Electrons reaching amplifiers and processed to produce a black-and-white image on the
the objective lens form an image that is then magnified and finally monitor. The SEM shows only surface views of the coated specimen
projected on a fluorescent screen or a charge-coupled device but with a striking 3D, shadowed quality. The inside of organs or cells
(CCD) monitor and camera. can be analyzed after sectioning to expose their internal surfaces.

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 Autoradiography 9

shades of gray regions. These regions of an electron micro- layer of heavy metal (often gold) that reflects electrons in a
graph correspond to tissue areas through which electrons beam scanning the specimen. The reflected electrons are cap-

C H A P T E R
passed readily (appearing brighter or electron-lucent) and tured by a detector, producing signals that are processed to pro-
areas where electrons were absorbed or deflected (appearing duce a black-and-white image. SEM images are usually easy to
darker or more electron-dense). To improve contrast and reso- interpret because they present a three-dimensional view that
lution in TEM, compounds with heavy metal ions are often appears to be illuminated in the same way that large objects are
added to the fixative or dehydrating solutions used for tissue seen with highlights and shadows caused by light.
preparation. These include osmium tetroxide, lead citrate,

1
and uranyl compounds, which bind cellular macromolecules,
››AUTORADIOGRAPHY

Histology & Its Methods of Study  ■ Autoradiography

increasing their electron density and visibility.
Cryofracture and freeze etching are techniques that
Microscopic autoradiography is a method of localizing
allow TEM study of cells without fixation or embedding and
newly synthesized macromolecules in cells or tissue sections.
have been particularly useful in the study of membrane struc-
Radioactively labeled metabolites (nucleotides, amino acids,
ture. In these methods, very small tissue specimens are rap-
sugars) provided to the living cells are incorporated into spe-
idly frozen in liquid nitrogen and then cut or fractured with a
cific macromolecules (DNA, RNA, protein, glycoproteins, and
knife. A replica of the frozen exposed surface is produced in a
polysaccharides) and emit weak radiation that is restricted
vacuum by applying thin coats of vaporized platinum or other
to those regions where the molecules are located. Slides with
metal atoms. After removal of the organic material, the replica
radiolabeled cells or tissue sections are coated in a darkroom
of the cut surface can be examined by TEM. With membranes
with photographic emulsion in which silver bromide crystals
the random fracture planes often split the lipid bilayers, expos-
ing protein components whose size, shape, and distribution act as microdetectors of the radiation in the same way that
are difficult to study by other methods. they respond to light in photographic film. After an adequate
exposure time in lightproof boxes, the slides are developed
photographically. Silver bromide crystals reduced by the radia-
Scanning Electron Microscopy tion produce small black grains of metallic silver, which under
Scanning electron microscopy (SEM) provides a high- either the light microscope or TEM indicate the locations of
resolution view of the surfaces of cells, tissues, and organs. Like radiolabeled macromolecules in the tissue (Figure 1–9).
the TEM, this microscope produces and focuses a very narrow Much histological information becomes available by
beam of electrons, but in this instrument the beam does not autoradiography. If a radioactive precursor of DNA (such
pass through the specimen (Figure 1–8b). Instead, the surface as tritium-labeled thymidine) is used, it is possible to know
of the specimen is first dried and spray-coated with a very thin which cells in a tissue (and how many) are replicating DNA

FIGURE 1–9  Microscopic autoradiography.

G
G

a b

Autoradiographs are tissue preparations in which particles called (a) Black grains of silver from the light-sensitive material coating
silver grains indicate the cells or regions of cells in which specific the specimen are visible over cell regions with secretory granules
macromolecules were synthesized just prior to fixation. Shown and the duct indicating glycoprotein locations. (X1500)
here are autoradiographs from the salivary gland of a mouse (b) The same tissue prepared for TEM autoradiography shows sil-
injected with 3H-fucose 8 hours before tissue fixation. Fucose was ver grains with a coiled or amorphous appearance again localized
incorporated into oligosaccharides, and the free 3H-fucose was mainly over the granules (G) and in the gland lumen (L). (X7500)
removed during fixation and sectioning of the gland. Autoradio- (Figure 1–9b, used with permission from Drs Ticiano G. Lima and
graphic processing and microscopy reveal locations of newly syn- A. Antonio Haddad, School of Medicine, Ribeirão Preto, Brazil.)
thesized glycoproteins containing that sugar.

01_Mescher_ch01_p001-016.indd 9 26/04/18 11:10 am

 10 CHAPTER 1  ■  Histology & Its Methods of Study

and preparing to divide. Dynamic events may also be analyzed.

For example, if one wishes to know where in the cell protein is
››ENZYME HISTOCHEMISTRY
produced, if it is secreted, and its path in the cell before being Enzyme histochemistry (or cytochemistry) is a method for
secreted, several animals are injected with a radioactive amino localizing cellular structures using a specific enzymatic activ-
acid and tissues collected at different times after the injections. ity present in those structures. To preserve the endogenous
Autoradiography of the tissues from the sequential times will enzymes, histochemical procedures usually use unfixed or
indicate the migration of the radioactive proteins. mildly fixed tissue, which is sectioned on a cryostat to avoid
adverse effects of heat and organic solvents on enzymatic
activity. For enzyme histochemistry: (1) tissue sections are
››CELL & TISSUE CULTURE immersed in a solution containing the substrate of the enzyme
to be localized; (2) the enzyme is allowed to act on its sub-
Live cells and tissues can be maintained and studied outside strate; (3) the section is then put in contact with a marker
the body in culture (in vitro). In the organism (in vivo), cells compound that reacts with a product of the enzymatic action
are bathed in fluid derived from blood plasma and containing on the substrate; and (4) the final product from the marker,
many different molecules required for survival and growth. which must be insoluble and visible by light or electron
Cell culture allows the direct observation of cellular behavior microscopy, precipitates over the site of the enzymes, identify-
under a phase-contrast microscope, and many experiments ing their location.
technically impossible to perform in the intact animal can be Examples of enzymes that can be detected histochemi-
accomplished in vitro. cally include the following:
The cells and tissues are grown in complex solutions of
known composition (salts, amino acids, vitamins) to which ■■ Phosphatases, which remove phosphate groups from
serum or specific growth factors are added. Cells to be cultured macromolecules (Figure 1–10).
are dispersed mechanically or enzymatically from a tissue or ■■ Dehydrogenases, which transfer hydrogen ions from
organ and placed with sterile procedures in a clear dish to one substrate to another, such as many enzymes of the
which they adhere, usually as a single layer (see Figure 1–5). citric acid (Krebs) cycle, allowing histochemical identifi-
Such preparations are called primary cell cultures. Some cation of such enzymes in mitochondria.
cells can be maintained in vitro for long periods because they ■■ Peroxidase, which promotes the oxidation of sub-
become immortalized and constitute a permanent cell line. strates with the transfer of hydrogen ions to hydrogen
Most cells obtained from normal tissues have a finite, geneti- peroxide.
cally programmed life span. However, certain changes (some
related to oncogenes; see Chapter 3) can promote cell immor-
tality, a process called transformation, and are similar to › ›› MEDICAL APPLICATION
the initial changes in a normal cell’s becoming a cancer cell.
Many enzyme histochemical procedures are used in the
Improvements in culture technology and use of specific growth
medical laboratory, including Perls’ Prussian blue reaction for
factors now allow most cell types to be maintained in vitro.
iron (used to diagnose the iron storage diseases, hemochro-
As shown in Chapter 2, incubation of living cells in vitro
matosis and hemosiderosis), the PAS-amylase and alcian blue
with a variety of new fluorescent compounds that are seques-
reactions for polysaccharides (to detect glycogenosis and
tered and metabolized in specific compartments of the cell
mucopolysaccharidosis), and reactions for lipids and sphin-
provides a new approach to understanding these compart-
golipids (to detect sphingolipidosis).
ments both structurally and physiologically. Other histologic
techniques applied to cultured cells have been particularly
important for understanding the locations and functions of
microtubules, microfilaments, and other components of the
cytoskeleton.
››VISUALIZING SPECIFIC MOLECULES
A specific macromolecule present in a tissue section may also
be identified by using tagged compounds or macromolecules
› ›› MEDICAL APPLICATION that bind specifically with the molecule of interest. The com-
pounds that interact with the molecule must be visible with
Cell culture is very widely used to study molecular changes
the light or electron microscope, often by being tagged with a
that occur in cancer; to analyze infectious viruses, myco-
detectible label. The most commonly used labels are fluores-
plasma, and some protozoa; and for many routine genetic or
cent compounds, radioactive atoms that can be detected with
chromosomal analyses. Cervical cancer cells from a patient
autoradiography, molecules of peroxidase or other enzymes
later identified as Henrietta Lacks, who died from the disease
that can be detected with histochemistry, and metal (usually
in 1951, were used to establish one of the first cell lines,
gold) particles that can be seen with light and electron micros-
called HeLa cells, which are still used in research on cellular
copy. These methods can be used to detect and localize specific
structure and function throughout the world.
sugars, proteins, and nucleic acids.

01_Mescher_ch01_p001-016.indd 10 26/04/18 11:10 am

 Visualizing Specific Molecules 11

Examples of molecules that interact specifically with

FIGURE 1–10  Enzyme histochemistry.
other molecules include the following:

C H A P T E R
■■ Phalloidin, a compound extracted from mushroom,
Amanita phalloides, interacts strongly with the actin pro-
tein of microfilaments.
L ■■ Protein A, purified from Staphylococcus aureus bacte-
ria, binds to the Fc region of antibody molecules, and
can therefore be used to localize naturally occurring or

1
applied antibodies bound to cell structures.

Histology & Its Methods of Study  ■  Visualizing Specific Molecules

■■ Lectins, glycoproteins derived mainly from plant seeds,
bind to carbohydrates with high affinity and specificity.
Different lectins bind to specific sugars or sequences of
sugar residues, allowing fluorescently labeled lectins to
be used to stain specific glycoproteins or other macro-
molecules bearing specific sequences of sugar residues.

Immunohistochemistry
A highly specific interaction between macromolecules is that
between an antigen and its antibody. For this reason, labeled
antibodies are routinely used in immunohistochemistry
L L to identify and localize many specific proteins, not just
those with enzymatic activity that can be demonstrated by
histochemistry.
aa The body’s immune cells interact with and produce anti-
bodies against other macromolecules—called antigens—that
are recognized as “foreign,” not a normal part of the organism,
and potentially dangerous. Antibodies belong to the immu-
noglobulin family of glycoproteins and are secreted by lym-
phocytes. These molecules normally bind specifically to their
provoking antigens and help eliminate them.
Ly Widely applied for both research and diagnostic pur-
poses, every immunohistochemical technique requires an
antibody against the protein that is to be detected. This means
that the protein must have been previously purified using bio-
Ly
chemical or molecular methods so that antibodies against it
can be produced. To produce antibodies against protein x of a
certain animal species (eg, a human or rat), the isolated pro-
tein is injected into an animal of another species (eg, a rabbit
or a goat). If the protein’s amino acid sequence is sufficiently
b N different for this animal to recognize it as foreign—that is, as
an antigen—the animal will produce antibodies against the
protein.
(a) Micrograph of cross sections of kidney tubules treated Different groups (clones) of lymphocytes in the injected
histochemically to demonstrate alkaline phosphatases (with animal recognize different parts of protein x and each clone
maximum activity at an alkaline pH) showing strong activity of produces an antibody against that part. These antibodies are
this enzyme at the apical surfaces of the cells at the lumens (L)
of the tubules. (X200)
collected from the animal’s plasma and constitute a mixture
of polyclonal antibodies, each capable of binding a different
(b) TEM image of a kidney cell in which acid phosphatase has
been localized histochemically in three lysosomes (Ly) near the region of protein x.
nucleus (N). The dark material within these structures is lead It is also possible, however, to inject protein x into a
phosphate that precipitated in places with acid phosphatase mouse and a few days later isolate the activated lymphocytes
activity. (X25,000) and place them into culture. Growth and activity of these cells
(Figure 1–10b, used with permission from Dr Eduardo
can be prolonged indefinitely by fusing them with lymphocytic
Katchburian, Department of Morphology, Federal University of
São Paulo, Brazil.) tumor cells to produce hybridoma cells. Different hybridoma
clones produce different antibodies against the several parts of

01_Mescher_ch01_p001-016.indd 11 26/04/18 11:10 am

 12 CHAPTER 1  ■  Histology & Its Methods of Study

protein x, and each clone can be isolated and cultured sepa- The indirect method is used more widely in research and
rately so that the different antibodies against protein x can be pathologic tests because it is more sensitive, with the extra
collected separately. Each of these antibodies is a monoclo- level of antibody binding serving to amplify the visible signal.
nal antibody. An advantage to using a monoclonal antibody Moreover, the same preparation of labeled secondary antibody
rather than polyclonal antibodies is that it can be selected to can be used in studies with different primary antibodies (spe-
be highly specific and to bind strongly to the protein to be cific for different antigens) as long as all these are made in the
detected, with less nonspecific binding to other proteins that same species. There are other indirect methods that involve the
are similar to the one of interest. use of other intermediate molecules, such as the biotin-avidin
In immunohistochemistry, a tissue section that one technique, which are also used to amplify detection signals.
believes contains the protein of interest is incubated in a solu- Examples of indirect immunocytochemistry are shown in
tion containing antibody (either monoclonal or polyclonal) Figure 1–12, demonstrating the use of this method with cells
against this protein. The antibody binds specifically to the in culture or after tissue sectioning for both light microscopy
protein and after a rinse the protein’s location in the tissue or and TEM.
cells can be seen with either the light or electron microscope
by visualizing the antibody. Antibodies are commonly tagged
with fluorescent compounds, with peroxidase or alkaline › ›› MEDICAL APPLICATION
phosphatase for histochemical detection, or with electron- Because cells in some diseases, including many cancer cells,
dense gold particles for TEM. often produce proteins unique to their pathologic condition,
As Figure 1–11 indicates, there are direct and indirect immunohistochemistry can be used by pathologists to diag-
methods of immunocytochemistry. The direct method just nose many diseases, including certain types of tumors and
involves a labeled antibody that binds the protein of interest. some virus-infected cells. Table 1–1 shows some applications
Indirect immunohistochemistry involves sequential of immunocytochemistry routinely used in clinical practice.
application of two antibodies and additional washing steps. The
(primary) antibody specifically binding the protein of interest
is not labeled. The detectible tag is conjugated to a second- Hybridization Techniques
ary antibody made in an animal species different (“foreign”) Hybridization usually implies the specific binding between
from that which made the primary antibody. For example, pri- two single strands of nucleic acid, which occurs under appro-
mary antibodies made by mouse lymphocytes (such as most priate conditions if the strands are complementary. The greater
monoclonal antibodies) are specifically recognized and bound the similarities of their nucleotide sequences, the more read-
by antibodies made in a rabbit or goat injected with mouse ily the complementary strands form “hybrid” double-strand
antibody immunoglobulin. molecules. Hybridization at stringent conditions allows the

FIGURE 1–11  Immunocytochemistry techniques.

Labeled
secondary
Labeled Unlabeled antibody
antibody primary
antibody
Antigen Antigen

Tissue section

Glass slide

Direct Indirect

Immunocytochemistry (or immunohistochemistry) can be direct labeled secondary antibody is obtained that was (1) made in
or indirect. Direct immunocytochemistry (left) uses an antibody another species against immunoglobulin proteins (antibodies)
made against the tissue protein of interest and tagged directly from the species in which the primary antibodies were made and
with a label such as a fluorescent compound or peroxidase. When (2) labeled with a fluorescent compound or peroxidase. When
placed with the tissue section on a slide, these labeled antibod- the labeled secondary antibody is applied to the tissue section, it
ies bind specifically to the protein (antigen) against which they specifically binds the primary antibodies, indirectly labeling the
were produced and can be visualized by the appropriate method. protein of interest on the slide. Because more than one labeled
Indirect immunocytochemistry (right) uses first a primary secondary antibody can bind each primary antibody molecule,
antibody made against the protein (antigen) of interest and labeling of the protein of interest is amplified by the indirect
applied to the tissue section to bind its specific antigen. Then a method.

01_Mescher_ch01_p001-016.indd 12 26/04/18 11:10 am

 Visualizing Specific Molecules 13

FIGURE 1–12  Cells and tissues stained by immunohistochemistry.

1 C H A P T E R
Histology & Its Methods of Study  ■  Visualizing Specific Molecules
c
a

the cytoplasm. Primary antibodies against the filament pro-

tein desmin and fluorescein isothiocyanate (FITC)-labeled
secondary antibodies were used in the indirect staining
technique, with the nucleus counterstained blue with DAPI.
(X650)
(b) A section of small intestine treated with an antibody against
the enzyme lysozyme. The secondary antibody labeled with
peroxidase was then applied and the localized brown color
produced histochemically with the peroxidase substrate
3,3′-diamino-azobenzidine (DAB). The method demonstrates
lysozyme-containing structures in scattered macrophages and
in the large clusters of cells. Nuclei were counterstained with
hematoxylin. (X100)
(c) A section of pancreatic cells in a TEM preparation incubated
with an antibody against the enzyme amylase and then with
protein A coupled with gold particles. Protein A has high affin-
ity toward antibody molecules and the resulting image reveals
the presence of amylase with the gold particles localized as very
b
small black dots over dense secretory granules and developing
granules (left). With specificity for immunoglobulin molecules,
labeled protein A can be used to localize any primary antibody.
Immunocytochemical methods to localize specific proteins can
(X5000)
be applied to either light microscopic or TEM preparations using
(Figure 1–12c, used with permission from Dr Moise Bendayan,
a variety of labels.
Departments of Pathology and Cell Biology, University of Montreal,
(a) A single cultured uterine cell stained fluorescently to reveal Montreal, Canada.)
a meshwork of intermediate filaments (green) throughout

TABLE 1–1    Examples of specific antigens with diagnostic importance.

Antigens Diagnosis

Specific cytokeratins Tumors of epithelial origin

Protein and polypeptide hormones Certain endocrine tumors
Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the digestive tract and breast
Steroid hormone receptors Breast duct cell tumors
Antigens produced by viruses Specific virus infections

01_Mescher_ch01_p001-016.indd 13 26/04/18 11:10 am

 14 CHAPTER 1  ■  Histology & Its Methods of Study

specific identification of sequences in genes or RNA. This can

occur with cellular DNA or RNA when nucleic acid sequences
› ›› MEDICAL APPLICATION
in solution are applied directly to prepared cells and tissue sec- Warts on the skin of the genitals and elsewhere are due to
tions, a procedure called in situ hybridization (ISH). infection with the human papillomavirus (HPV) which causes
This technique is ideal for (1) determining if a cell has the characteristic benign proliferative growth. As shown in
a specific sequence of DNA, such as a gene or part of a gene Figure 1–12, such virus-infected cells can often be demon-
(Figure 1–13), (2) identifying the cells containing specific mes- strated by ISH. Certain cancer cells with unique or elevated
senger RNAs (mRNAs) (in which the corresponding gene is expression of specific genes are also localized in tumors and
being transcribed), or (3) determining the localization of a gene studied microscopically by ISH.
in a specific chromosome. DNA and RNA of the cells must be
initially denatured by heat or other agents to become completely
single-stranded, and the nucleotide sequences of interest are
detected with probes consisting of single-stranded comple-
››INTERPRETATION OF STRUCTURES
mentary DNA (cDNA). The probe may be obtained by cloning,
IN TISSUE SECTIONS
by polymerase chain reaction (PCR) amplification of the target In studying and interpreting stained tissue sections, it is
sequence, or by chemical synthesis if the desired sequence is important to remember that microscopic preparations are the
short. The probe is tagged with nucleotides containing a radio- end result of a series of processes that began with collecting
active isotope (localized by autoradiography) or modified with the tissue and ended with mounting a coverslip on the slide.
a small compound such as digoxigenin (identified by immuno- Certain steps in this procedure may distort the tissues slightly,
cytochemistry). A solution containing the probe is placed over producing minor structural abnormalities called artifacts not
the specimen under conditions allowing hybridization and after present in the living tissue.
the excess unbound probe is washed off, the localization of the One such distortion is minor shrinkage of cells or tissue
hybridized probe is revealed through its label. regions produced by the fixative, by the ethanol, or by the heat
needed for paraffin embedding. Shrinkage can create artificial
spaces between cells and other tissue components. Such spaces
FIGURE 1–13  In situ hybridization (ISH). can also result from the loss of lipids or low-molecular-weight
substances not preserved by the fixative or removed by the
dehydrating and clearing fluids. Slight cracks in sections may
also appear as large spaces in the tissue.
Other artifacts may include small wrinkles in the section
(which the novice may confuse with linear structures in tissue)
and precipitates from the stain (which may be confused with
cellular structures such as cytoplasmic granules). Students must
be aware of the existence of artifacts and able to recognize them.
Another difficulty in the study of histologic sections is the
impossibility of differentially staining all tissue components on
one slide. A single stain can seldom demonstrate well nuclei,
mitochondria, lysosomes, basement membranes, elastic fibers,
etc. With the light microscope, it is necessary to examine prepa-
rations stained by different methods before an idea of the whole
composition and structure of a cell or tissue can be obtained. The
TEM allows the observation of cells with all its internal structures
and surrounding ECM components, but only a few cells in a tis-
sue can be conveniently studied in these very small samples.
Finally, when a structure’s three-dimensional volume is
cut into very thin sections, the sections appear microscopically
to have only two dimensions: length and width. When examin-
In situ hybridization of this tissue section with probes for the
ing a section under the microscope, the viewer must always keep
human papillomavirus (HPV) reveals the presence of many
cells containing the virus. The section was incubated with a in mind that components are missing in front of and behind
solution containing a digoxigenin-labeled complementary what is being seen because many tissue structures are thicker
DNA (cDNA) probe for the HPV DNA. The probe was then than the section. Round structures seen microscopically may
visualized by direct immunohistochemistry using peroxidase- actually be portions of spheres or tubes. Because structures in
labeled antibodies against digoxigenin. This procedure stains
a tissue have different orientations, their two-dimensional (2D)
brown only those cells containing HPV. (X400; H&E)
(Used with permission from Dr Jose E. Levi, Virology Lab, Insti- appearance will also vary depending on the plane of section. A
tute of Tropical Medicine, University of São Paulo, Brazil.) single convoluted tube will appear in a tissue section as many
separate rounded or oval structures (Figure 1–14).

01_Mescher_ch01_p001-016.indd 14 26/04/18 11:10 am

 Interpretation of Structures in Tissue Sections 15

FIGURE 1–14  Interpretation of 3D structures in 2D sections.

1 C H A P T E R
Histology & Its Methods of Study  ■  Interpretation of Structures in Tissue Sections
In thin sections 3D structures appear to have only two dimensions.
Such images must be interpreted correctly to understand the actual
structure of tissue and organ components. For example, blood ves-
sels and other tubular structures appear in sections as round or oval
shapes whose size and shape depend on the transverse or oblique
angle of the cut. A highly coiled tube will appear as several round
and oval structures. In TEM sections of cells, round structures may
represent spherical organelles or transverse cuts through tubular
organelles such as mitochondria. It is important to develop such
interpretive skill to understand tissue and cell morphology in micro-
scopic preparations.

Histology & Its Methods of Study   SUMMARY OF KEY POINTS

Preparation of Tissues for Study Autoradiography

■■ Chemical fixatives such as formalin are used to preserve tissue ■■ This process localizes cell components synthesized using radioactive
structure by cross-linking and denaturing proteins, inactivating precursors by detecting silver grains produced by weakly emitted radia-
enzymes, and preventing cell autolysis or self-digestion. tion in a photographic emulsion coating the tissue section or cells.
■■ Dehydration of the fixed tissue in alcohol and clearing in organic ■■ With either light microscopy or TEM, autoradiography permits
solvents prepare it for embedding and sectioning. unique studies of processes such as tissue growth (using radioactive
■■ Embedding in paraffin wax or epoxy resin allows the tissue to be DNA precursors) or cellular pathways of macromolecular synthesis.
cut into very thin sections (slices) with a microtome.
Cell & Tissue Culture
■■ Sections are mounted on glass slides for staining, which is
required to reveal specific cellular and tissue components with the ■■ Cells can be grown in vitro from newly explanted tissues (primary
microscope. cultures) or as long-established cell lines and can be examined in the
■■ The most commonly used staining method is a combination of the living state by phase-contrast light microscopy.
stains H&E, which act as basic and acidic dyes, respectively. Enzyme Histochemistry
■■ Cell substances with a net negative (anionic) charge, such as DNA ■■ Histochemical (or cytochemical) techniques use specific enzy-
and RNA, react strongly with hematoxylin and basic stains; such matic activities in lightly fixed or unfixed tissue sections to produce
material is said to be “basophilic.” visible products in the specific enzyme locations.
■■ Cationic substances, such as collagen and many cytoplasmic pro- ■■ Fixation and paraffin embedding denatures most enzymes, so histo-
teins react with eosin and other acidic stains and are said to be chemistry usually uses frozen tissue sectioned with a cryostat.
“acidophilic.” ■■ Enzyme classes for which histochemical study is useful include
phosphatases, dehydrogenases, and peroxidases, with peroxidase
Light Microscopy
often conjugated to antibodies used in immunohistochemistry.
■■ Bright-field microscopy, the method most commonly used by
both students and pathologists, uses ordinary light and the colors Visualizing Specific Molecules
are imparted by tissue staining. ■■ Some substances specifically bind certain targets in cells.
■■ Fluorescence microscopy uses UV light, under which only fluo- ■■ Immunohistochemistry is based on specific reactions between an anti-
rescent molecules are visible, allowing localization of fluorescent gen and antibodies labeled with visible markers, often fluorescent com-
probes which can be much more specific than routine stains. pounds or peroxidase for light microscopy and gold particles for TEM.
■■ Phase-contrast microscopy uses the differences in refractive ■■ If the cell or tissue antigen of interest is detected by directly binding
index of various natural cell and tissue components to produce an a labeled primary antibody specific for that antigen, the process is
image without staining, allowing observation of living cells. considered direct immunohistochemistry.
■■ Confocal microscopy involves scanning the specimen at succes- ■■ Indirect immunohistochemistry uses an unlabeled primary anti-
sive focal planes with a focused light beam, often from a laser, and body that is detected bound to its antigen with labeled secondary
produces a 3D reconstruction from the images. antibodies.

01_Mescher_ch01_p001-016.indd 15 26/04/18 11:10 am

 16 CHAPTER 1  ■  Histology & Its Methods of Study

■■ The indirect immunohistochemical method is more commonly used Interpretation of Structures in Tissue Sections
because the added level of antibody binding amplifies the signal ■■ Many steps in tissue processing, slide preparation, and staining can
detected and provides greater technical flexibility. introduce minor artifacts such as spaces and precipitates that are not
■■ Specific gene sequences or mRNAs of cells can be detected micro- normally present in the living tissue and must be recognized.
scopically using labeled cDNA probes in a technique called in situ ■■ Sections of cells or tissues are essentially 2D planes through 3D
hybridization (ISH). structures, and understanding this fact is important for their correct
interpretation and study.

Histology & Its Methods of Study   ASSESS YOUR KNOWLEDGE

1. In preparing tissue for routine light microscopic study, which 7. Microscopic autoradiography uses radioactivity and can be
procedure immediately precedes clearing the specimen with an employed to study what features in a tissue section?
organic solvent? a. The types of enzymes found in various cell locations
a. Dehydration b. Cellular sites where various macromolecules are synthesized
b. Fixation c. The sequences of mRNA made in the cells
c. Staining d. The dimensions of structures within the cells
d. Clearing e. The locations of genes transcribed for specific mRNA
e. Embedding
8. To identify and localize a specific protein within cells or the ECM,
2. Which of the following staining procedures relies on the cationic one would best use what approach?
and anionic properties of the material to be stained? a. Autoradiography
a. Enzyme histochemistry b. Enzyme histochemistry
b. PAS reaction c. Immunohistochemistry
c. H&E staining d. TEM
d. Immunohistochemistry e. Polarizing microscopy
e. Metal impregnation techniques
9. In situ hybridization is a histologic technique used to visualize what
3. In a light microscope used for histology, resolution and magnifica- type of macromolecule?
tion of cells are largely dependent on which component? a. Proteins
a. Condenser b. Carbohydrates
b. Objective lens c. Certain enzymes
c. Eyepieces or ocular lenses d. Nucleic acids
d. Specimen slide e. Lipids
e. The control for illumination intensity
10. Hospital laboratories frequently use unfixed, frozen tissue specimens
4. Cellular storage deposits of glycogen, a free polysaccharide, could sectioned with a cryostat for rapid staining, microscopic examina-
best be detected histologically using what procedure? tion, and diagnosis of pathologic conditions. Besides saving much
a. Autoradiography time by avoiding fixation and procedures required for paraffin
b. Electron microscopy embedding, frozen sections retain and allow study of what macro-
c. Enzyme histochemistry molecules normally lost in the paraffin procedure?
d. H&E staining a. Carbohydrates
e. PAS reaction b. Small mRNA
c. Basic proteins
5. Adding heavy metal compounds to the fixative and ultrathin sec- d. Acidic proteins
tioning of the embedded tissue with a glass knife are techniques e. Lipids
used for which histologic procedure?
a. Scanning electron microscopy
b. Fluorescent microscopy
c. Enzyme histochemistry
d. Confocal microscopy
e. TEM
6. Resolution in electron microscopy greatly exceeds that of light
microscopy due to which of the following?
a. The wavelength of the electrons in the microscope beam is
shorter than that of a beam of light.
b. The lenses of an electron microscope are of greatly improved
quality.
c. For electron microscopy the tissue specimen does not require
staining.
d. The electron microscope allows much greater magnification of
a projected image than a light microscope provides.
e. An electron microscope can be much more finely controlled Answers: 1a, 2c, 3b, 4e, 5e, 6a, 7b, 8c, 9d, 10e
than a light microscope.

01_Mescher_ch01_p001-016.indd 16 26/04/18 11:10 am

 C H A P T E R

CELL DIFFERENTIATION
2 The Cytoplasm

17 Proteasomes37
Mitochondria38
THE PLASMA MEMBRANE 17
Transmembrane Proteins & Membrane Transport 19 Peroxisomes39
Transport by Vesicles: Endocytosis & Exocytosis 21 THE CYTOSKELETON 42
Signal Reception & Transduction 23 Microtubules43
CYTOPLASMIC ORGANELLES 27 Microfilaments (Actin Filaments) 44
Ribosomes27 Intermediate Filaments 45
Endoplasmic Reticulum 28 INCLUSIONS47
Golgi Apparatus 31 SUMMARY OF KEY POINTS 51
Secretory Granules 33
ASSESS YOUR KNOWLEDGE 52
Lysosomes34

C ells and extracellular material together comprise the

tissues that make up animal organs. In all tissues,
cells are the basic structural and functional units, the
smallest living parts of the body. Animal cells are enclosed
by cell membranes and are eukaryotic, each with a distinct,
using these proteins to convert chemical energy into forceful
contractions.
Major cellular functions performed by specialized cells in the
body are listed in Table 2–1. It is important to understand that the
functions listed there can be performed by most cells of the body;
membrane-enclosed nucleus surrounded by cytoplasm, fluid specialized cells have greatly expanded their capacity for one or
containing a system of membranous organelles, nonmembra- more of these functions during differentiation. Changes in cells’
nous molecular assemblies, and a cytoskeleton. In contrast, microenvironments under normal and pathologic conditions
the smaller prokaryotic cells of bacteria typically have a cell can cause the same cell type to have variable features and activi-
wall and lack nuclei and membranous cytoplasmic structures. ties. Cells that appear similar structurally often have different
families of receptors for signaling molecules such as hormones

››CELL DIFFERENTIATION
and extracellular matrix (ECM) components, causing them to
behave differently. For example, because of their diverse arrays of
receptors, breast fibroblasts and uterine smooth muscle cells are
The average adult human body consists of nearly 40 trillion
exceptionally sensitive to female sex hormones, while most other
cells, according to the best available estimate. These cells exist
fibroblasts and smooth muscle cells are insensitive.
as hundreds of histologically distinct cell types, all derived
from the zygote, and the single cell formed by the merger of a
spermatozoon with an oocyte at fertilization. The first zygotic
cellular divisions produce cells called blastomeres, and as ››THE PLASMA MEMBRANE
part of the early embryo’s inner cell mass blastomeres give The plasma membrane (cell membrane or plasmalemma)
rise to all tissue types of the fetus. Explanted to tissue culture that envelops every eukaryotic cell consists of phospholipids,
cells of the inner cell mass are called embryonic stem cells. cholesterol, and proteins, with oligosaccharide chains covalently
Most cells of the fetus undergo a specialization process called linked to many of the phospholipids and proteins. This limiting
differentiation in which they predominantly express sets of membrane functions as a selective barrier regulating the passage
genes that mediate specific cytoplasmic activities, becoming of materials into and out of the cell and facilitating the transport
efficiently organized in tissues with specialized functions and of specific molecules. One important role of the cell membrane is
usually changing their shape accordingly. For example, muscle to keep constant the ion content of cytoplasm, which differs from
cell precursors elongate into long, fiber-like cells containing that of the extracellular fluid. Membrane proteins also perform a
large arrays of actin and myosin. All animal cells contain actin number of specific recognition and signaling functions, playing a
filaments and myosins, but muscle cells are specialized for key role in the interactions of the cell with its environment.

17

02_Mescher_ch02_p017-052.indd 17 27/04/18 6:48 pm

 18 CHAPTER 2  ■  The Cytoplasm

abundant in the outer half, while phosphatidylserine and

       
Differentiated cells typically
TABLE 2–1 specialize in one activity.
phosphatidylethanolamine are more concentrated in the inner
layer. Some of the outer layer’s lipids, known as glycolipids,
General Cellular Activity Specialized Cell(s) include oligosaccharide chains that extend outward from the
cell surface and contribute to a delicate cell surface coating
Movement Muscle and other contractile called the glycocalyx (Figures 2–1b and 2–2). With the trans-
cells mission electron microscope (TEM) the cell membrane—as
Form adhesive and tight Epithelial cells well as all cytoplasmic membranes—may exhibit a trilaminar
junctions between cells appearance after fixation in osmium tetroxide; osmium binds
Synthesize and secrete Fibroblasts, cells of bone and the polar heads of the phospholipids and the oligosaccharide
components of the extracellular cartilage chains, producing the two dark outer lines that enclose the
matrix light band of osmium-free fatty acids (Figure 2–1b).
Convert physical and chemical Neurons and sensory cells Proteins are major constituents of membranes (~50%
stimuli into action potentials by weight in the plasma membrane). Integral proteins are
Synthesis and secretion of Cells of digestive glands incorporated directly within the lipid bilayer, whereas periph-
degradative enzymes eral proteins are bound to one of the two membrane surfaces,
Synthesis and secretion of Cells of mucous glands
particularly on the cytoplasmic side (Figure 2–2). Peripheral
glycoproteins proteins can be extracted from cell membranes with salt solu-
tions, whereas integral proteins can be extracted only by using
Synthesis and secretion of Certain cells of the adrenal
steroids gland, testis, and ovary
detergents to disrupt the lipids. The polypeptide chains of
many integral proteins span the membrane, from one side to
Ion transport Cells of the kidney and the other, several times and are accordingly called multipass
salivary gland ducts
proteins. Integration of the proteins within the lipid bilayer
Intracellular digestion Macrophages and neutrophils is mainly the result of hydrophobic interactions between the
Lipid storage Fat cells lipids and nonpolar amino acids of the proteins.
Freeze-fracture electron-microscope studies of mem-
Metabolite absorption Cells lining the intestine
branes show that parts of many integral proteins protrude
from both the outer or inner membrane surface (Figure 2–2b).
Although the plasma membrane defines the outer limit of Like those of glycolipids, the carbohydrate moieties of glyco-
the cell, a continuum exists between the interior of the cell and proteins project from the external surface of the plasma mem-
extracellular macromolecules. Certain plasma membrane pro- brane and contribute to the glycocalyx (Figure 2–3). They are
teins, the integrins, are linked to both the cytoskeleton and important components of proteins acting as receptors, which
ECM components and allow continuous exchange of influ- participate in important interactions such as cell adhesion, cell
ences, in both directions, between the cytoplasm and material recognition, and the response to protein hormones. As with
in the ECM. lipids, the distribution of membrane polypeptides is different
Membranes range from 7.5 to 10 nm in thickness and in the two surfaces of the cell membranes. Therefore, all mem-
consequently are visible only in the electron microscope. The branes in the cell are asymmetric.
line between adjacent cells sometimes seen faintly with the Studies with labeled membrane proteins of cultured cells
light microscope consists of plasma membrane proteins plus reveal that many such proteins are not bound rigidly in place
extracellular material, which together can reach a dimension and are able to move laterally (Figure 2–4). Such observa-
visible by light microscopy. tions as well as data from biochemical, electron microscopic,
Membrane phospholipids are amphipathic, consisting of and other studies showed that membrane proteins comprise
two nonpolar (hydrophobic or water-repelling) long-chain a moveable mosaic within the fluid lipid bilayer, the well-
fatty acids linked to a charged polar (hydrophilic or water- established fluid mosaic model for membrane structure
attracting) head that bears a phosphate group (Figure 2–1a). (Figure 2–2a). Unlike the lipids, however, lateral diffusion of
Phospholipids are most stable when organized into a double many membrane proteins is often restricted by their cyto-
layer (bilayer) with the hydrophobic fatty acid chains located skeletal attachments. Moreover, in most epithelial cells tight
in a middle region away from water and the hydrophilic polar junctions between the cells (see Chapter 4) also restrict lat-
head groups contacting the water (Figure 2–1b). Molecules eral diffusion of unattached transmembrane proteins and
of cholesterol, a sterol lipid, insert at varying densities among outer layer lipids, producing different domains within the
the closely-packed phospholipid fatty acids, restricting their cell membranes.
movements and modulating the fluidity of all membrane Membrane proteins that are components of large enzyme
components. The phospholipids in each half of the bilayer are complexes are also usually less mobile, especially those
different. For example, in the well-studied membranes of red involved in the transduction of signals from outside the cell.
blood cells, phosphatidylcholine and sphingomyelin are more Such protein complexes are located in specialized membrane

02_Mescher_ch02_p017-052.indd 18 25/04/18 6:47 pm

 The Plasma Membrane 19

FIGURE 2–1  Lipids in membrane structure.

C H A P T E R
Polar head group Nonpolar fatty acid chains
(hydrophilic) (hydrophobic)

O
Saturated CH3
CH2 O C
fatty acid
O (straight) CH3

2
CH O C

The Cytoplasm  ■  The Plasma Membrane

O
Unsaturated
CH2 O P O X fatty acid OH
O– (bent)

General structure of a phospholipid Cholesterol

Sugar chains of a glycolipid

Phospholipids

Hydrophilic surface

Hydrophobic region
Extracellular fluid
Hydrophilic surface

Cholesterol
Cytoplasm

(a) Membranes of animal cells have as their major lipid com- throughout the lipid bilayer; cholesterol affects the packing of the
ponents phospholipids and cholesterol. A phospholipid is fatty acid chains, with a major effect on membrane fluidity. The
amphipathic, with a phosphate group charge on the polar head outer layer of the cell membrane also contains glycolipids with
and two long, nonpolar fatty acid chains, which can be straight extended carbohydrate chains.
(saturated) or kinked (at an unsaturated bond). Membrane cho- Sectioned, osmium-fixed cell membrane may have a faint trilami-
lesterol is present in about the same amount as phospholipid. nar appearance with the transmission electron microscope (TEM),
(b) The amphipathic nature of phospholipids produces the bilayer showing two dark (electron-dense) lines enclosing a clear (electron-
structure of membranes as the charged (hydrophilic) polar heads lucent) band. Reduced osmium is deposited on the hydrophilic phos-
spontaneously form each membrane surface, in direct contact phate groups present on each side of the internal region of fatty acid
with water, and the hydrophobic nonpolar fatty acid chains are chains where osmium is not deposited. The “fuzzy” material on the
buried in the membrane’s middle, away from water. Cholesterol outer surface of the membrane represents the glycocalyx of oligo-
molecules are also amphipathic and are interspersed less evenly saccharides of glycolipids and glycoproteins. (X100,000)

patches termed lipid rafts with higher concentrations of cho- small molecules cross the membrane by the general mecha-
lesterol and saturated fatty acids which reduce lipid fluidity. nisms shown schematically in Figure 2–5 and explained as
This together with the presence of scaffold proteins that main- follows:
tain spatial relationships between enzymes and signaling pro-
teins allows the proteins assembled within lipid rafts to remain
■■ Diffusion transports small, nonpolar molecules directly
through the lipid bilayer. Lipophilic (fat-soluble) mol-
in close proximity and interact more efficiently.
ecules diffuse through membranes readily, water very
slowly.
Transmembrane Proteins & MembraneTransport ■■ Channels are multipass proteins forming transmem-
The plasma membrane is the site where materials are brane pores through which ions or small molecules
exchanged between the cell and its environment. Most pass selectively. Cells open and close specific channels

02_Mescher_ch02_p017-052.indd 19 25/04/18 6:47 pm

 20 CHAPTER 2  ■  The Cytoplasm

FIGURE 2–2  Proteins associated with the membrane lipid bilayer.

Sugar chain Sugar chain

of glycolipid of glycoprotein

2 E face
Peripheral protein 1
Transmembrane protein

Lipid

P face

(a) The fluid mosaic model of membrane structure emphasizes (b) When cells are frozen and fractured (cryofracture), the lipid
that the phospholipid bilayer of a membrane also contains pro- bilayer of membranes is often cleaved along the hydrophobic
teins inserted in it or associated with its surface (peripheral pro- center. Splitting occurs along the line of weakness formed by
teins) and that many of these proteins move within the fluid lipid the fatty acid tails of phospholipids. Electron microscopy of such
phase. Integral proteins are firmly embedded in the lipid layers; cryofracture preparation replicas provides a useful method for
those that completely span the bilayer are called transmem- studying membrane structures. Most of the protruding mem-
brane proteins. Hydrophobic amino acids of these proteins inter- brane particles seen (1) are proteins or aggregates of proteins
act with the hydrophobic fatty acid portions of the membrane that remain attached to the half of the membrane adjacent to
lipids. Both the proteins and lipids may have externally exposed the cytoplasm (P or protoplasmic face). Fewer particles are found
oligosaccharide chains. attached to the outer half of the membrane (E or extracellular
face). Each protein bulging on one surface has a corresponding
depression (2) on the opposite surface.

for Na+, K+, Ca2+, and other ions in response to various down a concentration gradient due to its kinetic energy. In
physiological stimuli. Water molecules usually cross contrast, membrane pumps are enzymes engaged in active
the plasma membrane through channel proteins called transport, utilizing energy from the hydrolysis of adenos-
aquaporins. ine triphosphate (ATP) to move ions and other solutes across
■■ Carriers are transmembrane proteins that bind small membranes, against often steep concentration gradients.
molecules and translocate them across the membrane via Because they consume ATP pumps, they are often referred
conformational changes. to as ATPases.
Diffusion, channels, and carrier proteins operate pas- These transport mechanisms are summarized with addi-
sively, allowing movement of substances across membranes tional detail in Table 2–2.

02_Mescher_ch02_p017-052.indd 20 25/04/18 6:47 pm

 The Plasma Membrane 21

FIGURE 2–3  Membrane proteins.

C H A P T E R
Interstitial fluid

Phospholipid

2
Glycolipid Carbohydrate

The Cytoplasm  ■  The Plasma Membrane

Polar head of
phospholipid
molecule
Phospholipid
bilayer

Glycoprotein
Nonpolar tails
of phospholipid Cholesterol Protein
molecule
Integral protein

Peripheral protein

Filaments of
cytoskeleton
Cytosol

Functions of Plasma Membrane

1. Physical barrier: Establishes a flexible boundary, protects cellular contents, 3. Electrochemical gradients: Establishes and maintains an electrical
and supports cell structure. Phospholipid bilayer separates substances charge difference across the plasma membrane.
inside and outside the cell. 4. Communication: Contains receptors that recognize and respond to
2. Selective permeability: Regulates entry and exit of ions, nutrients, molecular signals.
and waste molecules through the membrane.

Both protein and lipid components often have covalently connections, and as selective gateways for molecules entering
attached oligosaccharide chains exposed at the external mem- the cell.
brane surface. These contribute to the cell’s glycocalyx, which Transmembrane proteins often have multiple hydrophobic
provides important antigenic and functional properties to the regions buried within the lipid bilayer to produce a channel or
cell surface. Membrane proteins serve as receptors for vari- other active site for specific transfer of substances through the
ous signals coming from outside cells, as parts of intercellular membrane.

Transport by Vesicles: Endocytosis & Exocytosis cytoskeletal changes. Fusion of the membranous folds
encloses the bacterium in an intracellular vacuole called
Macromolecules normally enter cells by being enclosed within
a phagosome, which then merges with a lysosome
folds of plasma membrane (often after binding specific mem-
for degradation of its contents as discussed later in this
brane receptors) which fuse and pinch off internally as spheri-
chapter.
cal cytoplasmic vesicles (or vacuoles) in a general process
known as endocytosis. Three major types of endocytosis are 2. Pinocytosis (“cell drinking”) involves smaller invagina-
recognized, as summarized in Table 2–2 and Figure 2–6. tions of the cell membrane which fuse and entrap extra-
cellular fluid and its dissolved contents. The resulting
1. Phagocytosis (“cell eating”) is the ingestion of particles pinocytotic vesicles (~80 nm in diameter) then pinch
such as bacteria or dead cell remnants. Certain blood- off inwardly from the cell surface and either fuse with
derived cells, such as macrophages and neutrophils, are lysosomes or move to the opposite cell surface where
specialized for this activity. When a bacterium becomes they fuse with the membrane and release their contents
bound to the surface of a neutrophil, it becomes sur- outside the cell. The latter process, called transcytosis,
rounded by extensions of plasmalemma and cytoplasm accomplishes bulk transfer of dissolved substances across
which project from the cell in a process dependent on the cell.

02_Mescher_ch02_p017-052.indd 21 25/04/18 6:47 pm

 22 CHAPTER 2  ■  The Cytoplasm

receptors causes these proteins to aggregate in special

FIGURE 2–4  Experiment demonstrating the membrane regions that then invaginate and pinch off
fluidity of membrane proteins.
internally as vesicles.
The formation and fate of vesicles in receptor-mediated
endocytosis also often depend on specific peripheral proteins
on the cytoplasmic side of the membrane (Figure 2–7). The
occupied cell-surface receptors associate with these cytoplas-
mic proteins and begin to invaginate as coated pits. The
electron-dense coating on the cytoplasmic surface of such pits
contains several polypeptides, the major one being clathrin.
Clathrin molecules interact like the struts of a geodesic dome,
a forming that region of cell membrane into a cage-like invagi-
nation that soon pinches off into the cytoplasm as a coated
vesicle (Figure 2–7b) with the receptor-bound ligands inside.
Another type of receptor-mediated endocytosis prominent
in very thin cells produces invaginations called caveolae
(L. caveolae, little caves) that involve a family of integral mem-
brane proteins called caveolins associated with diverse periph-
eral proteins called cavins.
In all these endocytotic processes, the vesicles or vacuoles
produced quickly enter and fuse with the endosomal com-
partment, a dynamic collection in the peripheral cytoplasm
b of membranous tubules and vacuoles (Figure 2–7). The clath-
rin molecules separate from the coated vesicles and recycle
back to the cell membrane for the formation of new coated
pits. Vesicle trafficking through the endosomal compartment
is directed largely through peripheral membrane G-proteins
called Rab proteins, small GTPases that bind guanine nucle-
otides and associated proteins.
As shown in Figure 2–7, phagosomes and pinocytotic
vesicles typically fuse with lysosomes within the endosomal
compartment for digestion of their contents, while molecules
entering by receptor-mediated endocytosis may be directed
down other pathways. The membranes of many late endo-
somes have ATP-driven H+ pumps that acidify their interior,
activating the hydrolytic enzymes of lysosomes, and in other
endosomes causing ligands to uncouple from their receptors,
c
after which the two molecules are sorted into separate endo-
(a) Two types of cells were grown in tissue cultures, one with fluo- somes. The receptors may be sorted into recycling endosomes
rescently labeled transmembrane proteins in the plasmalemma and returned to the cell surface for reuse. Low-density lipopro-
(right) and one without. tein receptors, for example, are recycled several times within
(b) Cells of each type were fused together experimentally into cells. Other endosomes may release their entire contents at a
hybrid cells. separate domain of the cell membrane (transcytosis), which
(c) Minutes after the fusion of the cell membranes, the fluorescent occurs in many epithelial cells.
proteins of the labeled cell spread to the entire surface of the Movement of large molecules from inside to outside
hybrid cells. Such experiments provide important data supporting
the cell usually involves vesicular transport in the process of
the fluid mosaic model. However, many membrane proteins show
more restricted lateral movements, being anchored in place by exocytosis. In exocytosis, a cytoplasmic vesicle containing
links to the cytoskeleton. the molecules to be secreted fuses with the plasma membrane,
resulting in the release of its contents into the extracellu-
lar space without compromising the integrity of the plasma
membrane (see “Transcytosis” in Figure 2–7a). Exocytosis
3. Receptor-mediated endocytosis: Receptors for many is triggered in many cells by a transient increase in cytosolic
substances, such as low-density lipoproteins and protein Ca2+. Membrane fusion during exocytosis is highly regulated,
hormones, are integral membrane proteins at the cell with selective interactions between several specific membrane
surface. High-affinity binding of such ligands to their proteins.

02_Mescher_ch02_p017-052.indd 22 25/04/18 6:47 pm

 The Plasma Membrane 23

FIGURE 2–5  Major mechanisms by which molecules cross membranes.

2 C H A P T E R
The Cytoplasm  ■  The Plasma Membrane
(a) Simple diffusion (b) Channel (c) Carrier/pump

Lipophilic and some small, uncharged molecules can cross mem- conformations and release the molecule to the other side of the
branes by simple diffusion (a). membrane.
Most ions cross membranes in multipass proteins called chan- Diffusion, channels and most carrier proteins translocate sub-
nels (b) whose structures include transmembrane ion-specific pores. stances across membranes using only kinetic energy. In contrast,
Many other larger, water-soluble molecules require binding pumps are carrier proteins for active transport of ions or other
to sites on selective carrier proteins (c), which then change their solutes and require energy derived from ATP.

Exocytosis of macromolecules made by cells occurs via are called exosomes, which can fuse with other cells transfer-
either of two pathways: ring their contents and membranes.
■■ Constitutive secretion is used for products that are
released from cells continuously, as soon as synthesis is Signal Reception & Transduction
complete, such as collagen subunits for the ECM. Cells in a multicellular organism communicate with one
■■ Regulated secretion occurs in response to signals com- another to regulate tissue and organ development, to control
ing to the cells, such as the release of digestive enzymes their growth and division, and to coordinate their functions.
from pancreatic cells in response to specific stimuli. Many adjacent cells form communicating gap junctions that
Regulated exocytosis of stored products from epithelial couple the cells and allow exchange of ions and small mol-
cells usually occurs specifically at the apical domains of ecules (see Chapter 4).
cells, constituting a major mechanism of glandular secre- Cells also use about 25 families of receptors to detect
tion (see Chapter 4). and respond to various extracellular molecules and physical
stimuli. Each cell type in the body contains a distinctive set
Portions of the cell membrane become part of the endo-
of cell surface and cytoplasmic receptor proteins that enable
cytotic vesicles or vacuoles during endocytosis; during exocy-
it to respond to a complementary set of signaling molecules
tosis, membrane is returned to the cell surface. This process
in a specific, programmed way. Cells bearing receptors for a
of membrane movement and recycling is called membrane
specific ligand are referred to as target cells for that molecule.
trafficking (see Figure 2–7a). Trafficking of membrane com-
The routes of signal molecules from source to target provide
ponents occurs continuously in most cells and is not only
one way to categorize the signaling process:
crucial for maintaining the cell but also for physiologically
important processes such as reducing blood lipid levels. ■■ In endocrine signaling, the signal molecules (here
In many cells subpopulations of vacuoles and tubules called hormones) are carried in the blood from their
within the endosomal compartment accumulate small vesicles sources to target cells throughout the body.
within their lumens by further invaginations of their limiting ■■ In paracrine signaling, the chemical ligand diffuses in
membranes, becoming multivesicular bodies. While multi- extracellular fluid but is rapidly metabolized so that its
vesicular bodies may merge with lysosomes for selective deg- effect is only local on target cells near its source.
radation of their content, this organelle may also fuse with the ■■ In synaptic signaling, a special kind of paracrine inter-
plasma membrane and release the intralumenal vesicles out- action, neurotransmitters act on adjacent cells through
side the cell. The small (<120 nm diameter) vesicles released special contact areas called synapses (see Chapter 9).

02_Mescher_ch02_p017-052.indd 23 25/04/18 6:47 pm

 24 CHAPTER 2  ■  The Cytoplasm

TABLE 2–2        Mechanisms of transport across the plasma membrane.

Process Type of Movement Example

PASSIVE PROCESSES Movement of substances down a concentration gradient due to the kinetic energy of the substance; no
expenditure of cellular energy is required; continues until equilibrium is reached (if unopposed)
Simple diffusion Unassisted net movement of small, nonpolar Exchange of oxygen and carbon dioxide between
substances down their concentration gradient blood and body tissues
across a selectively permeable membrane
Facilitated diffusion Movement of ions and small, polar molecules
down their concentration gradient; assisted across
a selectively permeable membrane by a transport
protein
 Channel-mediated Movement of ion down its concentration gradient Na+ moves through Na+ channel into cell
through a protein channel
 Carrier-mediated Movement of small, polar molecule down its Transport of glucose into cells by glucose carrier
concentration gradient by a carrier protein
Osmosis Diffusion of water across a selectively permeable Solutes in blood in systemic capillaries “pulls” fluid
membrane; direction is determined by relative from interstitial space back into the blood
solute concentrations; continues until equilibrium
is reached
ACTIVE PROCESSES Movement of substances requires expenditure of cellular energy
Active transport Transport of ions or small molecules across the
membrane against a concentration gradient by
transmembrane protein pumps
 Primary Movement of substance up its concentration Ca2+ pumps transport Ca2+ out of the cell Na+/K+
gradient; powered directly by ATP pump moves Na+ out of the cell and K+ into the cell
 Secondary Movement of a substance up its concentration
gradient is powered by harnessing the movement
of a second substance (eg, Na+) down its
concentration gradient
 Symport Movement of substance up its concentration Na+/glucose transport
gradient in the same direction as Na+
 Antiport Movement of substance up its concentration Na+/H+ transport
gradient in the opposite direction from Na+
Vesicular transport Vesicle formed or lost as material is brought into a
cell or released from a cell
 Exocytosis Bulk movement of substance out of the Release of neurotransmitter by nerve cells
cell by fusion of secretory vesicles with the
plasma membrane
 Endocytosis Bulk movement of substances into the cell by
vesicles forming at the plasma membrane
 Phagocytosis Type of endocytosis in which vesicles are formed White blood cell engulfing a bacterium
as particulate materials external to the cell are
engulfed by pseudopodia
 Pinocytosis Type of endocytosis in which vesicles are formed as Formation of small vesicles in capillary wall
interstital fluid is taken up by the cell to move substances
 Receptor-mediated Type of endocytosis in which plasma membrane Uptake of cholesterol into cells
endocytosis receptors first bind specific substances; receptor
and bound substance then taken up by the cell

02_Mescher_ch02_p017-052.indd 24 25/04/18 6:47 pm

 The Plasma Membrane 25

FIGURE 2–6  Three major forms of endocytosis.

C H A P T E R
Extracellular fluid
Receptors
Pseudopodia
Particle

Plasma

2
membrane

The Cytoplasm  ■  The Plasma Membrane

Plasma
membrane
Vacuole
Cytoplasmic
vesicle
Cytoplasm
a Phagocytosis
c Receptor-mediated endocytosis

There are three general types of endocytosis:

(a) Phagocytosis involves the extension from the cell of surface
folds or pseudopodia which engulf particles such as bacteria,
and then internalize this material into a cytoplasmic vacuole or
phagosome.
(b) In pinocytosis the cell membrane forms similar folds or
invaginates (dimples inward) to create a pit containing a drop of
Plasma extracellular fluid. The pit pinches off inside the cell when the cell
Vesicle membrane membrane fuses and forms a pinocytotic vesicle containing the
fluid.
(c) Receptor-mediated endocytosis includes membrane proteins
called receptors that bind specific molecules (ligands). When
many such receptors are bound by their ligands, they aggregate in
one membrane region, which then invaginates and pinches off to
create a vesicle or endosome containing both the receptors and
the bound ligands.
b Pinocytosis

■■ In autocrine signaling, signals bind receptors on the

same cells that produced the messenger molecule. › ›› MEDICAL APPLICATION
■■ In juxtacrine signaling, important in early embryonic Many diseases are caused by defective receptors. For exam-
tissue interactions, the signaling molecules are cell mem- ple, pseudohypoparathyroidism and one type of dwarfism
brane-bound proteins which bind surface receptors of the are caused by nonfunctioning parathyroid and growth hor-
target cell when the two cells make direct physical contact. mone receptors, respectively. In these two conditions, the
glands produce the respective hormones, but the target cells
Receptors for hydrophilic signaling molecules, includ- cannot respond because they lack normal receptors.
ing polypeptide hormones and neurotransmitters, are usu-
ally transmembrane proteins in the plasmalemma of target
Ligands binding such receptors in a cell membrane can
cells. Three important functional classes of such receptors are
be considered first messengers, beginning a process of signal
shown in Figure 2–8:
transduction by activating a series of intermediary enzymes
■■ Channel-linked receptors open associated channels downstream to produce changes in the cytoplasm, the
upon ligand binding to promote transfer of molecules or nucleus, or both. Channel-mediated ion influx or activation of
ions across the membrane. kinases can activate various cytoplasmic proteins, amplifying
■■ Enzymatic receptors, in which ligand binding induces the signal. Activated G-proteins target ion channels or other
catalytic activity in associated peripheral proteins. membrane-bound effectors that also propagate the signal
■■ G-protein-coupled receptors upon ligand binding further into the cell (Figure 2–8). One such effector protein
stimulate associated G-proteins which then bind the gua- is the enzyme adenyl cyclase which generates large quanti-
nine nucleotide GTP and are released to activate other ties of second messenger molecules, such as cyclic adenosine
cytoplasmic proteins. monophosphate (cAMP). Other second messengers include

02_Mescher_ch02_p017-052.indd 25 25/04/18 6:47 pm

 26 CHAPTER 2  ■  The Cytoplasm

FIGURE 2–7  Receptor-mediated endocytosis involves regulated membrane trafficking.

Receptor
Ligand ligand Clathrin
complexes coat

Receptors

Coated pit
Apical
domain Dynamin
of cell Adaptor
membrane protein Clathrin

Coat
proteins Coated CP CP
recycled vesicle
CV
Receptor
recycling
Early
endosome

Late
endosome
Transcytosis b
Lysosomal
degradation

Basolateral domain
of cell membrane

Major steps during and after endocytosis are indicated diagram- ■■ Receptors and ligands may be carried to late endosomes and
matically in part a. Ligands bind with high affinity to specific then to lysosomes for degradation.
surface receptors, which then associate with specific cytoplasmic ■■ Ligands may be released from the receptors and the empty
proteins, including clathrin and adaptor proteins, and aggregate receptors sequestered into recycling endosomes and
in membrane regions to form coated pits. Clathrin facilitates returned to the cell surface for reuse.
invagination of the pits, and another peripheral membrane pro- ■■ Other endosomal vesicles containing ligands may move
tein, dynamin, forms constricting loops around the developing to and fuse with another cell surface, where the ligands are
neck of the pit, causing the invagination to pinch off as a coated released again outside the cell in the process of transcytosis.
vesicle. The clathrin lattice of coated pits (CP) and vesicles (CV) is
(Figure 2–7b, used with permission from Dr John Heuser,
shown ultrastructurally in part b.
Department of Cell Biology and Physiology, Washington University
Internalized vesicles lose their clathrin coats, which are recy-
School of Medicine, St. Louis, MO.)
cled, and fuse with other endosomes that comprise the endo-
somal compartment. Ligands may have different fates within the
endosomal compartment:

1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate carrier proteins in the plasma for transport through the body.
(IP3). The ionic changes or second messengers amplify the Such hormones are lipophilic and pass by diffusion through
first signal and trigger a cascade of enzymatic activity, usu- cell membranes, binding to specific cytoplasmic receptor pro-
ally including kinases, leading to changes in gene expression teins in target cells. With many steroid hormones, receptor
or cell behavior. Second messengers may diffuse through the binding activates that receptor, enabling the complex to move
cytoplasm or be retained locally by scaffold proteins for more into the nucleus and bind with high affinity to specific DNA
focused amplification of activity. sequences. This generally increases the level of transcription of
Low-molecular-weight hydrophobic signaling molecules, those genes. Each steroid hormone is recognized by a different
such as steroids and thyroid hormones, bind reversibly to member of a family of homologous receptor proteins.

02_Mescher_ch02_p017-052.indd 26 25/04/18 6:47 pm

 Cytoplasmic Organelles 27

FIGURE 2–8  Major types of membrane receptors.

C H A P T E R
Channel open
Ions

Ligand g
Ligand

2
Channel
closed

The Cytoplasm  ■  Cytoplasmic Organelles

Inactive protein Active protein
kinase enzyme kinase enzyme
phosphorylates
other enzymes.
Ions
Phosphate Enzyme turned
on or turned off

a Channel-linked receptors b Enzymatic receptors

1 A ligand binds to a
receptor, causing a Ions
connfor
format
ma ion
mat ional
conformational al change
change
Ligand o activate
to acctiv
a tivate
a receptor.
recep
cep
ceptor
e tor
tor. Effector protein (eg, ion channel)

2 G protein binds to activated Inactive protein

receptor. kinase enzyme 5 Active protein
kinase enzyme
phosphorylates other
Second enzymes
messenger
Activated
Ac iva
Act ivated
ted
d
G protein
prot
ro
otein
ote
ei
GTP 3 GTP binds to G protein Phosphate
4 The activated effector protein
causing G-protein activation. makes secondary messenger
Activated G protein leaves the Effector protein
available within the cell, which
receptor. It attaches to and (eg, enzyme)
leads to protein kinase Enzyme turned
activates an effector protein. enzyme activation. on or turned off
(an ion channel or an enzyme).
c G-protein–coupled receptors

Protein and most small ligands are hydrophilic molecules that activated to phosphorylate (and usually activate) other proteins
bind transmembrane protein receptors to initiate changes in the upon ligand binding. (c) G-protein-coupled receptors bind
target cell. ligand, changing the conformation of its G-protein subunit,
(a) Channel-linked receptors bind ligands such as allowing it to bind GTP, and activating and releasing this pro-
neurotransmitters and open to allow influx of specific ions. tein to in turn activate other proteins such as ion channels and
(b) Enzymatic receptors are usually protein kinases that are adenyl cyclase.

››CYTOPLASMIC ORGANELLES energy. Oxygen, CO2, electrolytic ions, low-molecular-weight

substrates, metabolites, and waste products all diffuse through
Inside the cell membrane the fluid cytoplasm (or cytosol) cytoplasm, either freely or bound to proteins, entering or leav-
bathes metabolically active structures called organelles, ing organelles where they are used or produced.
which may be membranous (such as mitochondria) or non-
membranous protein complexes (such as ribosomes and pro-
teasomes). Most organelles are positioned in the cytoplasm by Ribosomes
movements along the polymers of the cytoskeleton, which Ribosomes are macromolecular machines, about 20 × 30 nm
also determines a cell’s shape and motility. in size, which assemble polypeptides from amino acids on
Cytosol also contains hundreds of enzymes, such as those molecules of transfer RNA (tRNA) in a sequence specified
of the glycolytic pathway, which produce building blocks for by mRNA. A functional ribosome has two subunits of differ-
larger molecules and break down small molecules to liberate ent sizes bound to a strand of mRNA. The core of the small

02_Mescher_ch02_p017-052.indd 27 25/04/18 6:47 pm

 28 CHAPTER 2  ■  The Cytoplasm

ribosomal subunit is a highly folded ribosomal RNA (rRNA) properly are conjugated to the protein ubiquitin that targets them
chain associated with more than 30 unique proteins; the core for breakdown by proteasomes (discussed below). As indicated
of the large subunit has three other rRNA molecules and in Figure 2–9, proteins synthesized for use within the cytosol (eg,
nearly 50 other basic proteins. glycolytic enzymes) or for import into the nucleus and certain
The rRNA molecules in the ribosomal subunits not only other organelles are synthesized on polyribosomes existing as iso-
provide structural support but also position transfer RNAs lated cytoplasmic clusters. Polyribosomes attached to membranes
(tRNA) molecules bearing amino acids in the correct “reading of the endoplasmic reticulum (ER) translate mRNAs coding for
frame” and catalyze the formation of the peptide bonds. The membrane proteins of the ER, the Golgi apparatus, or the cell
more peripheral proteins of the ribosome seem to function membrane; enzymes to be stored in lysosomes; and proteins to
primarily to stabilize the catalytic RNA core. undergo exocytosis from secretory vesicles.
These ribosomal proteins are themselves synthesized in
cytoplasmic ribosomes, but are then imported to the nucleus Endoplasmic Reticulum
where they associate with newly synthesized rRNA. The ribo- The cytoplasm of most cells contains a convoluted membranous
somal subunits thus formed then move from the nucleus to network called the endoplasmic reticulum (ER). As shown in
the cytoplasm where they are reused many times, for transla- Figure 2–10, this network (reticulum) extends from the surface
tion of any mRNA strand. of the nucleus throughout most of the cytoplasm and encloses
During protein synthesis many ribosomes typically bind a series of intercommunicating channels called cisternae
the same strand of mRNA to form larger complexes, called (L. cisternae, reservoirs). With a membrane surface up to 30
polyribosomes or polysomes (Figure 2–9). In stained prepa- times that of the plasma membrane, the ER is a major site for
rations of cells, polyribosomes are intensely basophilic because vital cellular activities, including biosynthesis of proteins and
of the numerous phosphate groups of the constituent RNA mol- lipids. Numerous polyribosomes attached to the membrane in
ecules that act as polyanions. Thus, cytoplasmic regions that some regions of ER allow two types of ER to be distinguished.
stain intensely with hematoxylin and basic dyes, such as methy-
lene and toluidine blue, indicate sites of active protein synthesis. Rough Endoplasmic Reticulum
Proper folding of new proteins is guided by protein chap- Rough endoplasmic reticulum (RER) is prominent in cells
erones. Denatured proteins or those that cannot be refolded specialized for protein secretion, such as pancreatic acinar

FIGURE 2–9  Polyribosomes: free or bound to the endoplasmic reticulum.

3′
FREE POLYRIBOSOMES 5′ ER-BOUND POLYRIBOSOMES 3′
5′
mRNA
Ribosome

Cisterna of
rough ER

Misfolded &
Proteins of cytosol denatured proteins
& cytoskeleton
Conjugated to ubiquitin Golgi apparatus processing & sorting

Specific proteins imported to Secretory vesicles Lysosomes

Mitochondria Peroxisomes Nucleus Proteasome Proteins secreted Proteins of cell

Protein degradation from cell membrane

Free polyribosomes (not attached to the endoplasmic reticulum, polysomes attached to the membranes of ER. The proteins pro-
or ER) synthesize cytosolic and cytoskeletal proteins and proteins duced by these ribosomes are segregated during translation into
for import into the nucleus, mitochondria, and peroxisomes. the interior of the ER’s membrane cisternae.
Proteins that are to be incorporated into membranes, stored In both pathways misfolded proteins are conjugated to
in lysosomes, or eventually secreted from the cell are made on ubiquitin and targeted for proteasomal degradation.

02_Mescher_ch02_p017-052.indd 28 25/04/18 6:47 pm

 Cytoplasmic Organelles 29

FIGURE 2–10  Rough and smooth endoplasmic reticulum.

C H A P T E R
Nucleus

2
The Cytoplasm  ■  Cytoplasmic Organelles
Cisternae
Ribosomes

a c

Ribosomes Rough ER
Smooth ER

Functions of Endoplasmic Reticulum

1. Synthesis: Provides a place for chemical reactions

a. Smooth ER is the site of lipid synthesis and carbohydrate metabolism
b. Rough ER synthesizes proteins for secretion, incorporation into the
plasma, membrane, and as enzymes within lysosomes
2. Transport: Moves molecules through cisternal space from one part of
the cell to another, sequestered away from the cytoplasm
3. Storage: Stores newly synthesized molecules
4. Detoxification: Smooth ER detoxifies both drugs and alcohol

(a) The endoplasmic reticulum is an anastomosing network The interconnected membranous cisternae of RER are flattened,
of intercommunicating channels or cisternae formed by a con- while those of SER are frequently tubular. (14,000X)
tinuous membrane, with some regions that bear polysomes (c) In a very thin cultured endothelial cell, both ER (green) and
appearing rough and other regions appearing smooth. While mitochondria (orange) can be visualized with vital fluorescent
RER is the site for synthesis of most membrane-bound proteins, dyes that are sequestered specifically into those organelles. This
three diverse activities are associated with smooth ER: (1) lipid staining method with intact cells clearly reveals the continuous,
biosynthesis, (2) detoxification of potentially harmful compounds, lacelike ER present in all regions of the cytoplasm.
and (3) sequestration of Ca++ ions. Specific cell types with well- (Figure 2–10c, © 2015 Thermo Fisher Scientific, Inc. Used under
developed SER are usually specialized for one of these functions. permission.)
(b) By TEM cisternae of RER appear separated, but they actually
form a continuous channel or compartment in the cytoplasm.

cells (making digestive enzymes), fibroblasts (collagen), and polypeptides, and the assembly of multichain proteins. These
plasma cells (immunoglobulins). The RER consists of saclike activities are mediated by resident enzymes of the RER and
as well as parallel stacks of flattened cisternae (Figure 2–10), by protein complexes that act as chaperones guiding the fold-
each limited by membranes that are continuous with the outer ing of nascent proteins, inhibiting aggregation, and generally
membrane of the nuclear envelope. The presence of polyribo- monitoring protein quality within the ER.
somes on the cytosolic surface of the RER confers basophilic Protein synthesis begins on polyribosomes in the cytosol.
staining properties on this organelle when viewed with the light The 5′ ends of mRNAs for proteins destined to be segregated
microscope. in the ER encode an N-terminal signal sequence of 15-40
The major function of RER is production of membrane- amino acids that includes a series of six or more hydrophobic
associated proteins, proteins of many membranous organelles, residues. As shown in Figure 2–11, the newly translated signal
and proteins to be secreted by exocytosis. Production here sequence is bound by a protein complex called the signal-
includes the initial (core) glycosylation of glycoproteins, cer- recognition particle (SRP), which inhibits further polypep-
tain other posttranslational modifications of newly formed tide elongation. The SRP–ribosome–nascent peptide complex

02_Mescher_ch02_p017-052.indd 29 25/04/18 6:47 pm

 30 CHAPTER 2  ■  The Cytoplasm

FIGURE 2–11  Movement of polypeptides into the RER.

mRNA tRNA

5′ Signal sequence
SRP binds to SRP is is removed from
SRP receptor liberated 3′
growing polypeptide

New polypeptide
with initial
signal peptide SRP bound to
signal peptide

SRP Ribosome receptor

Signal recognition receptor and protein Signal Growing
particle (SRP) translocator complex pepsidase polypeptide
Completed
RER membrane RER cisterna polypeptide

The newly translated amino terminus of a protein to be incorpo- ribosomal subunit, more firmly attaching the ribosome to the ER.
rated into membranes or sequestered into vesicles contains 15-40 The hydrophobic signal peptide is translocated through a protein
amino acids that include a specific sequence of hydrophobic pore (translocon) in the ER membrane, and the SRP is freed for
residues comprising the signal sequence or signal peptide. This reuse. The signal peptide is removed from the growing protein by
sequence is bound by a signal-recognition particle (SRP), which a peptidase and translocation of the growing polypeptide contin-
then recognizes and binds a receptor on the ER. Another recep- ues until it is completely segregated into the ER cisterna.
tor in the ER membrane binds a structural protein of the large

binds to SRP receptors on the ER membrane. SRP then releases products and how these differences determine a cell’s histo-
the signal sequence, allowing translation to continue with logic features.
the nascent polypeptide chain transferred to a translocator
complex (also called a translocon) through the ER membrane
(Figure 2–11). Inside the lumen of the RER, the signal sequence
› ›› MEDICAL APPLICATION
is removed by an enzyme, signal peptidase. With the ribosome Quality control during protein production in the RER and
docked at the ER surface, translation continues with the grow- properly functioning ERAD to dispose of defective proteins
ing polypeptide pushing itself while chaperones and other are extremely important and several inherited diseases result
proteins serve to “pull” the nascent polypeptide through the from malfunctions in this system. For example, in some forms
translocator complex. Upon release from the ribosome, post- of osteogenesis imperfecta bone cells synthesize and secrete
translational modifications and proper folding of the polypep- defective procollagen molecules that cannot assemble prop-
tide continue. erly and produce very weak bone tissue.
RER has a highly regulated system to prevent nonfunc-
tional proteins being forwarded to the pathway for secretion Smooth Endoplasmic Reticulum
or to other organelles. New proteins that cannot be folded
Regions of ER that lack bound polyribosomes make up the
or assembled properly by chaperones undergo ER-associated
smooth endoplasmic reticulum (SER), which is continuous
degradation (ERAD), in which unsalvageable proteins are
with RER but frequently less abundant (Figure 2–10). Lack-
translocated back into the cytosol, conjugated to ubiquitin,
ing polyribosomes, SER is not basophilic and is best seen with
and then degraded by proteasomes.
the TEM. Unlike the cisternae of RER, SER cisternae are more
As mentioned, proteins synthesized in the RER can have
tubular or saclike, with interconnected channels of various
several destinations: intracellular storage (eg, in lysosomes
shapes and sizes rather than stacks of flattened cisternae.
and specific granules of leukocytes), provisional storage in
SER has three main functions, which vary in importance
cytoplasmic vesicles prior to exocytosis (eg, in the pancreas,
in different cell types.
some endocrine cells), and as integral membrane proteins.
Diagrams in Figure 2–12 show a few cell types with dis- ■■ Enzymes in the SER perform synthesis of phospholipids
tinct differences in the destinations of their major protein and steroids, major constituents of cellular membranes.

02_Mescher_ch02_p017-052.indd 30 25/04/18 6:47 pm

 Cytoplasmic Organelles 31

FIGURE 2–12  Protein localization and cell morphology.

2 C H A P T E R
The Cytoplasm  ■  Cytoplasmic Organelles
(a) Erythroblast (b) Eosinophilic leukocyte (c) Plasma cell (d) Pancreatic acinar cell

The ultrastructure and general histologic appearance of a cell are (c) Cells with extensive RER and a well-developed Golgi apparatus
determined by the nature of the most prominent proteins the cell show few secretory granules because the proteins undergo exocytosis
is making. immediately after Golgi processing is complete. Many cells, especially
(a) Cells that make few or no proteins for secretion have very little those of epithelia, are polarized, meaning that the distribution of RER
RER, with essentially all polyribosomes free in the cytoplasm. and secretory vesicles is different in various regions or poles of the cell.
(b) Cells that synthesize, segregate, and store various proteins in (d) Epithelial cells specialized for secretion have distinct polarity,
specific secretory granules or vesicles always have RER, a Golgi with RER abundant at their basal ends and mature secretory gran-
apparatus, and a supply of granules containing the proteins ready ules at the apical poles undergoing exocytosis into an enclosed
to be secreted. extracellular compartment, the lumen of a gland.

These lipids are then transferred from the SER to other Golgi Apparatus
membranes by lateral diffusion into adjacent membranes,
The dynamic organelle called the Golgi apparatus, or Golgi
by phospholipid transfer proteins, or by vesicles which
complex, completes posttranslational modifications of pro-
detach from the SER for movement along the cytoskeleton
teins produced in the RER and then packages and addresses
and fusion with other membranous organelles. In cells
these proteins to their proper destinations. The organelle was
that secrete steroid hormones (eg, cells of the adrenal cor-
named after histologist Camillo Golgi who discovered it in
tex), SER occupies a large portion of the cytoplasm.
1898. The Golgi apparatus consists of many smooth membra-
■■ Other SER enzymes, including those of the cytochrome
nous saccules, some vesicular, others flattened, but all con-
P450 family, allow detoxification of potentially harmful
taining enzymes and proteins being processed (Figure 2–13).
exogenous molecules such as alcohol, barbiturates, and
In most cells the small Golgi complexes are located near the
other drugs. In liver cells, these enzymes also process
nucleus.
endogenous molecules such as the components of bile.
As shown in Figure 2–13, the Golgi apparatus has two dis-
■■ SER vesicles are also responsible for sequestration and
tinct functional sides or faces, formed by the complex traffic of
controlled release of Ca2+, which is part of the rapid
vesicles within cells. Material moves from the RER cisternae
response of cells to various stimuli. This function is par-
to the Golgi apparatus in small, membrane-enclosed carriers
ticularly well developed in striated muscle cells, where
called transport vesicles that are transported along cytoskel-
the SER has an important role in the contraction process
etal polymers by motor proteins. The transport vesicles merge
and assumes a specialized form called the sarcoplasmic
with the Golgi-receiving region, or cis face. On the opposite
reticulum (see Chapter 10).
side of the Golgi network, at its shipping or trans face, larger
saccules or vacuoles accumulate, condense, and generate other
› ›› MEDICAL APPLICATION vesicles that carry completed protein products to organelles
away from the Golgi (Figure 2–13).
Jaundice denotes a yellowish discoloration of the skin and is
Formation of transport vesicles and secretory vesicles
caused by accumulation in extracellular fluid of bilirubin and
is driven by assembly of various coat proteins (including
other pigmented compounds, which are normally metabo-
clathrin), which also regulate vesicular traffic to, through,
lized by SER enzymes in cells of the liver and excreted as bile.
and beyond the Golgi apparatus. Forward movement of ves-
A frequent cause of jaundice in newborn infants is an under-
icles in the cis Golgi network of saccules is promoted by the
developed state of SER in liver cells, with failure of bilirubin to
coat protein COP-II, while retrograde movements in that
be converted to a form that can be readily excreted.
region involve COP-I. Other membrane proteins important

02_Mescher_ch02_p017-052.indd 31 25/04/18 6:47 pm

 32 CHAPTER 2  ■  The Cytoplasm

FIGURE 2–13  Golgi apparatus.

TV Vacuole
shipping
region

CF Secretory
vesicles

SV

TF
SV
Transport
Transport
vesicle
vesicle
Lumen of cisterna
filled with secretory
TV product

ER

G
M

b c

The Golgi apparatus is a highly plastic, morphologically complex (b) Morphological aspects of the Golgi apparatus are revealed
system of membrane vesicles and cisternae in which proteins and more clearly by SEM, which shows a three-dimensional snapshot
other molecules made in the RER undergo further modification of the region between RER and the Golgi membrane compart-
and sorting into specific vesicles destined for different roles in ments. Cells may have multiple Golgi apparatuses, each with the
the cell. general organization shown here and typically situated near the
(a) TEM of the Golgi apparatus provided early evidence about cell nucleus. (X30,000)
how this organelle functions. To the left is a cisterna of RER and (c) The Golgi apparatus location can be clearly seen in intact cul-
close to it are many small vesicles at the cis face (CF), or receiving tured cells processed by immunocytochemistry using an antibody
face, of the Golgi apparatus, merging with the first of several flat- against golgin-97 to show the many complexes of Golgi vesicles
tened Golgi cisternae. In the center are the characteristic flattened, (green), all near the nucleus, against a background of microfila-
curved, and stacked medial cisternae of the complex. Cytological ments organized as stress fibers and stained with fluorescent
and molecular data suggest that other transport vesicles (TV) phalloidin (violet). Because of the abundance of lipids in its many
move proteins serially through the cisternae until at the trans membranes, the Golgi apparatus is difficult to visualize in typical
face (TF), or shipping region, larger condensing secretory vesicles paraffin-embedded, H&E-stained sections. In developing white
(SV) and other vacuoles emerge to carry the modified proteins blood cells with active Golgi complexes, the organelle can some-
elsewhere in the cell. Formation and fusion of the vesicles through times be seen as a faint unstained juxtanuclear region (sometimes
the Golgi apparatus is controlled by specific membrane proteins. called a “Golgi ghost”) surrounded by basophilic cytoplasm.
(X30,000) Inset: A small region of a Golgi apparatus in a 1 μm sec- (Figure 2–13b reproduced, with permission from Naguro T, Iino A.
tion from a silver-stained cell, demonstrating abundant glycopro- Prog Clin Biol Res. 1989;295:250; Figure 2–13c, © 2015 Thermo Fisher
teins within cisternae. Scientific, Inc. Used under permission.)

02_Mescher_ch02_p017-052.indd 32 25/04/18 6:47 pm

 Cytoplasmic Organelles 33

for directed vesicle fusion include various Rab proteins and and the control of protein processing are subjects of active
other enzymes, receptors and specific binding proteins, and research.

C H A P T E R
fusion-promoting proteins that organize and shape mem-
branes. Depending on the activity of these proteins, vesicles
are directed toward different Golgi regions and give rise to Secretory Granules
lysosomes or secretory vesicles for exocytosis. Originating as condensing vesicles in the Golgi apparatus,
As indicated in Figure 2–14, Golgi saccules at sequential secretory granules are found in cells that store a product
locations contain different enzymes at different cis, medial, until its release by exocytosis is signaled by a metabolic, hor-

2
and trans levels. Enzymes of the Golgi apparatus are impor- monal, or neural message (regulated secretion). The granules

The Cytoplasm  ■  Cytoplasmic Organelles

tant for glycosylation, sulfation, phosphorylation, and lim- are surrounded by the membrane and contain a concentrated
ited proteolysis of proteins. Along with these activities, the form of the secretory product (Figure 2–15). The contents of
Golgi apparatus initiates packaging, concentration, and storage some secretory granules may be up to 200 times more con-
of secretory products. Protein movements through the Golgi centrated than those in the cisternae of the RER. Secretory

FIGURE 2–14  Summary of functions within the Golgi apparatus.

Rough ER
In the RER
• New proteins are translocated into ER cisternae
• Preassembled mannose-rich oligosaccharides are added to
specific asparagine residues (N-linked)
• Proteins are folded, guided by chaperones, with strict
RER quality control
Polyribosome
• Disulfide bonds are formed between specific cysteine residues
Transporting vesicle
from RER to Golgi
In the cis Golgi network (CGN),
cis Golgi • Vesicle movement from RER and forward through the CGN is
promoted by the coat protein COP-II
• Similarly, COP-I controls retrograde vesicle movements
• Mannose-6-phophate is added to future lysosomal enzymes
• N-linked oligosaccharides are trimmed and other sugars added

medial Golgi cisternae Vesicles move to the medial Golgi cisternae where
• New glycosylation occurs on –OH groups of some lipids
Transporting and serine and threonine residues (O-linked)
vesicles • N-linked oligosaccharides on proteins are modified further
• Glycoproteins and glycolipids are sorted into specific vesicles

In the trans Golgi network

trans Golgi • Sialic acid is added as the terminal sugar to certain
oligosaccharides
• Sulfation of tyrosine residues and some sugars occurs
• Specific vesicles with different destinations are separated and
sorted

Lysosome

Secretory granule

Membrane
Exocytosis proteins

The main molecular processes are listed at the right, with the major proteins and glycoproteins combine with specific receptors which
compartments where they occur. In the trans Golgi network, the guide them to the next stages toward their destinations.

02_Mescher_ch02_p017-052.indd 33 25/04/18 6:47 pm

 34 CHAPTER 2  ■  The Cytoplasm

FIGURE 2–15  Secretory granules.

C
S
G

TEM of one area of a pancreatic acinar cell shows numerous condensed. In H&E-stained sections secretory granules are often
mature, electron-dense secretory granules (S) in association shown as intensely eosinophilic structures, which in polarized
with condensing vacuoles (C) of the Golgi apparatus (G). Such epithelial cells are concentrated at the apical region prior to exocy-
granules form as the contents of the Golgi vacuoles become more tosis. (X18,900)

granules with dense contents of digestive enzymes are also and visible with the light microscope, especially after histo-
referred to as zymogen granules. chemical staining.
Cytosolic components are protected from these enzymes
Lysosomes by the membrane surrounding lysosomes and because the
Lysosomes are sites of intracellular digestion and turnover of enzymes have optimal activity at an acidic pH (~5.0). Any
cellular components. Lysosomes (Gr. lysis, solution, + soma, leaked lysosomal enzymes are practically inactive at the pH of
body) are membrane-limited vesicles that contain about 40 cytosol (~7.2) and harmless to the cell.
different hydrolytic enzymes and are particularly abundant in Lysosomal hydrolases are synthesized and segregated in
cells with great phagocytic activity (eg, macrophages, neutro- the RER and then transferred to the Golgi apparatus, where
phils). Although the nature and activity of lysosomal enzymes the enzymes are further modified and packaged in vacuoles
vary depending on the cell type, the most common are acid that form lysosomes. The marker mannose-6-phosphate (M6P)
hydrolyases such as proteases, nucleases, phosphatase, phos- is added by a phosphotransferase in the cis Golgi only to the
pholipases, sulfatases, and β-glucuronidase. As can be seen N-linked oligosaccharides of the hydrolases destined for lyso-
from this list, lysosomal enzymes are capable of breaking somes. Membrane receptors for M6P-containing proteins in the
down most macromolecules. trans Golgi network then bind these proteins and divert them
Lysosomes, which are usually spherical, range in diam- from the secretory pathway for segregation into lysosomes.
eter from 0.05 to 0.5 μm and present a uniformly granular, Material taken from outside the cell by endocytosis is
electron-dense appearance in the TEM (Figure 2–16). In digested when the membrane of the phagosome or pinocy-
macrophages and neutrophils, lysosomes are slightly larger totic vesicle fuses with a lysosome. This mixes the endocytosed

02_Mescher_ch02_p017-052.indd 34 25/04/18 6:47 pm

 Cytoplasmic Organelles 35

FIGURE 2–16  Lysosomes.

2 C H A P T E R
N

The Cytoplasm  ■  Cytoplasmic Organelles

N

b
N

L G
L G
L

L
L
L

L
a c

Lysosomes are spherical membrane-enclosed vesicles that (b) Lysosomes in cultured vascular endothelial cells can be specifi-
function as sites of intracellular digestion and are particularly cally stained using fluorescent dyes sequestered into these organ-
numerous in cells active after the various types of endocytosis. elles (green), which are abundant around the blue Hoechst-stained
Lysosomes are not well shown on H&E-stained cells but can nucleus. Mitochondria (red) are scattered among the lysosomes.
be visualized by light microscopy after staining with toluidine (c) In the TEM lysosomes (L) have a characteristic very electron-
blue. dense appearance and are shown here near groups of Golgi
(a) Cells in a kidney tubule show numerous purple lysosomes (L) cisternae (G). The less electron-dense lysosomes represent het-
in the cytoplasmic area between the basally located nuclei (N) and erolysosomes in which digestion of the contents is under way. The
apical ends of the cells at the center of the tubule. Using endocy- cell is a macrophage with numerous fine cytoplasmic extensions
tosis, these cells actively take up small proteins in the lumen of the (arrows). (X15,000)
tubule, degrade the proteins in lysosomes, and then release the (Figure 2–16b, © 2015 Thermo Fisher Scientific, Inc. Used under
resulting amino acids for reuse. (X300) 35 permission.)

material with the lysosomal enzymes and activates proton Indigestible material is retained within a small vacuolar rem-
pumps in the lysosomal membrane that acidify the contents, nant called a residual body (Figure 2–17). In some long-lived
allowing digestion. The composite, active organelle is now cells (eg, neurons, heart muscle), residual bodies can accumu-
termed a secondary or heterolysosome. Heterolysosomes late over time as granules of lipofuscin.
are generally somewhat larger and have a more heterogeneous Besides degrading exogenous macromolecules lysosomes
appearance in the TEM because of the wide variety of materi- also function in a process called autophagy by which excess
als they may be digesting (Figure 2–16c). organelles or large aggregates of nonfunctional macromol-
During this digestion of macromolecules, released nutri- ecules in cytoplasm are degraded (Figures 2–17 and 2–18).
ents diffuse into the cytosol through the lysosomal membrane. During autophagy a lipid bilayer membrane forms around

02_Mescher_ch02_p017-052.indd 35 25/04/18 6:47 pm

 36 CHAPTER 2  ■  The Cytoplasm

FIGURE 2–17  Lysosomal functions.

Phagocytosis of
bacteria or debris
(by neutrophils)

Endocytotic
Phagocytic
vesicles
vacuole
Release of
nutrients during
digestion
Early
endosome

Late
endosome Secondary Residual body
lysosymes containing
indigestible material
Primary
lysosymes

Autophagosome
Golgi
apparatus
Secretion of
hydrolytic enzymes
into special
microenvironments
(eg, by osteoclasts)
Autophagic
vacuole forming
around
mitochondrion

Nucleus

RER

Synthesis of lysosomal enzymes occurs in the RER, with packaging after nonfunctional or surplus organelles become enclosed with
in the Golgi apparatus. Endocytosis produces vesicles that fuse membrane and the resulting structure fuses with a lysosome. The
with endosomes before merging with lysosomes. Phagocytic products of lysosomal digestion are recycled to the cytoplasm, but
vacuoles (or phagosomes) fuse with primary lysosomes to become indigestible molecules remain in a membrane-enclosed residual
secondary lysosomes (or heterolysosomes), in which ingested body, which may accumulate in long-lived cells as lipofuscin.
material is degraded. Autophagosomes, such as those depicted In some cells, such as osteoclasts, the lysosomal enzymes are
here with a mitochondrion in the process of digestion, are formed secreted into a restricted extracellular compartment.

02_Mescher_ch02_p017-052.indd 36 25/04/18 6:48 pm

 Cytoplasmic Organelles 37

other digestion products are released from autophagosomes

FIGURE 2–18  Autophagy.
for reuse as sources of energy or in new synthetic reactions.

C H A P T E R
› ›› MEDICAL APPLICATION
Diseases categorized as lysosomal storage disorders stem from
defects in one or more of the digestive enzymes present in
lysosomes, usually due to a mutation leading to a deficiency

2
of one of the enzymes, or defects due to faulty posttransla-

The Cytoplasm  ■  Cytoplasmic Organelles

tional processing. In cells that must digest the substrate of
the missing or defective enzyme following autophagy, the
lysosomes cannot function properly. Such cells accumulate
large secondary lysosomes or residual bodies filled with the
indigestible macromolecule. The accumulation of these vacu-
oles may eventually interfere with normal cell or tissue func-
tion, producing symptoms of the disease. A few lysosomal
storage diseases are listed in Table 2–3, with the enzyme
involved for each and the tissue affected.

Proteasomes
Proteasomes are very small abundant protein complexes that
are not associated with membrane, each approximately the size
of the small ribosomal subunit. They function to degrade dena-
RB tured or otherwise nonfunctional polypeptides. Proteasomes
also remove proteins no longer needed by the cell and provide
an important mechanism for restricting the activity of a specific
protein to a certain window of time. Whereas lysosomes digest
organelles or membranes by autophagy, proteasomes deal pri-
Autophagy is a process in which the cell uses lysosomes to marily with free proteins as individual molecules.
dispose of excess or nonfunctioning organelles or membranes. As shown in Figure 2–9, the proteasome is a cylindri-
Membrane that appears to emerge from the SER encloses the cal structure made of four stacked rings, each composed of
organelles to be destroyed, forming an autophagosome that seven proteins including proteases. At each end of the cylinder
then fuses with a lysosome for digestion of the contents. In this is a regulatory particle that contains ATPase and recognizes
TEM the two autophagosomes at the upper left contain portions
of RER more electron dense than the neighboring normal RER proteins with attached molecules of ubiquitin, an abundant
and one near the center contains what may be mitochondrial cytosolic 76-amino acid protein found in all cells. Misfolded
membranes plus RER. Also shown is a vesicle with features of a or denatured proteins, or short-lived proteins with oxidized
residual body (RB). (30,000X) amino acids, are recognized by chaperones and targeted for
destruction by other enzyme complexes that conjugate ubiq-
and isolates the cytoplasmic portion to be removed, produc- uitin to lysine residues of the protein, followed by formation
ing an autophagosome (Gr. autos, self, + phagein, to eat, of a polyubiquitin chain. Ubiquinated proteins are recog-
+ soma), which then fuses with a lysosome for digestion of nized by the regulatory particles of proteasomes, unfolded
the enclosed material. Autophagy is enhanced in times of by the ATPase using energy from ATP, and then translocated
starvation or nutrient stress. Amino acids, nucleotides, and into the core of the cylindrical structure and degraded by

TABLE 2–3   Examples of lysosomal storage diseases caused by defective lysosomal enzymes.

Disease Faulty Enzyme Main Organs Affected

Hurler syndrome (MPS I) α-L-Iduronidase Skeleton and nervous system

McArdle syndrome Muscle phosphorylase Skeletal muscles
Tay–Sachs GM2-gangliosidase Nervous system
Gaucher Glucocerebrosidase Liver and spleen
I-cell disease Phosphotransferase for M6P formation Skeleton and nervous system

02_Mescher_ch02_p017-052.indd 37 25/04/18 6:48 pm

 38 CHAPTER 2  ■  The Cytoplasm

endopeptidases. The ubiquitin molecules are released for reuse (Figure 2–19). Under the TEM each mitochondrion is seen
and the peptides produced may be broken down further to to have two separated and very different membranes that
amino acids or they may have other specialized destinations, together create two compartments: the innermost matrix
such as the antigen-presenting complexes of cells activating an and a narrow intermembrane space (Figure 2–20a). Both
immune response. mitochondrial membranes contain a higher density of pro-
tein molecules than other membranes in the cell and have
reduced fluidity. The outer membrane is sieve-like, contain-
› ›› MEDICAL APPLICATION ing many transmembrane proteins called porins that form
Failure of proteasomes or other aspects of a cell’s protein qual- channels through which small molecules such as pyruvate
ity control can allow large aggregates of protein to accumulate and other metabolites readily pass from the cytoplasm to the
in affected cells. Such aggregates may adsorb other macromol- intermembrane space.
ecules to them and damage or kill cells. Aggregates released The inner membrane has many long folds called
from dead cells can accumulate in the extracellular matrix of cristae, which project into the matrix and greatly increase
the tissue. In the brain this can interfere directly with cell func- the membrane’s surface area (Figure 2–20). The number
tion and lead to neurodegeneration. Alzheimer’s disease and of cristae in mitochondria also corresponds to the energy
Huntington’s disease are two neurologic disorders caused needs of the cell. The lipid bilayer of the inner membrane
initially by such protein aggregates. contains unusual phospholipids and is highly impermeable
to ions (Figure 2–20). Integral proteins include various
transport proteins that make the inner membrane selectively
Mitochondria permeable to the small molecules required by enzymes in the
Mitochondria (Gr. mitos, thread, + chondros, granule) are matrix. Mitochondrial matrix enzymes include those that
membrane-enclosed organelles with arrays of enzymes spe- oxidize pyruvate and fatty acids to form acetyl coenzyme A
cialized for aerobic respiration and production of adenosine (CoA) and those of the citric acid cycle that oxidize acetyl
triphosphate (ATP), with high-energy phosphate bonds, CoA, releasing CO2 as waste and small energy-rich mole-
which supplies energy for most cellular activities. Glycolysis cules that provide electrons for transport along the electron-
converts glucose anaerobically to pyruvate in the cytoplasm, transport chain (or respiratory chain). Enzymes and other
releasing some energy. The rest of the energy is captured when components of this chain are embedded in the inner mem-
pyruvate is imported into mitochondria and oxidized to CO2 brane and allow oxidative phosphorylation, which produces
and H2O. Mitochondrial enzymes yield 15 times more ATP most of the ATP in animal cells.
than is produced by glycolysis alone. Some of the energy Formation of ATP by oxidative phosphorylation
released in mitochondria is not stored in ATP but is dissipated enzymes occurs by a chemiosmotic process. Membrane
as heat that maintains body temperature. proteins guide the small electron carrier molecules through
Mitochondria are usually elongated structures with diam- closely packed enzyme complexes so that the electrons move
eters of 0.5-1 μm and lengths up to 10 times greater. They are sequentially along the chain. Electron transfer is coupled with
highly plastic, rapidly changing shape, fusing with one another oriented proton uptake and release, with protons accumulat-
and dividing, and are moved through the cytoplasm along ing in the intermembrane space (Figure 2–20) and producing
microtubules. The number of mitochondria is related to the an electrochemical gradient across the inner membrane.
cell’s energy needs: cells with a high-energy metabolism (eg, Membrane-associated proteins of the ATP synthase sys-
cardiac muscle, cells of some kidney tubules) have abundant tem form large (10 nm), multisubunit, globular complexes
mitochondria, whereas cells with a low-energy metabolism on stalk-like structures that project from the matrix side of
have few mitochondria. Similarly, mitochondria of differen- the inner membrane (Figure 2–20). This enzyme complex
tiated cells are concentrated in cytoplasmic regions where contains a channel through which protons flow down the
energy utilization is more intense. electrochemical gradient, crossing the membrane back into
the matrix. Passage of protons through this channel causes
rotation of specific polypeptides in the globular ATP syn-
› ›› MEDICAL APPLICATION thase complex, converting the energy of proton flow into the
Myoclonic epilepsy with ragged-red fibers (MERRF) is a mechanical energy of protein movement. Mechanical energy
rare disease occurring in individuals in whom cells of specific is stored in the new phosphate bond of ATP by other sub-
tissues, notably regions of skeletal muscle, inherit mitochon- unit polypeptides binding adenosine diphosphate (ADP)
drial DNA with a mutated gene for lysine-tRNA, leading to and inorganic phosphate (Figure 2–20). A steady torrent of
defective synthesis of respiratory chain proteins that can pro- protons along the gradient allows each of these remarkable
duce structural abnormality in muscle fibers and other cells. synthase complexes to produce more than 100 molecules of
ATP per second.
Another role for mitochondria occurs at times of cell
Mitochondria are often sufficiently large to be visible stress when the protein cytochrome c is released from the
with the light microscope as numerous discrete organelles inner membrane’s electron-transport chain. In the cytoplasm,

02_Mescher_ch02_p017-052.indd 38 25/04/18 6:48 pm

 Cytoplasmic Organelles 39

FIGURE 2–19  Mitochondria in the light microscope.

2 C H A P T E R
The Cytoplasm  ■  Cytoplasmic Organelles
a b

(a) In certain sectioned cells stained with H&E, mitochondria parallel along microtubules. Such preparations also show that
appear throughout the cytoplasm as numerous eosinophilic mitochondrial shape can be quite plastic and variable. Specific
structures. The mitochondria usually appear round or slightly elon- mitochondrial staining such as that shown here involves incu-
gated and are more numerous in cytoplasmic regions with higher bating living cells with specific fluorescent compounds that are
energy demands, such as near the cell membrane in cells under- specifically sequestered into these organelles, followed by fixation
going much active transport. The central nuclei are also clearly and immunocytochemical staining of the microtubules. In this
seen in these cells. preparation, microtubules are stained green and mitochondria
(b) Entire mitochondria can be shown in cultured cells, such as the appear yellow or orange, depending on their association with
endothelial cells shown here, and often appear as the elongated the green microtubules. The cell nucleus was stained with DAPI
structures (shown in yellow or orange here), usually arrayed in (4′,6-diamidino-2-phenylindole).

this protein activates sets of proteases that degrade all cellular aerobic prokaryote that lived symbiotically within an ances-
components in a regulated process called apoptosis which tral eukaryotic host cell.
results in rapid cell death (see Chapter 3).
New mitochondria originate by growth and division
(fission) of preexisting mitochondria. During cell mitosis each Peroxisomes
daughter cell receives approximately half the mitochondria in Peroxisomes are spherical organelles enclosed by a single
the parent cell. membrane and named for their enzymes producing and
Unlike most organelles mitochondria are partly auton- degrading hydrogen peroxide, H2O2 (Figure 2–21). Oxidases
omous of nuclear genes and activities. The mitochondrial located here oxidize substrates by removing hydrogen atoms
matrix contains a small circular chromosome of DNA, ribo- that are transferred to molecular oxygen (O2), producing H2O2.
somes, mRNA, and tRNA, all with similarities to the cor- Peroxidases such as catalase immediately break down H2O2,
responding bacterial components. Protein synthesis occurs which is potentially damaging to the cell. These enzymes also
in mitochondria, but because of the reduced amount of inactivate various potentially toxic molecules, including some
mitochondrial DNA, only a small subset of mitochondrial prescription drugs, particularly in the large and abundant per-
proteins is produced locally. Most are encoded by nuclear oxisomes of liver and kidney cells.
DNA and synthesized on free polyribosomes of the cytosol. Other diverse enzymes in peroxisomes complement cer-
These proteins have short terminal amino acid sequences tain functions of the SER and mitochondria in the metabo-
that serve as signals for their uptake across the mitochon- lism of lipids and other molecules. Thus, the β-oxidation of
drial membranes. The observation that mitochondria have long-chain fatty acids (18 carbons and longer) is preferen-
certain bacterial characteristics led with later work to the tially accomplished by peroxisomal enzymes that differ from
understanding that mitochondria evolved from an ancestral their mitochondrial counterparts. Certain reactions leading

02_Mescher_ch02_p017-052.indd 39 25/04/18 6:48 pm

 40 CHAPTER 2  ■  The Cytoplasm

FIGURE 2–20  Mitochondrial structure and ATP formation (Legend Opposite).

a b
Outer
mitochondrial
membrane
Inner
mitochondrial
membrane
Cristae
Matrix

Fatty acids

ATP CO2
β Oxidation

Glucose Pyruvate Pyruvate Acetyl CoA

Glycolysis
NADH CO2
NADH
Citric
3NADH acid
e− CO2
Cytosol cycle
FADH2
ATP
Outer membrane ½O2
e−
H+ Matrix
H+ H2O
Inner membrane e− H+ H+

H+ 2H+
H+
II Electron transport
H+ H+ H+ chain complexes
H+ I III
Intermembrane space H+ IV
H+ H+
H+ H+ H+
H+ H+ H+ H+
H+ H+ H+ H+
H+ H+ H+ H+
H+ H+
H+ H+

H+

H+ ATP synthase
H+ H+
H+
H+

ATP ATP
H+ ATP ADP + Pi ADP + Pi
ADP + Pi

ATP synthesis
H+ H+

H+

02_Mescher_ch02_p017-052.indd 40 25/04/18 6:48 pm

 Cytoplasmic Organelles 41

(a) The two mitochondrial membranes and the innermost matrix the directed movement of protons (H+) from the matrix into the

C H A P T E R
can be seen in the TEM and diagram. The outer membrane is intermembranous space, producing an electrochemical gradi-
smooth and the inner membrane has many sharp folds called ent across the membrane. Other membrane-associated proteins
cristae that increase its surface area greatly. The matrix is a gel make up the ATP synthase systems, each of which forms a globu-
with a high concentration of enzymes. lar complex on a stalk-like structure projecting from the matrix
(b) Metabolites such as pyruvate and fatty acids enter mitochon- side of the inner membrane. A channel in this enzyme complex
dria via membrane porins and are converted to acetyl CoA by allows proton flow down the electrochemical gradient and
matrix enzymes of the citric acid cycle (or Krebs cycle), yielding across the membrane back into the matrix. The flow of protons
causes rapid spinning of specific polypeptides in the globular

2
some ATP and NADH (nicotinamide adenine dinucleotide), a
major source of electrons for the electron-transport chain. The ATP synthase complex, converting the energy of proton flow into

The Cytoplasm  ■  Cytoplasmic Organelles

movement of electrons through the protein complexes of the mechanical energy, which other subunit proteins store in the new
inner membrane’s electron-transport system is accompanied by phosphate bond of ATP.

to the formation of bile acids and cholesterol also occur in peroxisomes. Peroxisomal proteins are synthesized on free
peroxisomes. polyribosomes and have targeting sequences of amino acids at
Peroxisomes form in two ways: budding of precursor either terminus recognized by receptors located in the peroxi-
vesicles from the ER or growth and division of preexisting somal membrane for import into the organelle.

FIGURE 2–21  Peroxisomes.

P G

a b c

Peroxisomes are small spherical, membranous organelles, containing (c) A cultured endothelial cell processed by immunocytochemis-
enzymes that use O2 to remove hydrogen atoms from fatty acids, in a try shows many peroxisomes (green) distributed throughout the
reaction that produces hydrogen peroxide (H2O2) that must be broken cytoplasm among the vitally stained elongate mitochondria (red)
down to water and O2 by another enzyme, catalase. around the DAPI-stained nucleus (blue). Peroxisomes shown here
(a) By TEM peroxisomes (P) generally show a matrix of moderate were specifically stained using an antibody against the membrane
electron density. Aggregated electron-dense particles represent protein PMP70, a transmembrane protein which targets proteins
glycogen (G). (X30,000) into this organelle during peroxisome formation.
(Figure 2–21c, © 2015 Thermo Fisher Scientific, Inc. Used under
(b) Peroxisomes (P) in most species are characterized by a cen- permission.)
tral, more electron-dense crystalloid aggregate of constituent
enzymes, as shown here. (X60,000)

02_Mescher_ch02_p017-052.indd 41 25/04/18 6:48 pm

 42 CHAPTER 2  ■  The Cytoplasm

› ›› MEDICAL APPLICATION
Several fairly rare disorders arise from defective peroxisomal
››THE CYTOSKELETON
The cytoplasmic cytoskeleton is a complex array of (1)
proteins. Neonatal adrenoleukodystrophy is caused by microtubules, (2) microfilaments (also called actin filaments),
a defective integral membrane protein needed for trans- and (3) intermediate filaments. These protein polymers deter-
port of very long-chain fatty acids into the peroxisome for mine the shapes of cells, play an important role in the move-
β-oxidation. Accumulation of these fatty acids in body fluids ments of organelles and cytoplasmic vesicles, and also allow
can disrupt the myelin sheaths in nerve tissue, causing severe the movement of entire cells. Important properties, functions,
neurologic symptoms. Deficiencies of peroxisomal enzymes and locations of the cytoskeletal components are summarized
cause Zellweger syndrome that affects the structure and in Table 2–4.
functions of several organ systems.

TABLE 2–4    Properties of cytoskeletal components (microtubules, microfilaments, and

intermediate filaments).

Microfilament
General Function of Cytoskeleton

1. Structural: Provides structural support to cell;

Intermediate filament stabilizes junctions between cells
2. Movement: Assists with cytosol streaming and
cell motility; helps move organelles and materials
throughout cell; helps move chromosomes during
Microtubule cell division
Centrosome

  Microtubules Microfilaments Intermediate Filaments

Polymer Protofibril

Protofilament
N C
α β
C N

Subunit Heterodimers of αβ-tubulin G–actin monomers Antiparallel tetramers of two rod-like

dimers
Overall Hollow tube with a wall of 13 parallel Two intertwined filaments of F-actin Cable of four intertwined protofibrils,
structure protofilaments each consisting of bundled tetramers
associated end-to-end
Diameter 25 nm 5-7 nm 8-10 nm
Monomeric α and β tubulin (54 kDa) Globular G-actin (42 kDa) Various α-helical rod-like proteins
proteins (~55 kDa, Table 2–5)
Polarity + and − ends + and − ends No apparent polarity
Relative Dynamic in cytoplasm; stable in Dynamic Stable
stability axonemes
General Radiating through cytoplasm from Concentrated beneath cell Arrayed throughout cytoplasm; at
locations concentration at centrosomes; axonemes membrane; in cell extensions like desmosomes; inside nuclear envelop
microvilli
Key Maintain cell’s shape and polarity; provide Contract and move cells; change Strengthen cell and tissue structure;
functions tracks for organelle and chromosome cell shape; cytokinesis; cytoplasmic maintain cell shape; maintain nuclear
movement; move cilia and flagella transport and streaming shape (lamins)

02_Mescher_ch02_p017-052.indd 42 25/04/18 6:48 pm

 The Cytoskeleton 43

Microtubules in the cytoplasmic extensions called cilia (discussed in Chap-

ter 4) and flagella. Each microtubule is hollow, with an outer

C H A P T E R
Within the cytoplasm of all eukaryotic cells are fine tubular
diameter of 25 nm and a wall 5-nm thick, a structure that con-
structures known as microtubules (Table 2–4; Figure 2–22),
fers significant rigidity to help maintain cell shape. Microtu-
most of which are highly dynamic in length. Microtubules are
bules vary in length, but can become many micrometers long.
also organized into larger, more stable arrays called axonemes
Two or more microtubules are often linked side-by-side by
protein arms or bridges, which are particularly important in
FIGURE 2–22  Microtubules and actin filaments in the axonemes of cilia and flagella.

2
cytoplasm. As indicated in Table 2–4, the protein subunit of a micro-

The Cytoplasm  ■  The Cytoskeleton

tubule is a heterodimer of α and β tubulin, each with a molec-
ular mass of about 50 kDa. Under appropriate conditions the
MF tubulin heterodimers polymerize to form the microtubules,
MF which have a slightly spiral organization. The tubulin subunits
align lengthwise as protofilaments, with 13 parallel protofila-
ments forming the circumference of each microtubule wall.
Polymerization of tubulins is directed by microtubule
MT organizing centers (MTOCs), which contain short assem-
blies of tubulin that act as nucleating sites for further polym-
MT erization. Microtubules are polarized structures, with growth
a (polymerization) occurring more rapidly at the (+) end
(Figure 2–23). Microtubules show dynamic instability, with
continuous cycles of polymerization and depolymerization at
steady-state conditions, which depend on concentrations of
tubulin, Ca2+, Mg2+, and the presence of various microtubule-
associated proteins (MAPs). Energy for assembly is derived
from GTP bound to incoming tubulin subunits. Individual
microtubules shorten as depolymerization exceeds growth.
Microtubule stability varies greatly with cellular location and
function; microtubules of cilia are very stable, while those of
the mitotic spindle are short-lived.
The dominant MTOC in most cells is the centrosome,
which is organized around two cylindrical centrioles, each
about 0.2 μm in diameter and 0.3-0.5 μm in length. Each
centriole is composed of nine highly organized microtubule
b
triplets (Figure 2–24). With their long axes at right angles,
the paired centrioles organize nearby tubulin complexes and
(a) Microtubules (MT) and actin microfilaments (MF) can both other proteins as a pericentriolar matrix found close to the
be clearly distinguished in this TEM of fibroblast cytoplasm, which nucleus of nondividing cells. Before cell division, more specifi-
provides a good comparison of the relative diameters of these two
cally during the period of DNA replication, each centrosome
cytoskeletal components. (X60,000)
duplicates itself so that now each centrosome has two pairs of
(b) Arrays of microfilaments and microtubules are easily demonstrated
by immunocytochemistry using antibodies against their subunit
centrioles. During mitosis, the centrosome divides into halves,
proteins, as in this cultured cell. Actin filaments (red) are most con- which move to opposite poles of the cell, and become organiz-
centrated at the cell periphery, forming prominent circumferential ing centers for the microtubules of the mitotic spindle.
bundles from which finer filaments project into cellular extensions Microtubules also form part of the system for intracel-
and push against the cell membrane. Actin filaments form a dynamic lular transport of membranous vesicles, macromolecular
network important for cell shape changes such as those during cell
division, locomotion, and formation of cellular processes, folds, pseu-
complexes, and organelles. Well-studied examples include
dopodia, lamellipodia, microvilli, etc, which serve to change a cell’s axoplasmic transport in neurons, melanin transport in pig-
surface area or give direction to a cell’s crawling movements. ment cells, chromosome movements by the mitotic spindle,
Microtubules (green/yellow) are oriented in arrays that gener- and vesicle movements among different cell compartments.
ally extend from the centrosome area near the nucleus into the In each of these examples, movement is suspended if micro-
most peripheral extensions. Besides serving to stabilize cell shape,
microtubules form the tracks for kinesin-based transport of vesicles
tubules are disrupted. Transport along microtubules is under
and organelles into the cell periphery and dynein-based transport the control of proteins called motor proteins, which use
toward the cell nucleus. ATP in moving the larger structures. Kinesins carry material
(Figure 2–22b, used with permission from Dr Albert Tousson, away from the MTOC near the nucleus toward the plus end of
University of Alabama—Birmingham High Resolution Imaging microtubules (anterograde transport); cytoplasmic dyneins
Facility, Birmingham.)
carry material along microtubules in the opposite direction

02_Mescher_ch02_p017-052.indd 43 25/04/18 6:48 pm

 44 CHAPTER 2  ■  The Cytoplasm

(retrograde transport), generally toward the nucleus. Impor-

FIGURE 2–23  Dynamic instability of microtubules.
tant roles for this system include extending the ER from the
nuclear envelope to the plasmalemma and moving vesicles to
and through the Golgi apparatus.
GTP cap

(+) Elongation by
adding GTP tubulin
› ›› MEDICAL APPLICATION
GTP Several inhibitory compounds used by cell biologists to
tubulin study details of microtubule dynamics are also widely used in
cancer chemotherapy to block activity of the mitotic spindle
GDP in rapidly growing neoplastic cells. Such drugs include vin-
tubulin blastine, vincristine, and paclitaxel, all of which were origi-
nally discovered as plant derivatives.

Microfilaments (Actin Filaments)

Microfilaments are composed of actin subunits and allow
(−)
motility and most contractile activity in cells, using reversible
assembly of the actin filaments and interactions between these
High Low filaments and associated myosin family proteins. Actin fila-
concentration concentration
of free of free ments are thin (5-7 nm diameter), polarized polymers, shorter
GTP tubulin GTP tubulin and more flexible than microtubules (Figure 2–22). They are
composed of globular G-actin monomers that assemble in
the presence of K+ and Mg2+ into a double-stranded helix of
GDP cap filamentous F-actin (Table 2–4). G-actin is generally added to
preexisting filaments, but new filaments can be formed from
a pool of G-actin by the action of nucleating proteins, such as
Stable or Unstable, formin. Another nucleating factor, a complex of polypeptides
continued depolymerization
growth of protofilaments called Arp2/3, also binds to the side of preexisting actin fila-
ments and induces a new F-actin branch, a process which can
lead to formation of a microfilament network.
Like microtubules actin filaments are highly dynamic.
Monomers are added rapidly at the (+) or barbed end, with
ATP hydrolysis at each addition; at the same time monomers
dissociate at the (−) or pointed end. This leads to migration of
subunits through the polymer, which occurs rapidly in a pro-
cess called treadmilling (Figure 2–25). In cells both the assem-
bly and disassembly of subunits from F-actin are promoted by
other proteins, such as profilin and cofilin, respectively.
At stable tubulin concentrations some microtubules grow Actin is very abundant in all cells and is concentrated
while others shrink, each existing in a condition called dynamic in networks of actin filaments and as free G-actin subunits
instability. In cytoplasmic areas where the tubulin concentra- near the cell membrane, a cytoplasmic region often called
tion is high, tubulin GTP is added at a microtubule’s (+) end the cell cortex. Arp2/3 activity produces an important
faster than the incorporated GTP can be hydrolyzed. The
resulting “GTP cap” stabilizes that end of the microtubule and
branched network of microfilaments in this region. Micro-
promotes further rapid growth. As free tubulin concentrations filament-rich cellular extensions, including surface folds or
decrease, the rate of growth also decreases, thereby allow- ruffles, are important for endocytosis and microfilaments of
ing GTP hydrolysis to catch up. The resulting “GDP cap” at the the cortex underlie endosomal trafficking. Cell movements
microtubule end is unstable and favors rapid depolymerization on firm substrates involve sheet-like protrusions, or lamel-
(termed “catastrophe”). This increases the local concentration of
free, monomeric tubulin that “rescues” the microtubule before
lipodia, in which the concentrated actin filaments are con-
it completely disappears and produces another short period of tinuous with deeper parallel F-actin bundles called stress
microtubule elongation. fibers (Figure 2–13c).
Dynamic instability allows the growing ends of microtu- Actin-binding proteins, such as formin and others just
bules to explore the cytoplasm and become stabilized when mentioned, change the dynamic physical properties of micro-
they contact stabilizing structures, such as kinetochores on
chromosomes early in mitosis (see Chapter 3).
filaments, particularly their lengths and interactions with
other structures, and this determines the viscosity and other
mechanical properties of the local cytoplasm. Cross-linking

02_Mescher_ch02_p017-052.indd 44 25/04/18 6:48 pm

 The Cytoskeleton 45

FIGURE 2–24  Centrosome.

C H A P T E R
Microtubule triplet

Centriole
Microtubule

Longitudinal section Centrosome

of centriole

2
The Cytoplasm  ■  The Cytoskeleton
TEM 120,000X

Cross section of centriole

Centriole

Protein
links

The centrosome is the microtubule-organizing center for the and the pair is divided equally during a cell’s interphase, each half
mitotic spindle and consists of paired centrioles. The TEM reveals having a duplicated centriole pair. At the onset of mitosis, the
that the two centrioles in a centrosome exist at right angles to two daughter centrosomes move to opposite sides of the nucleus
each other in a dense matrix of free tubulin subunits and other and become the two poles of the mitotic spindle of microtubules
proteins. Each centriole consists of nine microtubular triplets. attaching to chromosomes.
In a poorly understood process, the centrosome duplicates itself

within networks of F-actin increases cytoplasmic viscosity, ■■ Membrane-associated molecules of myosin I whose
while severing (and capping) the filaments tends to decrease movements along microfilaments produce the cell sur-
viscosity. The lengths and other physical properties of actin fil- face changes during endocytosis
aments are controlled by various other types of actin-binding
Stabilized arrays of actin filaments integrated with arrays
proteins, including those indicated in Figure 2–26.
of thicker (16 nm) myosin filaments permit very forceful
Just as the molecular motors kinesin and dynein move
contractions in specialized cells such as those of muscle (see
structures along microtubules, various myosin motors use
Chapter 10).
ATP to transport cargo along F-actin. Movement is usually
toward the barbed (+) ends of actin filaments; myosin VI is the Intermediate Filaments
only known myosin that moves in the other direction. Interac-
The third class of cytoskeletal components includes filaments
tions between F-actin and myosins form the basis for various
intermediate in size between the other two, with a diameter
cell movements:
averaging 10 nm (Table 2–4). Unlike microtubules and actin
■■ Transport of organelles, vesicles, and granules in the pro- filaments, these intermediate filaments are stable, confer
cess of cytoplasmic streaming increased mechanical stability to cell structure, and are made
■■ Contractile rings of microfilaments with myosin II up of different protein subunits in different cell types. More
constricting to produce two cells by cytokinesis during than a dozen proteins, ranging in size from 40 to 230 kDa,
mitosis serve as subunits of various intermediate filaments and can be

02_Mescher_ch02_p017-052.indd 45 25/04/18 6:48 pm

 46 CHAPTER 2  ■  The Cytoplasm

FIGURE 2–25  Actin filament treadmilling. FIGURE 2–26  Roles of major actin-binding
proteins in regulating the organization of
microfilaments.

G-actin monomers

Promote Promote
polymerization filament
(eg, profilin) disassembly
(eg, cofilin)
(–) (+)

Sever and
cap filaments
(eg, gelsolin)
Cross-link
filaments
(eg, filamin,
Arp 2/3 complex) Actin
(–) (+) filament

Cap stable Anchor filaments

filaments at membrane
(–) (+) (eg, cap Z) (eg, spectrin)

Bundle parallel filaments

(eg, villin, fimbrin, α-actinin)

A large number of proteins regulate the assembly of microfila-

(–) (+) ments and the interactions of these filaments with one another.
By altering microfilament length and cross-linking, such proteins
greatly influence physical properties of the local cytoplasm.
b

(a) Actin filaments or microfilaments are helical two-stranded

polymers assembled from globular actin subunits.
Intermediate filament proteins with particular biological,
histological, or pathological importance include the following:
(b) Assembly of actin filaments (F-actin) is polarized, with
G-actin subunits added to the plus (+) end and removed at the ■■ Keratins (Gr. keras, horn) or cytokeratins are a
minus (−) end. Even actin filaments of a constant length are diverse family of acidic and basic isoforms that com-
highly dynamic structures, balancing G-actin assembly and
disassembly at the opposite ends, with a net movement or flow pose heterodimer subunits of intermediate filaments in
along the polymer known as treadmilling. all epithelial cells (see Chapter 4). They are encoded by
(Figure 2–25a, used with permission from John Heuser, over 30 related genes and produce filaments with differ-
Washington University School of Medicine, St. Louis, MO.) ent chemical and immunologic properties for various
functions. Intermediate filaments of keratins form large
localized by immunohistochemistry in various cells. As indi- bundles (tonofibrils) that attach to certain junctions
cated in Table 2–4, nearly all these subunits are coiled, rod-like between epithelial cells (Figure 2–27). In skin epidermal
dimers that form antiparallel tetramers, which self-assemble cells, cytokeratins accumulate during differentiation in
into large cable-like bundles or protofibrils stabilized by fur- the process of keratinization, producing an outer layer
ther lateral interactions. Table 2–5 lists six classes of inter- of nonliving cells that reduces dehydration. Keratiniza-
mediate filament proteins forming rod-like subunits, their tion of skin made terrestrial life possible in the course
sizes and cell distributions, and diseases that result from their of evolution. Keratinization also provides some protec-
disruption. tion from minor abrasions and produces various hard

02_Mescher_ch02_p017-052.indd 46 25/04/18 6:48 pm

 Inclusions 47

    Major classes and representatives of intermediate filament proteins, their sizes

TABLE 2–5

C H A P T E R
and locations.

Class Protein Size (kDa) Cell Distribution Disease Involvement (If Known)

I Acidic cytokeratin 40-65 Epithelial cells Certain skin-blistering disorders

II Basic cytokeratin 51-68 Epithelial cells Keratoderma; corneal dystrophy

2
III Desmin 53 Muscle cells Myopathies

The Cytoplasm  ■ Inclusions

Synemin 190 Muscle cells
GFAP 50 Astrocytes (less in other glial cells) Alexander disease
Peripherin 57 Neurons
Vimentin 54 Mesenchymal cells
IV NF-L 68 Neurons
NF-M 110 Neurons
NF-H 130 Neurons
α-Internexin 55 Embryonic neurons
V Lamins 62-72 Nuclei of all cells Cardiomyopathy; muscular dystrophies; progeria
VI Nestin 230 Neural stem cells

protective structures of skin, such as nails (as well as

FIGURE 2–27  Intermediate filaments of keratin. feathers, beaks, horns, and the scales of reptiles).
■■ Vimentin is the most common class III intermediate
filament protein and is found in most cells derived from
embryonic mesenchyme. Important vimentin-like pro-
teins include desmin found in almost all muscle cells
and glial fibrillar acidic protein (GFAP) found espe-
cially in astrocytes, supporting cells of central nervous
system tissue. Desmin filaments of a cultured cell are
shown immunocytochemically in Figure 1–12a.
■■ Neurofilament proteins of three distinct sizes make
heterodimers that form the subunits of the major inter-
mediate filaments of neurons.
IF ■■ Lamins are a family of seven isoforms present in the cell
nucleus, where they form a structural framework called
IF
the nuclear lamina just inside the nuclear envelope
(see Chapter 3).
J
› ›› MEDICAL APPLICATION
J The presence of a specific type of intermediate filament in
tumors can often reveal the cellular origin of the tumor, infor-
mation important for diagnosis and treatment of the cancer.
Identification of intermediate filament proteins by means of
Intermediate filaments (IF) display an average diameter of immunocytochemical methods is a routine procedure. One
8-10 nm, between that of actin filaments and microtubules, example is the use of GFAP to identify astrocytomas, the
and serve to provide mechanical strength or stability to cells. most common type of brain tumor.
A large and important class of intermediate filaments is com-

››INCLUSIONS
posed of keratin subunits, which are prominent in epithelial
cells. Bundles of keratin filaments called tonofibrils associate
with certain classes of intercellular junctions (J) common in
epithelial cells and are easily seen with the TEM, as shown here Cytoplasmic inclusions contain accumulated metabolites
in two extensions in an epidermal cell bound to a neighboring or other substances, but unlike organelles have little or no
cell. (60,000X)
metabolic activity themselves. Most inclusions are transitory

02_Mescher_ch02_p017-052.indd 47 25/04/18 6:48 pm

 48 CHAPTER 2  ■  The Cytoplasm

FIGURE 2–28  Cellular inclusions.

PD

a b c

Inclusions are cytoplasmic structures or deposits filled with stored Glycogen is a ready source of energy, and such granules are often
macromolecules and are not present in all cells. abundant in cells with high metabolic activity. (X30,000)
(a) Lipid droplets are abundant in cells of the adrenal cortex and (c) Pigment deposits (PD) occur in many cell types and may con-
appear with the TEM as small spherical structures with homog- tain various complex substances, such as lipofuscin or melanin.
enous matrices (L). Mitochondria are also seen here. As aggregates Lipofuscin granules represent an accumulating by-product of lyso-
of hydrophobic lipid molecules these inclusions are enclosed somal digestion in long-lived cells, but melanin granules serve to
by a single monolayer of phospholipids with various peripheral protect cell nuclei from damage to DNA caused by light. Many cells
proteins, including enzymes for lipid metabolism. In routine pro- contain pigmented deposits of hemosiderin granules containing
cessing of tissue for paraffin sections, fat droplets are generally the protein ferritin, which forms a storage complex for iron. Hemo-
removed, leaving empty spaces in the cells. Common fat cells have siderin granules are very electron dense, but with the light micro-
cytoplasm essentially filled with one large lipid droplet. (X19,000) scope they appear brownish and resemble lipofuscin. The liver
(b) TEM of a liver cell cytoplasm shows numerous individual or cells shown have large cytoplasmic regions filled with pigment
clustered electron-dense particles representing glycogen gran- deposits, which probably represent iron-containing hemosiderin.
ules, although these granules lack membrane. Glycogen granules (X400; Giemsa)
usually form characteristic aggregates such as those shown.

structures not enclosed by membrane. Features of important with many atoms of bound iron, prominent in phago-
and commonly seen inclusions are shown in Figure 2–28 and cytic cells of the liver and spleen, where it results from
include the following: phagocytosis of red blood cells.
■■ Lipid droplets, accumulations of lipid-filling adipocytes
(fat cells) and present in various other cells.
■■ Glycogen granules, aggregates of the carbohydrate
› ›› MEDICAL APPLICATION
polymer in which glucose is stored, visible as irregu- A condition termed hemosiderosis, in which the iron-
lar clumps of periodic acid–Schiff (PAS)—positive or containing inclusion hemosiderin occurs in cells of organs
electron-dense material in several cell types, notably throughout the body, may be seen with increased uptake of
liver cells. dietary iron, impaired iron utilization, or with excessive lysis
■■ Pigmented deposits of naturally colored material, includ- of red blood cells. Hemosiderosis itself does not damage cell
ing melanin, dark brown granules which in skin serve to or organ function. However, extreme accumulations of iron
protect cells from ultraviolet radiation; lipofuscin, a pale in cellular hemosiderin can lead to disorders, such as hemo-
brown granule found in many cells, especially in stable chromatosis and iron overload syndrome, in which tissues of
nondividing cells (eg, neurons, cardiac muscle), contain- the liver and other organs are damaged.
ing a complex mix of material partly derived from resid-
ual bodies after lysosomal digestion; and hemosiderin, A summary of the major structural and functional fea-
a dense brown aggregate of denatured ferritin proteins tures of all cytoplasmic components is presented in Table 2–6.

02_Mescher_ch02_p017-052.indd 48 25/04/18 6:48 pm

 Inclusions 49

TABLE 2–6      Summary of cellular structural components.

C H A P T E R
Component Structure Major Function    Appearance

Plasma Phospholipid bilayer containing Acts as a physical barrier to enclose cell Plasma membrane
membrane cholesterol and proteins (integral contents; regulates material movement
and peripheral) and some into and out of the cell; establishes and
carbohydrates (externally); forms a maintains an electrical charge difference
selectively permeable boundary of across the plasma membrane; functions

2
the cell in cell communication

The Cytoplasm  ■ Inclusions

Cilia Short, numerous membrane Move substances (eg, mucus and Cilia
extensions supported by dissolved materials) over the cell surface
microtubules, which occur on
exposed membrane surfaces of
some cells
Flagellum Long, singular membrane extension Propels sperm
supported by microtubules; present
on sperm cells Flagellum

Microvilli Numerous thin membrane folds Increase membrane surface area for Microvilli
projecting from the free cell surface; greater absorption
supported by microfilaments
Nucleus Large structure enclosed within Houses the DNA that serves as the
a double membrane; contains genetic material for directing protein
chromatin, nucleolus, and synthesis Nucleus
nucleoplasm
 Nuclear Double membrane boundary Separates nucleus from cytoplasm Nuclear
envelope between cytoplasm and nuclear pores
contents; continuous with rough Nucleolus
Nuclear
endoplasmic reticulum envelope Chromatin

 Nuclear Openings through the nuclear Allow passage of materials between the
pores envelope cytoplasm and nucleoplasm, including
ribonucleic acid (RNA), protein, ions, and
small water-soluble molecules
 Nucleolus Large, prominent structure within Functions in synthesis of ribosomes
the nucleus
Cytoplasm Contents of cells between the Responsible for many cellular processes Cytoplasm
plasma membrane and nuclear Cytosol Organelles
envelope
 Cytosol Viscous fluid medium with Provides support for organelles; serves as
dissolved solutes (eg, ions, proteins, the viscous fluid medium through which
carbohydrates, lipids) diffusion occurs
 Organelles Membrane-bound and Carry out specific metabolic activities of
nonmembrane-bound structures the cell Inclusions

Rough Extensive interconnected Modifies, transports, and stores proteins

endoplasmic membrane network that varies produced by attached ribosomes;
reticulum in shape (eg, cisternae, tubules); these proteins are secreted, become
(rough ER) ribosomes attached on cytoplasmic components of the plasma membrane, or
surface serve as enzymes of lysosomes

Smooth Extensive interconnected Synthesizes, transports, and stores lipids

endoplasmic membrane network lacking (eg, steroids); metabolizes carbohydrates;
reticulum ribosomes detoxifies drugs, alcohol, and poisons;
(smooth ER) forms vesicles and peroxisomes

(Continued )

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 50 CHAPTER 2  ■  The Cytoplasm

TABLE 2–6    Summary of cellular structural components. (Continued)

Component Structure Major Function    Appearance

Golgi Series of several elongated, Modifies, packages, and sorts materials

apparatus flattened saclike membranous that arrive from the ER in transport
structures vesicles; forms secretory vesicles and
lysosomes

Vesicles Spherical-shaped membrane- Transport cellular material

bound sacs; contain various types
of materials to be transported
through the cell

Lysosomes Spherical-shaped membrane- Digest microbes or materials (eg, ingested

bound organelles formed from the by the cell, worn-out cellular components,
Golgi apparatus; contain digestive or the entire cell)
enzymes

Peroxisomes Smaller, spherical-shaped Detoxify specific harmful substances

membrane-bound organelles either produced by the cell or taken into
formed from the ER or through the cell; engage in beta oxidation of fatty
fission; contain oxidative enzymes acids to acetyl CoA
Mitochondria Double membrane-bound Synthesize most ATP during aerobic
organelles containing a circular cellular respiration by digestion of fuel
strand of DNA (genes for producing molecules (eg, glucose) in the presence
mitochondrial proteins) of oxygen

Ribosomes Organelles composed of both Engage in protein synthesis: Bound Bound

protein and ribosomal RNA (rRNA) ribosomes produce proteins that are secreted, ribosomes
that are organized into both a large incorporated into plasma membrane, and
and small subunit; may be bound within lysosomes; free ribosomes produce Free
to a membrane or free in cytosol proteins used within the cell ribosomes

Cytoskeleton Organized network of protein Maintains intracellular structural support Cytoskeleton

filaments and hollow tubules, and organization of cells; participates in
including microfilaments, cell division; facilitates movement Intermediate filament Microfilament
intermediate filaments, and Microtubule
microtubules
Microfilaments Actin protein monomers organized Maintain cell shape; support microvilli;
into two thin, intertwined protein separate two cells during cytokinesis
filaments (actin filaments) (a process of cell division); facilitate change in
cell shape; participate in muscle contraction
Intermediate Various protein components Provide structural support; stabilize
filaments junctions between cells
Microtubules Hollow cylinders composed of Maintain cell shape and rigidity; organize
tubulin protein and move organelles; support cilia and
flagella; participate in vesicular transport;
separate chromosomes during the
process of cell division
Centrosome Amorphous region adjacent to Organizes microtubules; participates
nucleus; contains a pair of centrioles in mitotic spindle formation during cell
division Centriole
Centrosome

Proteasomes Large, barrel-shaped protein Degrade and digest damaged or

complexes located in both the unneeded proteins; ensure quality of
cytosol and nucleus exported proteins
Inclusions Aggregates of specific types of Serve as temporary storage for these Variable appearance
molecules (eg, melanin protein, molecules
glycogen, or lipid)

02_Mescher_ch02_p017-052.indd 50 25/04/18 6:48 pm

 Inclusions 51

The Cytoplasm   SUMMARY OF KEY POINTS

C H A P T E R
■■ Cell differentiation is the process by which cells of an embryo ■■ Vesicle movement through the Golgi apparatus is guided by specific
become specialized structurally to augment specific cytoplasmic coat proteins such as COPII and COPI.
activities for functions at the level of tissues and organs. ■■ Important protein modifications in the Golgi apparatus include sul-
■■ Organelles are metabolically active structures or complexes, with fation and many glycosylation reactions.
or without membranes, in the cytoplasm of eukaryotic cells. ■■ Modified proteins leave the Golgi apparatus after packaging in
vesicles with coat proteins that direct movement to lysosomes, the

2
Plasma Membrane
plasma membrane, or secretion by exocytosis.
■■ The plasma membrane (cell membrane or plasmalemma) is the

The Cytoplasm  ■ Inclusions

lipid bilayer with embedded proteins that surrounds a cell and is Lysosomes
seen only with the TEM. ■■ Primary lysosomes emerge from the Golgi apparatus containing
■■ The lipid bilayer forms from amphipathic phospholipids, stabi- inactive acid hydrolases specific for degrading a wide variety of cel-
lized by cholesterol, and contains many embedded (integral) pro- lular macromolecules.
teins and many peripheral proteins on its cytoplasmic surface. ■■ Secondary lysosomes are more heterogeneous, having fused with
■■ Membrane proteins move laterally within the lipid bilayer, with vesicles produced by endocytosis that contain material to be digested
less movement in areas referred to as lipid rafts, which have by the hydrolytic enzymes.
higher concentrations of cholesterol and saturated fatty acids. ■■ During autophagy, lysosomes digest unneeded or nonfunctional
■■ Integral membrane proteins include receptors for external organelles after these are surrounded by membrane that then fuses
ligands, channels for passive or active movement of molecules with a lysosome.
across the membrane, and pumps for active membrane transport. ■■ Products of digestion in secondary lysosomes are released to the
■■ Endocytosis is cellular uptake of macromolecules or fluid by cytoplasm for reuse; final condensed vesicles containing any indi-
plasma membrane engulfment or invagination, followed by the gestible molecules are called residual bodies.
“pinching off ” of a filled membranous vesicle in the cytoplasm.
■■ Major types of endocytosis include phagocytosis (uptake of par- Proteasomes
ticulate material), pinocytosis (uptake of dissolved substances), ■■ Proteasomes are small cytoplasmic protein complexes which
and receptor-mediated endocytosis (uptake of specific mol- degrade improperly folded proteins after they are tagged with the
ecules bound to integral membrane receptor proteins). polypeptide ubiquitin.
■■ Exocytosis is a type of cellular secretion in which cytoplasmic
Mitochondria
membrane vesicles fuse with the plasma membrane and release
their contents to the extracellular space. ■■ Mitochondria are the major sites of ATP synthesis and are abun-
■■ All types of cell signaling use membrane receptor proteins that are dant in cells or cytoplasmic regions where large amounts of energy
often linked to enzymes such as kinases or adenylyl cyclase whose are expended.
activities initiate intracellular signaling pathways. ■■ Mitochondria are usually elongated organelles and form by fission
of preexisting mitochondria.
Ribosomes ■■ Mitochondria have two membranes: a porous outer membrane
■■ The two ribosomal subunits, each a complex of rRNA and many encloses the intermembrane space and an inner membrane with
proteins, attach to mRNA and translate that message into protein. many folds (cristae) enclosing a gel-like matrix.
■■ Multiple ribosomes on the same mRNA make up a polyribosome ■■ The mitochondrial matrix contains enzymes for β-oxidation of fatty
(polysome), and an abundance of these produces basophilic cyto- acids and the citric acid (Krebs) cycle.
plasm after H&E staining. ■■ The inner membrane includes enzyme assemblies of the electron-
transport system and ATP synthase.
Endoplasmic Reticulum ■■ Mitochondria of stressed cells may release cytochrome c from the
■■ The ER is a convoluted network of membrane enclosing continu- inner membrane, triggering a regulated series of events culminating
ous spaces called cisternae and extending from the nucleus to the in cell death (apoptosis).
plasma membrane.
■■ Rough ER has a granular, basophilic cytoplasmic surface due to Peroxisomes
the presence of polysomes making most membrane proteins, pro- ■■ Peroxisomes are small spherical organelles containing enzymes for
teins in certain other organelles, or for exocytosis; RER is always various metabolic reactions, notably for oxidation and detoxifica-
well developed in cells actively secreting proteins. tion, and catalase that breaks down the H2O2 resulting from those
■■ Proteins to be processed through the RER contain initial signal reactions.
peptides which bind receptors in the ER membrane, localizing
Cytoskeleton
them to that organelle.
■■ After translocation across the membrane into the cisterna, the ■■ The cytoskeleton contains three types of polymers: (1) microtubules
proteins undergo posttranslational modification and folding in 25 nm in diameter; (2) actin filaments or microfilaments (5-7 nm);
a process monitored by RER molecular chaperones and enzymes. and (3) intermediate filaments (8-10 nm).
■■ Smooth ER (SER) lacks ribosomes, but includes enzymes for ■■ Microtubules are semirigid tubular structures with walls composed
lipid and glycogen metabolism, for detoxification reactions, of polymerized tubulin heterodimers; their structure is often very
and for temporary Ca2+ sequestration. dynamic, with steady addition and dissociation of tubulin.
■■ Microtubules are important in maintaining cell shape and as tracks
Golgi Apparatus for transport of vesicles and organelles by the motor proteins kine-
■■ The Golgi apparatus is a dynamic organelle consisting of stacked sin and dynein.
membranous cisternae in which proteins made in RER are pro- ■■ Microfilaments are short, flexible, highly dynamic filaments of actin
cessed further and packaged for secretion or other roles. subunits, in which changes in length and interactions with binding
■■ Proteins in transport vesicles enter the cis or receiving face of the proteins regulate cytoplasmic viscosity and movement.
Golgi, move through medical cisternae of the Golgi network for enzy- ■■ Myosins are motor proteins that bind and move along actin fila-
matic modifications, and are released in other vesicles at the trans face. ments, carrying vesicles or producing cytoplasmic movement.

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 52 CHAPTER 2  ■  The Cytoplasm

■■ Movements of cytoplasm produced by actin filaments and myosins neurofilament proteins, and keratins, which are especially impor-
are important for endocytosis, cell cleavage after mitosis, and cell tant in epithelial cells.
locomotion on substrates.
■■ Intermediate filaments are the most stable cytoskeletal component, Inclusions
conferring strong mechanical stability to cells. ■■ Unlike organelles, inclusions are not metabolically active and are
■■ Intermediate filaments are composed of various protein sub- primarily storage sites, such as lipid droplets, glycogen granules,
units in different cells; they include vimentin, nuclear lamins, pigment granules, or residual bodies (also called lipofuscin).

The Cytoplasm   ASSESS YOUR KNOWLEDGE

7. Vesicles of a Golgi apparatus that are destined to become part of

1. In transmission EM preparations of cells the cell membrane often
other organelles most likely have which of the following on their
appears as a trilaminar structure having two parallel dark-staining
membranes?
components on either side of an unstained middle layer. This cen-
tral poorly stained region of the membrane is primarily respon- a. Channel proteins
sible for which of the following functions? b. Clathrin
c. COP II
a. Creation of a barrier to water-soluble molecules
d. Actin
b. Binding by cellular receptions to specific ligands
e. GTP
c. Catalyzing membrane-associated activities
d. Transport of ions 8. About 3 years ago, a 39-year-old construction worker became increas-
e. Connections to the cytoskeleton ingly uncoordinated. His wife describes bouts of depression and apa-
thy beginning about a decade ago. Laboratory tests are normal. MRI
2. Chaperonins are cytoplasmic proteins most likely to be found in
and CT reveal striatal and caudate atrophy with “boxcar ventricles.”
which of the following organelles?
His mini-mental status examination score is 24/30. The cranial nerve
a. Lysosomes examination shows dysarthria, saccadic extraocular eye movements,
b. Golgi complexes and a hyperactive gag reflex. There is increased tone in all extremities.
c. Rough endoplasmic reticulum Polymerase chain reaction reveals one normal band with 20 CAG (tri-
d. Smooth endoplasmic reticulum nucleotide) repeats and the other with 49 CAG repeats. Modulation of
e. Mitochondria respiration and mitochondrial membrane potential, and bioenergetic
3. Which of the following best defines the term “exocytosis”? failure are associated with the abnormal gene in this disease. Which of
the following mechanisms used to establish the mitochondrial electro-
a. The discharge of ions or small molecules from a cell by protein
chemical gradient may be altered in this disease?
pumps in the cell membrane
b. The uptake of material at one domain of a cell’s surface and its a. The action of ATP synthase
discharge from the opposite side of the cell b. Transfer of electrons from NADH to O2 in the intermembrane
c. The process by which proteins move from one cytoplasmic space
compartment to another c. Pumping of protons into the mitochondrial matrix by respiratory
d. The discharge of proteins in cytoplasmic vesicles from a cell chain activity
following fusion of the vesicles with the plasmalemma d. Proton-translocating activity in the inner membrane
e. Diffusion of lipid-soluble molecules from a cell across the cell e. Transport of ATP out of the matrix compartment by a specific
membrane transporter

4. Cytoplasm often stains poorly because its lipid content is removed 9. A 56-year-old man has been taking atorvastatin because of a poor
by the organic solvents used in the clearing step in routine his- lipid profile and a family history of cardiovascular disease. The statin
tological preparations. This problem is most likely to occur with family of drugs enhances endocytosis of low-density lipoprotein
cytoplasmic regions rich in which of the following organelles? (LDL) from the blood. Endocytosis of LDL differs from phagocytosis
of bacterial cells in which of the following ways?
a. Free polysomes
b. Mitochondria a. Use of membrane-enclosed vesicles in the uptake process
c. Lysosomes b. Coupling with the lysosomal system
d. Smooth endoplasmic reticulum c. Dependence on acidification
e. Rough endoplasmic reticulum d. Use of clathrin-coated pits
e. Use of hydrolases
5. Polarity in microtubules is important in determining which of the
following? 10. A 14-year-old boy is diagnosed with epidermolysis bullosa simplex
(EBS). His skin blisters easily with rubbing or scratching. Blisters
a. The strength of vinblastine binding to microtubules
occur primarily on his hands and feet and heal without leaving scars.
b. The velocity of transport along microtubules with myosin motors
Genetic analysis shows mutations in the KRT5 and KRT14 genes,
c. The overall dynamic instability of the microtubules
which code keratin 5 and keratin 14. What is the primary function
d. The linkage of microtubules to intermediate filaments
of those proteins?
e. The direction of vesicular transport along microtubules
a. Generate movement
6. Which of the following proteins is/are most likely to have initially b. Provide mechanical stability
contained a “signal peptide” that bound a “signal recognition par- c. Carry out nucleation of microtubules
ticle” during its translation? d. Stabilize microtubules against disassembly
a. An enzyme of the respiratory chain e. Transport organelles within the cell
b. Lamins
c. Proteins in secretory granules
d. F-actin
e. Proteins in the large ribosomal subunit Answers: 1a, 2c, 3d, 4d, 5e, 6c, 7c, 8d, 9d, 10b

02_Mescher_ch02_p017-052.indd 52 25/04/18 6:49 pm

 C H A P T E R

Nuclear Envelope
3
COMPONENTS OF THE NUCLEUS 53
53
The Nucleus

STEM CELLS & TISSUE RENEWAL 65

MEIOSIS 65
Chromatin 53
APOPTOSIS 67
Nucleolus 56
THE CELL CYCLE 58 SUMMARY OF KEY POINTS 69

MITOSIS 61 ASSESS YOUR KNOWLEDGE 70

C ontaining the code for all of a cell’s enzymes and

other proteins, the nucleus is the command center
of the cell. The nucleus also contains the molecular
machinery to replicate the DNA and to synthesize and pro-
cess all types of RNA. During interphase, pore complexes in
which stabilizes the nuclear envelope. Major components of
this layer are the class of intermediate filament proteins called
lamins that bind to membrane proteins and associate with
chromatin in nondividing cells.
The inner and outer nuclear membranes are bridged at
the membrane enclosing the nucleus regulate macromolecular nuclear pore complexes (Figures 3–2 through 3–6). Vari-
transfer between the nuclear and cytoplasmic compartments. ous core proteins of a nuclear pore complex, called nucleo-
Mature RNA molecules pass into the cytoplasm for their roles porins, display eightfold symmetry around a lumen. Ions and
in protein synthesis, while proteins needed for nuclear activi- small solutes pass through the lumen by simple diffusion, but
ties are imported there from the cytoplasm. Restricting pro- the nucleoporin complex regulates the movement of macro-
tein synthesis to the cytoplasm helps ensure that newly made molecules between nucleus and cytoplasm. A growing cell
RNA molecules do not become involved in translation before has 3000-4000 such channels, each providing passage for up
processing is complete. to 1000 macromolecules per second. Individual pores permit
molecular transfer in both directions simultaneously. Mac-
romolecules shipped out of the nucleus include ribosomal
››COMPONENTS OF THE NUCLEUS subunits and other RNAs associated with proteins, while
inbound traffic consists of chromatin proteins, ribosomal
The nucleus usually appears as a large rounded or oval struc- proteins, transcription factors, and enzymes. Using mecha-
ture, often near the cell’s center (Figure 3–1). Typically the larg- nisms similar to that by which specific proteins are recog-
est structure within a cell, it consists of a nuclear envelope nized and translocated across the RER membrane, proteins
containing chromatin, the mass of DNA and its associated of complexes destined for the cytoplasm have specific nuclear
proteins, with one or more specialized regions of chromatin export sequences and proteins to be imported have nuclear
called nucleoli. In specific tissues, the size and shape of nuclei localization sequences. Such sequences bind specifically to
normally tend to be uniform. transport proteins (importins) that in turn interact with pro-
teins of the pore complexes for transfer across the nuclear
Nuclear Envelope envelope. Energy for the transport is derived from guanosine
5′-triphosphate (GTP), with specific GTPases helping provide
The nuclear envelope forms a selectively permeable barrier
directionality to the transfer.
between the nuclear and cytoplasmic compartments. Electron
microscopy reveals that the envelope has two concentric mem-
branes separated by a narrow (30-50 nm) perinuclear space Chromatin
(Figures 3–2 and 3–3). This space and the outer nuclear mem- Chromatin consists of DNA and all of the associated proteins
brane are continuous with the extensive cytoplasmic network involved in the organization and function of DNA. In humans,
of the rough endoplasmic reticulum (RER). Closely associated each cell’s chromatin (except that of eggs and sperm) is divided
with the inner nuclear membrane is a highly organized mesh- among 46 chromosomes (23 pairs). After DNA replication
work of proteins called the nuclear lamina (Figure 3–4), but before cell division, each chromosome consists of two

53

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 54 CHAPTER 3  ■  The Nucleus

and rearrangement of the histones allows temporary unwrap-

FIGURE 3–1  Nuclei of large, active cells.
ping of the DNA and arrival of enzymes and other proteins
required for replication and gene transcription.
DNA wrapped around nucleosomes is coiled further
for greater compaction within the nucleus and for general
regulation of gene activity. The 10-nm fiber of nucleosomes
and DNA undergoes helical folding to yield a fiber with a
diameter of 30 nm (Figure 3–8). Beyond the 30-nm fiber
chromatin structure is much less well understood, but it
does form nearly ubiquitous kilobase-sized and larger loops
of coiled DNA, some of which are unstable and may reflect
gene activity rather than a distinct hierarchical level of chro-
matin structure. Many such loops appear tethered to central
scaffold-like arrays containing large protein complexes (con-
densins) capable of compacting chromatin. Further packag-
ing during the first phase of cell division causes chromosomes
to become visible as discrete structures by light microscopy
(Figure 3–8).
Microscopically two categories of chromatin can be dis-
tinguished in nuclei of most nondividing cells (Figure 3–3).
Euchromatin is visible as finely dispersed granular material
in the electron microscope and as lightly stained basophilic
areas in the light microscope. Heterochromatin (Gr. heteros,
other + chroma, color) appears as coarse, electron-dense mate-
rial in the electron microscope and as intensely basophilic
clumps in the light microscope.
DNA in the more open structure of euchromatin is rich
with genes, although not all of the genes are transcribed in all
cells. Heterochromatin is always more compact than euchro-
matin, shows little or no transcriptional activity, and includes
at least two types of genomic material called constitutive and
Liver cells have large, central nuclei. One or more highly
facultative heterochromatin. Constitutive heterochromatin is
basophilic nucleoli are visible within each nucleus, indicating
intense protein synthesis by these cells. Most of the chroma- generally similar in all cell types and contains mainly repeti-
tin is light staining or euchromatic, with small areas of more tive, gene-poor DNA sequences, including the large chromo-
darkly stained heterochromatin scattered throughout the somal regions called centromeres and telomeres, which
nucleus and just inside the nuclear envelope. This superficial are located near the middle (most often) and at the ends of
heterochromatin allows the boundary of the organelle to be
chromosomes, respectively. Facultative heterochromatin con-
seen more easily by light microscopy. One cell here has two
nuclei, which is fairly common in the liver. (X500; Pararosani- tains other regions of DNA with genes where transcription
line–toluidine blue) is variably inactivated in different cells by epigenetic mecha-
nisms and can undergo reversible transitions from compact,
transcriptionally silent states to more open, transcriptionally
identical chromatin units called chromatids held together by active conformations.
complexes of cohesin proteins. The ratio of heterochromatin to euchromatin seen with
DNA of each human cell is approximately 2 m long, nuclear staining can provide a rough indicator of a cell’s meta-
with 3.2 billion base pairs (bp), and therefore must be exten- bolic and biosynthetic activity (Figure 3–3). Euchromatin
sively packaged within the nucleus. This occurs initially by predominates in active cells such as large neurons, while het-
the DNA associating with sets of small basic proteins called erochromatin is more abundant in cells with little synthetic
histones. The structural unit of DNA and histones is called activity such as circulating lymphocytes. Facultative hetero-
the nucleosome (Figures 3–7 and 3–8), which has a core of chromatin also occurs in the small, dense “sex chromatin” or
eight histones (two copies each of histones H2A, H2B, H3, Barr body which is one of the two large X chromosomes pres-
and H4), around which is wrapped about 150 bp of DNA. ent in human females but not males. The Barr body remains
Each nucleosome also has a larger histone (H1) associated tightly coiled, while the other X chromosome is uncoiled,
with both the wrapped DNA and the surface of the core. In transcriptionally active, and not visible. Cells of males have
the EM the series of nucleosomes on DNA resembles “beads one X chromosome and one Y chromosome; like the other
on a string,” with 50-80 bp of linker DNA separating each chromosomes, the single X chromosome remains largely
bead. Nucleosomes are structurally dynamic; modification euchromatic.

03_Mescher_ch03_p053-070.indd 54 25/04/18 6:52 pm

 Components of the Nucleus 55

FIGURE 3–2  Relationship of nuclear envelope to the rough ER (RER).

C H A P T E R
Nucleus

Nuclear
Nuclear
pores
envelope

3
Nucleolus

The Nucleus  ■  Components of the Nucleus

Chromatin

Ribosome

Rough endoplasmic
reticulum

Functions of the Nucleus

1. Cellular regulation: Houses genetic material, which directs

all cellular activities and regulates cellular structure
2. Production: Produces ribosomal subunits in nucleolus and
exports them into cytoplasm for assembly into ribosomes

Three-dimensional representation of a cell nucleus shows a single nuclear envelope. The outer membrane of the nuclear envelope is
large nucleolus and the distribution of the nuclear pores in the continuous with the RER. (TEM X20,000)

of each chromosomal pair are called homologous because,

› ›› MEDICAL APPLICATION although from different parents, they contain forms (alleles) of
Barr bodies or gender chromatin permit gender to be deter- the same genes. Cells of most tissues (somatic cells) are consid-
mined microscopically in patients whose external sex organs ered diploid because they contain these pairs of chromosomes.
do not permit that determination, as in hermaphroditism and Geneticists refer to diploid cells as 2n, where n is the number of
pseudohermaphroditism. Sex chromatin analysis also helps unique chromosomes in a species, 23 in humans. Sperm cells
reveal other anomalies involving the sex chromosomes, such and mature oocytes (germ cells) are haploid, with half the dip-
as the presence of XXY chromosomes (Klinefelter syndrome), loid number of chromosomes, each pair having been separated
which causes testicular abnormalities and azoospermia during meiosis (described later in the chapter).
(absence of sperm). Microscopic analysis of chromosomes usually begins with
cultured cells arrested in mitotic metaphase by colchicine or
other compounds that disrupt microtubules. After process-
Although much heterochromatin tends to be concen- ing and staining the cells, the condensed chromosomes of one
trated near the nuclear lamina, evidence for spatial orga- nucleus are photographed by light microscopy and rearranged
nization of chromatin is not normally seen. Recent in situ digitally to produce a karyotype in which stained chromo-
hybridization studies of cultured human fibroblast nuclei, somes can be analyzed (Figure 3–10).
using differently labeled fluorescent probes for sequences on
each individual chromosome, have revealed that these struc-
tures occupy discrete chromosomal territories within dis- › ›› MEDICAL APPLICATION
persed chromatin (Figure 3–9). Such studies show further that
Karyotyping is important for many prenatal diagnoses, in
chromosomal domains with few genes form a layer beneath
which chromosomal analysis of cultured cells from the fetus
the nuclear envelope, while domains with many active genes
or amnion can detect certain genetic anomalies. As with
are located deeper in the nucleus.
karyotypes of adults, missing or extra chromosomes and chro-
The X and Y sex chromosomes contain genes determining
mosomal deletions or translocations are readily seen. New
whether an individual will develop as a female or a male. In
methods of chromosomal staining and molecular techniques,
addition to the pair of sex chromosomes, cells contain 22 pairs
such as fluorescence in situ hybridization (FISH), are continu-
of autosomes. Each pair contains one chromosome originally
ously being developed and used for cytogenetic diagnosis.
derived from the mother and one from the father. The members

03_Mescher_ch03_p053-070.indd 55 25/04/18 6:52 pm

 56 CHAPTER 3  ■  The Nucleus

FIGURE 3–3  Ultrastructure of a nucleus.

Regions of euchromatin and heterochromatin display variable associated with the nuclear lamina. The more heterogeneous
electron densities with the transmission electron microscope electron-dense subdomain is the nucleolus (N), the site of rRNA
(TEM). An active nucleus typically has much diffuse, light-staining synthesis, and ribosomal subunit assembly. (X25,000)
euchromatin and smaller subdomains of electron-dense hetero-
chromatin (H), with many of these associated at the periphery

Nucleolus ribosomal subunits are then exported back to the cytoplasm

through those same nuclear pores.
The nucleolus is a generally spherical, highly basophilic sub-
domain of nuclei in cells actively engaged in protein synthesis
(Figures 3–1 through 3–3). The intense basophilia of nucleoli
is due not to heterochromatin but to the presence of densely › ›› MEDICAL APPLICATION
concentrated ribosomal RNA (rRNA) that is transcribed, pro-
Tissues with either stable or rapidly renewing cell popula-
cessed, and assembled into ribosomal subunits. Chromosomal
tions can include cells that become transformed to grow
regions with the genes for rRNA organize one or more nucle-
at a higher rate and in an uncoordinated manner. Such
oli in cells requiring intense ribosome production for protein
neoplastic proliferation typically follows damage to the
synthesis during growth or secretion. Ultrastructural analysis
DNA of proto-oncogenes and failure of the cells to be
of an active nucleolus reveals fibrillar and granular subregions
eliminated. Neoplastic growth can be either benign (with
with different staining characteristics that reflect stages of
slow growth and no invasiveness to neighboring organs) or
rRNA maturation, as described in Figure 3–11. Molecules of
malignant (with rapid growth and great capacity to invade
rRNA are processed in the nucleolus and very quickly asso-
other organs). Cancer is the common term for all malignant
ciate with the ribosomal proteins imported from the cyto-
tumors.
plasm via nuclear pores. The newly organized small and large

03_Mescher_ch03_p053-070.indd 56 25/04/18 6:52 pm

 Components of the Nucleus 57

FIGURE 3–4  The nuclear envelope, nuclear lamina, and nuclear pore complexes.

C H A P T E R
Perinuclear Rough ER Nuclear pore
space complex
Inner and outer
nuclear membrane
NL

3
The Nucleus  ■  Components of the Nucleus
NPC

NL
a b

Nuclear lamina Chromatin

(a) Bound to the inner membrane of the nuclear envelope is the the cytoplasm. Nuclear pores simultaneously transport macromol-
nuclear lamina, a meshwork assembled from lamins (class V inter- ecules in both directions, utilizing energy derived locally from GTP,
mediate filament proteins). The 100-nm wide nuclear pore com- and unlike most cell membrane channels or transporters do not
plexes (NPC) each contain approximately 34 nucleoporin proteins open and close while regulating transport.
forming a symmetric core of inner and outer rings which stabilize (b) Scanning EM of the inner nuclear membrane (nucleoplasmic
the extreme curvature of the nuclear envelope’s inner and outer face) showing portions of the nuclear lamina (NL) meshwork with
membranes where they abut the pore. Proteins in the cytoplasm many embedded nuclear pore complexes (NPC). The preparation
that are destined for the nucleus bind to importin proteins, which is from an actively growing amphibian oocyte. Nuclei of these
in turn bind filamentous polypeptides that extend from the NPC very large cells can be isolated manually, facilitating ultrastructural
outer ring and facilitate import into the nucleus. On the inside of studies of the nuclear envelope. (X100,000)
the NPC a basket-like assembly of other nucleoporins interacts (Used with permission from Dr M.W. Goldberg, Department of
with the transcriptional machinery in chromatin and facilitates Biological and Biomedical Sciences, Durham University, UK.)
export of newly made messenger ribonucleoprotein particles into

FIGURE 3–5  Nuclear pores.

C
N
a

b
(a) A TEM section through the nuclear envelope between
nucleus (N) and cytoplasm (C) shows its two-membrane struc-
ture. The electron-dense nuclear pore complexes bridging the (b) A tangential section through a nuclear envelope shows
nuclear envelope can also be seen (arrows). Electron-dense het- the nuclear pore complexes (arrows) and the electron-lucent
erochromatin is adjacent to the envelope, except at the nuclear patches in the peripheral heterochromatin, which represent
pores. the areas just inside pores. (X80,000)

03_Mescher_ch03_p053-070.indd 57 25/04/18 6:52 pm

 58 CHAPTER 3  ■  The Nucleus

FIGURE 3–6  Cryofracture of nuclear envelope showing nuclear pores.

An electron micrograph obtained by freeze-fractured cell shows membranes (left) but mostly just inside the envelope with the
the two layers of the nuclear envelope and nuclear pores. The chromatin removed. The size and distribution of the nuclear pore
fracture plane occurs partly between the two nuclear envelope complexes are clearly seen. (X60,000)

› ›› MEDICAL APPLICATION G2 (the gap between DNA duplication and the next mitosis).
The approximate durations of these phases in rapidly dividing
Certain mutations in the gene coding for lamin A are associ- human cells are illustrated in Figure 3–12. The G1 phase, usu-
ated with a subtype of the disorder progeria, which causes ally the longest and most variable part of the cycle, is a period
premature aging. In this and other rare “laminopathies,” the of active RNA and protein synthesis, including proteins con-
nuclear envelope is abnormal, but how this is linked to the trolling progress through the cell cycle. Also in G1, the cell
disorder is unclear. Laminopathies affect some tissues much volume, reduced by half during mitosis, returns to its previ-
more than others, although the lamins involved are in all the ous size. The S phase is characterized by DNA replication, his-
body’s cells. tone synthesis, and the beginning of centrosome duplication.
In the relatively short G2 phase, proteins required for mitosis
accumulate. As new postmitotic cells specialize and differen-

››THE CELL CYCLE tiate, cell cycle activities may be temporarily or permanently
suspended, with the cells sometimes referred to as being in
Before differentiation, most cells undergo repeated cycles of the G0 phase. Some differentiated cells, such as those of the
macromolecular synthesis (growth) and division (mitosis). liver, renew cycling under certain conditions; others, includ-
The regular sequence of events that produce new cells is termed ing most muscle and nerve cells, are terminally differentiated.
the cell cycle. Improved knowledge about how each phase of Cycling is activated in postmitotic G0 cells by protein sig-
the cell cycle is controlled and how the quality of molecular nals from the extracellular environment called mitogens or
synthesis, particularly DNA replication, is monitored has led growth factors that bind to cell surface receptors and trigger
to understanding the causes of many types of cancer, in which a cascade of kinase signaling in the cells. The cells are then
cells proliferate without those controls. maintained at the restriction point at the G1/S “boundary” until
The cell cycle has four distinct phases: mitosis and peri- sufficient nutrients and enzymes required for DNA synthesis
ods termed G1 (the time gap between mitosis and the begin- have accumulated, and when all is ready DNA replication
ning of DNA replication), S (the period of DNA synthesis), and (S phase) begins.

03_Mescher_ch03_p053-070.indd 58 25/04/18 6:52 pm

 The Cell Cycle 59

FIGURE 3–7  Components of a nucleosome. FIGURE 3–8  From DNA to chromatin.

C H A P T E R
Core of 8 histone molecules: H1 histone
H2A, H2B, H3, and H4
DNA 2 nm

Nucleosomes

3
(DNA and core 11 nm
6 nm histones)

The Nucleus  ■  The Cell Cycle

Central DNA
Packed
Link DNA nucleosomes
30 nm
in 30-nm
chromatin fiber
10 nm

Extended loops of
transcriptionally
active chromatin, 300 nm
Nucleosome is a structure that produces the initial organiza- tethered to
tion of free double-stranded DNA into chromatin. Each nucleo- protein scaffold
some has an octomeric core complex made up of four types
of histones, two copies each of H2A, H2B, H3, and H4. Around
this core is wound DNA approximately 150 base pairs in Heterochromatin
length. One H1 histone is located outside the DNA on the sur-
face of each nucleosome. DNA associated with nucleosomes Condensed
in vivo thus resembles a long string of beads. Nucleosomes are heterochromatin 700 nm
very dynamic structures, with H1 loosening and DNA unwrap- and dispersed
ping at least once every second to allow other proteins, includ- euchromatin Euchromatin
ing transcription factors and enzymes, access to the DNA.
Centromere

Entire
chromosome 1400 nm
As shown in Figure 3–13, entry or progression through at metaphase
other phases of the cycle is also monitored at other specific
checkpoints, where certain conditions must be met before the Chromatids
cell continues cycling. Overall cycling is regulated by a family
of cytoplasmic proteins called cyclins. With different cyclins Several orders of DNA packing occur in chromatin and dur-
present during different cell cycle phases, each activates one or ing chromatin condensation of mitotic prophase. The top
drawing shows the 2-nm DNA double helix, followed by the
more specific cyclin-dependent kinases (CDKs). Each acti-
association of DNA with histones to form 11-nm filaments of
vated CDK then phosphorylates specific proteins, including nucleosomes connected by the DNA (“beads on a string”).
enzymes, transcription factors for specific sets of genes, and Nucleosomes on the DNA then interact in a manner not well
cytoskeletal subunits, triggering the activities that characterize understood to form a more compact 30-nm fiber. DNA in such
the next phase of the cycle. When each successive set of activi- fibers forms various loops, some of which in euchromatin
involve gene transcription. Loops remain tethered to and sta-
ties is complete, the cyclin controlling that cell cycle phase is
bilized by interactions with protein scaffolds that eventually
ubiquitinated and removed rapidly by proteasomes and a new make up a central framework at the long axis of each chromo-
cyclin that promotes activities for the next phase takes over. In some. Heterochromatin is not transcribed and remains more
this way diverse cellular activities are coordinated with specific highly condensed. The bottom drawing shows a metaphase
phases of the cell cycle. The major cyclins, CDKs, and impor- chromosome, with maximum packing of DNA. The chromo-
some consists of two chromatids held together at a constric-
tant target proteins are summarized in Table 3–1.
tion called the centromere.

› ›› MEDICAL APPLICATION
Many mitogenic growth factors for research are produced factor (G-CSF), which stimulates neutrophil production in
commercially from microorganisms or cells with recombinant immunocompromised patients, and erythropoietin, which
DNA, and some have important medical uses. Important can stimulate red blood cell formation in patients with
examples include analogs of granulocyte colony-stimulating anemia.

03_Mescher_ch03_p053-070.indd 59 25/04/18 6:52 pm

 60 CHAPTER 3  ■  The Nucleus

FIGURE 3–9  Chromosome territories of a human FIGURE 3–10  Human karyotype.

fibroblast nucleus.

1 2 3 4 5

6 7 8 9 10 11 12

a
13 14 15 16 17 18

19 20 21 22 XY

Karyotypes provide light microscopic information regarding

the number and morphology of chromosomes in an organism.
Such preparations are made by staining and photographing
the chromosomes of a cultured cell arrested with colchicine
during mitosis, when chromosomes are maximally condensed.
From the image individual chromosomes are typically placed
together in pairs. With certain stains each chromosome has a
particular pattern of banding that facilitates its identification
and shows the relationship of the banding pattern to genetic
anomalies. Hybridization with fluorescent probes specific for
each chromosome (FISH) followed by karyotyping yields an
image like that shown here. Note that the 22 pairs of auto-
somes, as well as the X and Y chromosomes, differ in size, mor-
phology, and location of the centromere.
b

repair of the damage before the cell enters S phase, so that the
Fluorescence in situ hybridization (FISH) can be used with a damaged DNA does not reproduce gene defects during rep-
combination of labeled probes, each specific for sequences
on different chromosomes. A nucleus of a cultured human lication. If the problem encountered at any checkpoint can-
fibroblast was processed by 24-color FISH, photographed by not be corrected fairly quickly while cycling is halted, proteins
confocal microscopy in appropriate channels, and the results encoded by tumor suppressor genes are activated and that
superimposed to form an RGB (red-green-blue) image (a) of cell’s activity is redirected toward cell suicide or apoptosis.
the 24 differently labeled chromosome types (1-22, X, and Y).

› ›› MEDICAL APPLICATION
Individual chromosome territories in the image were identified
and false-colored after classification by software developed for
such analyses (b).
Many genes coding for proteins important in the control of cell
(Used with permission from Dr Thomas Cremer, Department
of Biology II, Anthropology and Human Genetics, Ludwig Maxi- proliferation and differentiation are often called proto-
milian University, Munich, Germany.) oncogenes; changes in the structure or expression of these can
convert them to oncogenes causing uncontrolled cell growth
and a potential for cancer. Altered proto-oncogenes are associ-
Progression through the cell cycle is halted by adverse con- ated with many types of tumors and hematologic cancers. Proto-
ditions such as inadequate nutrition (nutrient stress), inappro- oncogenes can encode almost any protein involved in the control
priate cellular microenvironments, or DNA damage. Nuclear of mitotic activity, including various specific growth factors, the
DNA is monitored very closely, and damage here can arrest the receptors for growth factors, and various kinases and other pro-
cell cycle not only at the G1 restriction point but also during teins involved in intracellular signaling of growth factors.
S or at a checkpoint in G2 (Figure 3–13). G1 arrest may permit

03_Mescher_ch03_p053-070.indd 60 25/04/18 6:52 pm

 Mitosis 61

cellular changes found in the various types of cancer. In many

FIGURE 3–11  Regions within a nucleolus.
forms of human cancer, the gene for the key tumor suppres-

C H A P T E R
sor p53 is itself mutated, thus reducing the ability to eliminate
cells with damaged DNA and facilitating proliferation of cells
NE C
H with new genetic defects.

››MITOSIS

3
The period of cell division, or mitosis (Gr. mitos, a thread),

The Nucleus  ■ Mitosis

is the only cell cycle phase that can be routinely distinguished
with the light microscope. During mitosis, a parent cell
F divides and each of the two daughter cells receives a chro-
G
mosomal set identical to that of the parent cell. The chromo-
somes replicated during the preceding S phase are distributed
H FC to the daughter cells. The long period between mitoses (the
G1, S, and G2 phases) is also commonly called interphase.
The events of mitosis are subdivided into four major stages
(Figure 3–12). Important details of each mitotic phase are
E included in Figure 3–14 and summarized here. During the
relatively long prophase, several changes occur:
■■ The nucleolus disappears and the replicated chroma-
TEM reveals morphologically distinct regions within a nucleo- tin condenses into discrete threadlike chromosomes,
lus. Small, light-staining areas are fibrillar centers (FC), con- each consisting of duplicate sister chromatids joined at
taining the DNA sequences for the rRNA genes (the nucleolar the centromere.
organizers). The darker fibrillar material (F) surrounding the ■■ The two centrosomes with their now-duplicated centri-
fibrillar centers consists of accumulating rRNA transcripts.
More granular material (G) of the nucleolus contains mainly
oles separate and migrate to opposite poles of the cell
the large and small ribosomal subunits being assembled from and organize the microtubules of the mitotic spindle.
rRNA and ribosomal proteins synthesized in the cytoplasm. ■■ Late in prophase, lamins and inner nuclear membrane
Various amounts of heterochromatin (H) are also typically are phosphorylated, causing the nuclear lamina and
found near the nucleolus, scattered in the euchromatin (E), nuclear pore complexes to disassemble and disperse in
and adjacent to the nuclear envelope (NE) that separates chro-
matin from cytoplasm (C). (X35,000)
cytoplasmic membrane vesicles.
During metaphase, chromosomes condense further and
large protein complexes called kinetochores (Gr. kinetos,
moving), located at the centromere DNA region of each chro-
› ›› MEDICAL APPLICATION mosome, attach to the mitotic spindle (Figure 3–15). The cell
Retinoblastoma is a type of cancer occurring in the eyes, usu-
is now more spherical and microtubules move the chromo-
ally in young children. One form of the disease is inherited or
somes into alignment at the equatorial plate.
familial. Research on the genetic basis of this disease led to
In anaphase, sister chromatids (now called chromo-
the discovery of Rb, a gene coding for a key protein active at
somes themselves) separate and move toward opposite spindle
the G1 restriction point that blocks cell cycle progression until
poles by a combination of microtubule motor proteins and
a mitogenic stimulus arrives. A kinase activated by a growth
dynamic changes in the lengths of the microtubules as the
factor receptor phosphorylates the Rb protein, causing it to
spindle poles move farther apart.
release the E2F transcription factor. This factor then activates
At telophase, the following occur:
genes needed for DNA replication. ■■ The two sets of chromosomes are at the spindle poles and
begin reverting to their uncondensed state.
■■ Microtubules of the spindle depolymerize and the
DNA changes (mutations) resulting from damage are nuclear envelope begins to reassemble around each set of
not always detected and corrected (or eliminated). If such a daughter chromosomes.
change occurs in a gene important for cell cycle activities, such ■■ A belt-like contractile ring of actin filaments associated
as genes for certain growth factors, their receptors or signaling with myosins develops in the cortical cytoplasm at the
kinases, normal controls on the cell cycle may be affected and cell’s equator. During cytokinesis at the end of telophase,
growth may occur in a less-regulated manner usually detected constriction of this ring produces a cleavage furrow and
by the tumor suppressor proteins such as p53. Failure to detect progresses until the cytoplasm and its organelles are
unregulated cell cycling can lead to additional defects and the divided into two daughter cells, each with one nucleus.

03_Mescher_ch03_p053-070.indd 61 25/04/18 6:52 pm

 62 CHAPTER 3  ■  The Nucleus

FIGURE 3–12  The cell cycle.

Preparation for DNA DNA replication

synthesis
S

8h
Prophase
( ± 1 h)
G2
2.5-3 h +
Quiescence or G
0
differentiation Interphase Mitosis M Metaphase
(< 1 h)

Anaphase
25 h (< 1/2 h)

G1
Telophase
Growth in size (minutes)

The ability to recognize microscopically cells during both mitosis mitosis, and M includes all phases of mitosis itself. In rapidly growing
and DNA replication (by autoradiography after administering human tissues, the cell cycle varies from 24 to 36 hours. The length
radiolabeled thymidine) led to the concept of the cell “cycle” with of G1 depends on many factors and is usually the longest and most
the phases occurring as shown here. In rapidly dividing cells, G1 is variable period; the length of S is largely a function of the genome
a period in which cells accumulate the enzymes and nucleotides size. G2 and mitosis together normally last only 2-3 hours. Differen-
required for DNA replication, S is the period devoted primarily tiating cells in growing tissues may have very long G1 periods and
to DNA replication, G2 is a usually short period of preparation for such cells are often said to be in the G0 phase of the cell cycle.

FIGURE 3–13  Controls at cell cycle checkpoints.

Start (G1/S) checkpoint:

• Are cell nutrition, size,
and environment favorable? S
• Is all DNA intact?
Progress driven by shifts
Prepare for DNA replication
G1 in active kinases, each
and enter S phase
targeting different sets
of proteins

Metaphase/anaphase checkpoint: M G2
• Is all DNA intact?
• Are all chromosomes attached
to the mitotic spindle? G2/M checkpoint:
• Is DNA completely replicated?
Begin chromatid separation and
prepare for cytokinesis Enter mitosis

Each phase of the cell cycle has one or more checkpoints where ■■ The metaphase spindle checkpoint that ensures that all chro-
the quality of specific cell activities is checked. Progression to the mosomes will be segregated
next phase of the cycle does not occur until all activities of the
Overall progression in the cycle is regulated by proteins called
preceding phase are completed satisfactorily. Three important
cyclins and cyclin-dependent kinases (CDKs) that phosphory-
checkpoints are shown here, including
late/activate enzymes and other proteins needed for phase-
■■ The start or restriction checkpoint just before the start of S specific functions. Major cyclins, their CDKs, and important protein
■■ The G2/M checkpoint that ensures that DNA replication is complete targets are summarized in Table 3–1.

03_Mescher_ch03_p053-070.indd 62 25/04/18 6:52 pm

 Mitosis 63

Major cyclin and cyclin-dependent kinase complexes regulating the human cell cycle
TABLE 3–1

C H A P T E R
and important target proteins.
 

Cycle Phase or Active Cyclin–CDK

Checkpoint Complex Examples of Target Proteins

Early G1 Cyclin D–CDK4 or 6 Phosphorylates Rb protein, releasing E2F, a transcription factor that activates genes
for many G1 activities and for cyclin A

3
Late G1 /entry of S Cyclin E–CDK2 Further activation E2F-mediated gene transcription; protein p53; other kinases

The Nucleus  ■ Mitosis

Progression through S Cyclin A–CDK2 DNA polymerase and other proteins for DNA replication
G2/entry of M Cyclin A–CDK1 Specific phosphatases and cyclin B
Progression through M Cyclin B-CDK1 Nuclear lamins; histone H1; chromatin- and centrosome-associated proteins

Most tissues undergo cell turnover with slow cell divi- regeneration. The cell turnover rate is rapid in the epithe-
sion and cell death. Nerve tissue and cardiac muscle are lium lining the digestive tract and uterus or covering the
exceptions because their differentiated cells cannot undergo skin. Mitotic cells are usually difficult to identify conclu-
mitosis. As discussed in later chapters, a capacity for mitosis sively in sections of adult organs but may often be detected
within a tissue, either by differentiated cells or by a reserve in rapidly growing tissues by their condensed chromatin
of stem cells, largely determines the tissue’s potential to (Figure 3–16).

FIGURE 3–14  Phases of mitosis.

Sister chromatids
Kinetochore Chromosome (two sister chromatids being pulled apart
surrounding joined at centromeres) Equatorial plate
centromeres Reforming
of both nuclear envelope
chromatids Cleavage furrow

Kinetochore
Centrosome microtubules
(pair of Nucleolus
centrioles) Centrosome
Developing (spindle pole)
spindle moving outward

Nucleus with Chromosomes aligned

dispersed chromosomes on equatorial plate Mitotic spindle Sister chromatids being pulled apart Cytokinesis occurring

Astral microtubules Mitotic spindle Cleavage furrow

a Prophase b Metaphase c Anaphase d Telophase

The chromosomal changes that occur during mitosis are easily spindle organized by the centrosomes. The spindle consists of
and commonly studied in the large cells of very early embryos kinetochore microtubules, polar microtubules which interdigi-
of fish after sectioning, such as the mitotic cells shown here. tate near the equatorial plate, and shorter astral microtubules
(a) During the relatively long prophase, the centrosomes move anchoring the spindle to the cell membrane. (c) During anaphase,
to opposite poles, the nuclear envelope disappears by fragmenta- the kinetochores separate and the chromatids (now called chro-
tion, and the chromosomes condense and become visible. Having mosomes themselves) are pulled on their microtubules toward
undergone DNA replication, each chromosome consists of two each centrosome. (d) In telophase, the cell pinches itself in two by
chromatids joined at their centromere regions by kinetochore contraction of the F-actin bundle in the cell cortex, after which the
protein complexes. (b) At the short metaphase, the chromo- chromosomes decondense, transcription resumes, nucleoli reap-
somes have become aligned at the equatorial plate as a result pear, and the nuclear lamina and nuclear envelope reassemble.
of their attachments to the dynamic microtubules of the mitotic (All X500; H&E)

03_Mescher_ch03_p053-070.indd 63 25/04/18 6:52 pm

 64 CHAPTER 3  ■  The Nucleus

FIGURE 3–15  Mitotic spindle and metaphase chromosomes.

TEM of a sectioned metaphase cell shows several features of the

mitotic apparatus, including the very electron-dense chromo-
somes bound at kinetochores (arrows) to the microtubules of
the spindle. The microtubules converge on the centrosomes (C),
each containing a pair of centrioles. The flattened membrane
vesicles near the mitotic spindle may represent fragments of the
nuclear envelope, which begin to reassemble during late telo-
phase. (X19,000)
(Used with permission from Richard McIntosh, Department of
Molecular, Cellular and Developmental Biology, University of Colo-
rado, Boulder.)

FIGURE 3–16  Mitotic cells in adult tissues.

a b c d

Dividing cells in recognizable stages of mitosis are rarely observed region of the intestinal crypts. Condensed chromosomes of cells
in adult tissues because they are rare and the cells are small, with in late anaphase and prophase phase can be distinguished here.
variable shapes and orientations. However, mitotic figures, nuclei (b) Metaphase cell in a gland of proliferating uterine endometrium.
with clumped, darkly stained chromatin, can sometimes be identi- (c) Telophase cell in the esophagus lining. (d) Metaphase in the
fied, as shown here in various rapidly renewing tissues. basal layer of epidermis. (X400; H&E)
(a) In the lining of the small intestine, many mitotic transit
amplifying cells can be found in the area above the most basal

03_Mescher_ch03_p053-070.indd 64 25/04/18 6:52 pm

 Meiosis 65

››STEM CELLS & TISSUE RENEWAL lining the digestive tract. Most mitotic cells here are not stem
cells but the more rapidly dividing progeny of the cells com-

C H A P T E R
Throughout an individual’s lifetime, many tissues and organs mitted to differentiation (Figure 3–17). They are commonly
contain a small population of undifferentiated stem cells called progenitor cells or transit amplifying cells because
whose cycling serves to renew the differentiated cells of tis- they are in transit along the path from the stem cell niche to a
sues as needed. Many stem cells divide infrequently and the differentiated state, while still amplifying by mitosis the num-
divisions are asymmetric; that is, one daughter cell remains as ber of new cells available for the differentiated tissue. Cells
a stem cell, while the other becomes committed to a path that formed by progenitor cells may become terminally differenti-

3
leads to differentiation (Figure 3–17). Stem cells of many tis- ated, meaning that renewed cycling cannot occur and the spe-

The Nucleus  ■ Meiosis

sues are found in specific locations or niches where the micro- cialized cells exist for only a short time.
environment helps maintain their uniquely undifferentiated In tissues with stable cell populations, such as most con-
properties; they are often rare and inconspicuous by routine nective tissues, smooth muscle, and the cells lining blood ves-
histologic methods. sels, stem cells are not readily apparent and differentiated cells
Stem cells are best studied in tissues with rapidly renew- appear to undergo slow and episodic division to maintain tis-
ing cell populations, including blood cells, skin cells, and cells sue integrity.

FIGURE 3–17  Stem cells.

Stem cell ››MEIOSIS

Meiosis is a specialized process involving two unique and
closely associated cell divisions that occurs only in the cells
that will form sperm and egg cells. Differentiation of these two
“germ cells” or gametes is discussed fully in Chapters 21 and 22,
but the chromosomal aspects of meiosis are described here for
comparison with the events of mitosis (Figure 3–18).
Two key features characterize meiosis. (1) Early in the
process the homologous chromosomes of each pair (one from
the mother, one from the father) come together in an activ-
Committed ity termed synapsis. During synapsis double-stranded breaks
progenitor cell
or transit and repairs occur in the DNA, some of which result in recip-
amplifying cell rocal DNA exchanges called crossovers between the aligned
homologous chromosomes. This produces new combinations
of genes in the chromosomes so that few if any chromosomes
in the germ cells are exactly the same as those in the mother
and father. (2) The cells produced are haploid, having just
one chromosome from each pair present in the body’s somatic
cells. The union of haploid eggs and sperm at fertilization
forms a new diploid cell (the zygote) that can develop into a
new individual.
As shown in Figure 3–18, the important events of meiosis
unfold as follows:
■■ A cell approaching the first meiotic division has just
Differentiated completed a typical S phase and replicated its DNA;
cells each chromosome contains the two identical DNA
molecules called sister chromatids bound by cohesin
proteins.
In rapidly growing adult tissues and perhaps in other tissues, ■■ During a greatly elongated prophase of the first mei-
there are slowly dividing populations of stem cells. Stem cells otic division (prophase I), the partially condensed
divide asymmetrically, producing one cell that remains as a stem chromatin of homologous chromosomes begins to
cell and another that becomes committed to a differentiative
come together and physically associate along their
pathway but divides a few more times at a more rapid rate. Such
cells have been termed progenitor cells, or “transit amplifying lengths during synapsis. Because each of the paired
cells,” each of which eventually stops dividing and becomes fully chromosomes has two chromatids, geneticists refer
differentiated. to synaptic chromosomes as tetrads to emphasize

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 66 CHAPTER 3  ■  The Nucleus

FIGURE 3–18  Mitosis and meiosis.

MITOSIS

Mitosis adds
and replaces
identical cells.

Interphase Prophase Metaphase Anaphase/Telophase

Chromosomes Chromosomes Genetically identical
condense. line up single file. daughter cells produced.

MEIOSIS

Meiosis II

Meiosis I

Meiosis produces
haploid cells with
new genetic
combinations.

Late Interphase Prophase I Metaphase I Anaphase I/ Metaphase II Anaphase II/

Synapsis and Crossing over Homologous Telophase I Chromosomes Telophase II
crossing-over continues. Paired chromosomes Homologs line up single file Sister chromatids
begin. chromosomes line up double separate into in haploid cells. separate into
condense. file. haploid daughter nonidentical
cells; sister haploid cells.
chromatids
remain joined.

Mitosis and meiosis share many aspects of chromatin condensa- align in synapsis and regions are exchanged during crossing over
tion and separation but differ in key ways. Mitosis produces two or genetic recombination. This is followed by two meiotic divi-
diploid cells that are the same genetically. In meiosis, the two sions with no intervening S phase, producing four haploid cells.
homologous maternal and paternal chromosomes physically

that four copies of each genetic sequence are pres- generation. In human, spermatogenesis prophase I
ent. During synapsis recombination or crossing over normally lasts for 3 weeks; oocytes arrest in this mei-
occurs among the four chromatids, which mixes up otic phase from the time of their formation in the fetal
the genes inherited from each parent and yields a new ovary through the woman’s reproductive maturity, that
and different set of genes to be passed on to the next is, for about 12 years to nearly five decades!

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 Apoptosis 67

■■ When synapsis and crossing over are completed, the FIGURE 3–19  Apoptotic cells.
chromosomes become fully condensed and undergo

C H A P T E R
metaphase, anaphase, and telophase as the cell divides.
This first meiotic division separates the homologous
A
chromosomes that paired during synapsis; each of the
separated chromosomes still contains two chromatids
held together by cohesins at the centromeric DNA.
■■ Each of the two new cells now divides again, much more

3
rapidly and without an intervening S phase. In the sec- A A

The Nucleus  ■ Apoptosis

ond meiotic division, cohesin proteins are lost and the A
chromatids separate to opposite poles as individual chro-
mosomes. In each new cell, a nuclear envelope forms A
A A
around this new haploid set of chromosomes. A

In summary, meiosis and mitosis share many aspects of

chromatin condensation and separation (see Figure 3–18), but a b
differ in key ways:
■■ Mitosis is a cell division that produces two diploid cells.
Meiosis involves two cell divisions and produces four
haploid cells.
■■ During meiotic crossing over, new combinations of
genes are produced and every haploid cell is genetically
unique. Lacking synapsis and the opportunity for DNA
recombination, mitosis yields two cells that are the same
genetically. A
A

A
› ›› MEDICAL APPLICATION
In humans chromosome 21 is a very small chromosome and A
c d
the one most likely to be “overlooked” at the metaphase/
anaphase checkpoint. Failure of these homologous chro-
mosomes to separate (nondisjunction) in the first meiotic
division also occurs with greater frequency in older oocytes Apoptotic cells in adult tissues are rare because the process is
completed very rapidly. Moreover, with their highly condensed
(or sperm progenitor cells). A gamete retaining this chromo-
chromatin in pyknotic nuclei and rounded shape, cells early in
some pair forms a viable zygote after fertilization, but the apoptosis may superficially resemble some mitotic cells. Shown
developing trisomy 21 individual has morphologic and cog- here are apoptotic cells (A) in the epithelium covering an intes-
nitive impairments associated with Down syndrome. tinal villus (a), in a corpus luteum beginning to undergo involu-
tion (b), in the epithelium of a uterine endometrial gland at the
onset of menstruation (c), and in the liver (d). (X400; H&E)

››APOPTOSIS avoided when cells are rapidly eliminated by apoptosis follow-

Less evident, but no less important than cell proliferation for ing cell cycle arrest or as part of normal organ development.
body functions, is the process of cell suicide called apoptosis
(Gr. apo, off + ptosis, a falling). Apoptosis is a rapid, highly
regulated cellular activity that shrinks and eliminates defec- › ›› MEDICAL APPLICATION
tive and unneeded cells (Figure 3–19). It results in small
Cancer cells often deactivate the genes that control the
membrane-enclosed apoptotic bodies, which quickly undergo
apoptotic process, thus preventing their elimination in this
phagocytosis by neighboring cells or cells specialized for debris
type of cell death and allowing progression toward a more
removal. Apoptotic cells do not rupture and release none of
malignant state. The Bcl-2 family of proteins that controls the
their contents, unlike cells that die as a result of injury and
onset of apoptosis was first identified by a genetic mutation
undergo necrosis. This difference is highly significant because
in a specific B-cell lymphoma, which provided the name for
release of cellular components triggers a local inflammatory
the original protein.
reaction and immigration of leukocytes. Such a response is

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 68 CHAPTER 3  ■  The Nucleus

Apoptosis is controlled by cytoplasmic proteins in the

Bcl-2 family, which regulate the release of death-promoting
FIGURE 3–20  Late apoptosis—apoptotic bodies.
factors from mitochondria. Activated by either external sig-
nals or irreversible internal damage, specific Bcl-2 proteins
induce a process with the following features:
■■ Loss of mitochondrial function and caspase activation:
Bcl-2 proteins associated with the outer mitochondrial
membrane compromise membrane integrity, stopping
normal activity and releasing cytochrome c into the
cytoplasm where it activates proteolytic enzymes called
caspases. The initial caspases activate a cascade of other
caspases, resulting in protein degradation throughout
the cell.
■■ Fragmentation of DNA: Endonucleases are activated,
which cleave DNA between nucleosomes into small frag-
ments. (The new ends produced in the fragmented DNA
allow apoptotic cells to be stained histochemically using
an appropriate enzyme that adds labeled nucleotides at
these sites.)
■■ Shrinkage of nuclear and cell volumes: Destruction of
the cytoskeleton and chromatin causes the cell to shrink
quickly, producing small structures with dense, darkly
stained pyknotic nuclei that may be identifiable with
the light microscope (see Figure 3–19).
■■ Cell membrane changes: The plasma membrane of the
shrinking cell undergoes dramatic shape changes, such
as “blebbing,” as membrane proteins are degraded and
lipid mobility increases.
■■ Formation and phagocytic removal: Membrane-bound
TEM of a cell in late apoptosis shows radical changes in cell
remnants of cytoplasm and nucleus separate as very shape, with membrane blebbing and the formation of many
small apoptotic bodies (Figure 3–20). Newly exposed membrane-bound cytoplasmic regions. These apoptotic bodies
phospholipids on these bodies induce their phagocytosis may separate from one another but remain enclosed by plasma
by neighboring cells or white blood cells. membrane so that no contents are released into the extracel-
lular space. The membrane changes are recognized by neigh-
boring cells, and macrophages and apoptotic bodies are very
› ›› MEDICAL APPLICATION rapidly phagocytosed. (X10,000)

Nuclei of cells in malignant tumors are often enlarged, abnor-

mally shaped, and extremely dark staining, with abnormal
organs or body regions, such as the free spaces between embry-
nucleoli, in comparison with nuclei of normal cells. Such
onic fingers and toes. Apoptosis of excess nerve cells plays an
changes are useful to pathologists looking for evidence of
important role in the final development of the central nervous
cancer during microscopic examinations of biopsies.
system.
Triggered by p53 and other tumor suppressor proteins,
A few examples of apoptosis emphasize its significance. apoptosis is the method for eliminating cells whose survival is
In the ovary, apoptosis is the mechanism for both the monthly blocked by lack of nutrients or by damage caused by free radi-
loss of luteal cells and the removal of excess oocytes and their cals or radiation. In all these examples, apoptosis occurs very
follicles. Apoptosis was first discovered as programmed cell rapidly, in less time than required for mitosis, and the affected
death in embryos, where it is important in shaping developing cells are removed without a trace.

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 Apoptosis 69

The Nucleus  SUMMARY OF KEY POINTS

C H A P T E R
Nuclear Envelope Mitosis
■■ Cytoplasm is separated from nucleoplasm by the nuclear envelope, ■■ Stages of mitotic cell divisions include prophase, when chromo-
a double set of membranes with a narrow perinuclear space; the somes condense, the nuclear envelope disassembles, and the micro-
outer membrane binds ribosomes and is continuous with the RER. tubular spindle forms; metaphase, when chromosomes are aligned;
■■ The nuclear envelope is penetrated by nuclear pore complexes, large anaphase, when they begin to separate toward the two centrosomes;
assemblies of nucleoporins with eightfold symmetry through which and telophase, when nuclear envelope re-forms around the sepa-

3
proteins and protein–RNA complexes move in both directions. rated chromosomes.
■■ The nuclear envelope is supported internally by a meshwork, the ■■ Telophase ends with cytokinesis or cell cleavage into two daughter

The Nucleus  ■ Apoptosis

nuclear lamina, composed of intermediate filament subunits called cells by a contractile ring of actin filaments and myosin.
lamins.
Stem Cells & Tissue Renewal
Chromatin ■■ Stem cells occur in all tissues with rapid cell turnover; they divide
■■ Chromatin is the combination of DNA and its associated proteins. slowly in an asymmetric manner, with one daughter cell remaining
■■ Chromatin with DNA that is active in transcription stains lightly a stem cell and one becoming committed toward differentiation.
and is called euchromatin; inactive chromatin stains more darkly ■■ Cells committed to differentiate (transit amplifying or progenitor
and is called heterochromatin. cells) typically divide more rapidly than stem cells before slowing or
■■ The DNA molecule initially wraps around complexes of basic pro- stopping division to differentiate.
teins called histones to form nucleosomes, producing a structure
resembling beads on a string. Meiosis
■■ Additional levels of chromatin fiber condensation are less well ■■ Meiosis is the process by which two successive cell divisions produce
understood and involve nonhistone proteins, including complexes cells called gametes containing half the number of chromosomes
of condensins. found in somatic cells.
■■ The extra X chromosome in cells of female mammals forms facul- ■■ Prophase of the first meiotic division is a unique, extended period in
tative heterochromatin and can be seen as the Barr body. which homologous chromosomes pair and undergo genetic recom-
bination during the process called synapsis.
Nucleolus ■■ Synaptic pairs separate toward two daughter cells at the first meiotic
■■ The nucleolus is a very basophilic or electron-dense area of chro- division.
matin localized where rRNA transcription and ribosomal sub- ■■ The second meiotic division occurs with no intervening S phase and
units assembly occur. separates the sister chromatids into two final cells that are haploid.
■■ By TEM, an active nucleolus is seen to have fibrous and granular
parts where rRNA forms and ribosomal subunits are assembled, Apoptosis
respectively. ■■ Apoptosis is the process by which redundant or defective cells are
rapidly eliminated in a manner that does not provoke a local inflam-
The Cell Cycle matory reaction in the tissue.
■■ The cell cycle is the sequence of events that controls cell growth ■■ Apoptosis involves a cascade of events controlled by the Bcl-2 family
and division. of proteins regulating the release of death-promoting factors from
■■ The G1 phase, the longest part of the cycle, begins immediately mitochondria.
after mitosis and includes all preparations for DNA replication. ■■ Cytochrome c from mitochondria activates cytoplasmic proteases
■■ The period of DNA (and histone) synthesis is the S phase. called caspases, which degrade proteins of the cytosol, cytoskeleton,
■■ In a short G2 phase the cell prepares for division during mitosis (M). and cell membrane.
■■ Cell cycling is controlled by the sequential appearance of key cyto- ■■ Endonucleases are activated, which degrade all nuclear DNA.
plasmic proteins, the cyclins, which bind CDKs. ■■ Cell and nuclear volumes shrink rapidly, and the cell membrane
■■ CDKs phosphorylate and activate the enzymes and transcription changes produce extensive blebbing of the cell surface.
factors whose functions characterize each phase of the cell cycle. ■■ Late in apoptosis, the cell breaks into many small apoptotic bodies
■■ Progress through the cell cycle stages is monitored at checkpoints, that undergo phagocytosis by neighboring cells.
including the G1 restriction point; only when each phase’s activities ■■ Apoptosis occurs rapidly, with little or no release of proteins that
are completed are the cyclins changed to trigger those of the next would trigger inflammation, unlike the death of injured cells by
phase. necrosis that typically induces local inflammation.

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