Cellular Membranes, Dynamics of

Anne K. Kenworthy,
Tennessee
doi: 10.1002/9780470048672.wecb064

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Types of Cell Membrane Dynamics Chemical Tools and Techniques to Study Cell Membrane Dynamics

Vanderbilt University School of Medicine, Nashville,

Cell membranes are two-dimensional uids that exhibit a wide range of dynamic behaviors. Recent technical advances have enabled unprecedented views of membrane dynamics in living cells. In this technical review, we provide a brief overview of three well-studied examples of membrane dynamics: lateral diffusion of proteins and lipids in the plane of the membrane, vesicular trafcking between intracellular compartments, and exchange of proteins on and off membranes. We then discuss experimental approaches to monitor membrane protein and lipid dynamics, and we place a special emphasis on the use of genetically encoded uorescent probes and live cell-imaging techniques.

The concept that membranes are uid, dynamic structures is now over 35 years old (1, 2). In this review, we describe three of the best-studied examples of cell membrane dynamicslateral diffusion of proteins and lipids within the plane of the bilayer, membrane trafcking between intracellular compartments, and cycling of proteins on and off membranesalong with the recent technical advances that have enabled researchers to visualize these motions directly within living cells. In particular, we describe how the use of green uorescent protein (GFP) from Aequorea Victoria and other, more recently developed labeling technologies can be used to mark molecules to study inside cells. We also summarize common uorescence microscopy techniques for live cell imaging, including conventional techniques such as wide-eld and confocal microscopy, and more specialized techniques such as total internal reection uorescence (TIRF) microscopy. Finally, we cover advanced methods used to study cell membrane dynamics, including single particle tracking (SPT), uorescence recovery after photobleaching (FRAP), photoactivation, uorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS).

Types of Cell Membrane Dynamics

Lateral diffusion
Membranes are two-dimensional uids whose protein and lipid components continuously exchange positions because of Brownian motion, a process commonly referred to as lateral diffusion. Lateral diffusion enables proteins and lipids to explore their environment, which encourages interactions between molecules. Thus, the speed of lateral diffusion is one of the limiting factors

that regulate intermolecular interactions, and, consequently, cellular function. Diffusional mobility of molecules can be estimated based on their size and the viscosity of the lipid bilayer and surrounding aqueous phase. As a result, deviations from this behavior can provide important insights into the environment that proteins and lipid encounter in biologic membranes. How rapidly diffusion occurs is characterized by the diffusion coefcient D , a parameter that provides a measure of the mean of the squared displacement x of a molecule per unit time t . For diffusion in two dimensions such as a membrane, uck model of this is given by <x2 > = 4D t. The Saffman-Delbr Brownian motion in biologic membranes describes the relationship between membrane viscosity, solvent viscosity, the radius R and height of the diffusing species, and D for both lateral and rotational diffusion of proteins in membranes (3, 4). This model predicts for example that for lateral diffusion, D should be relatively insensitive to the radius of the diffusing species, scaling with log (1/R ). Interestingly, the diffusional behavior of membrane proteins measured experimentally by FRAP, FCS, or single particle tracking in cells is more complex than predicted by this model. This technique is described best for the case of cell surface proteins, as assessed by FRAP. Such measurements indicate that diffusion is typically much slower than one would expect based on membrane viscosity. In cell membranes, typical values of D for transmembrane proteins are approximately 0.05 m2 /s or less, which is much slower than observed in articial membranes composed of puried lipids. In addition, a signicant fraction of proteins is often immobile over the timescale of diffusion experiments (4, 5). Furthermore, diffusional mobilities vary among proteins, and sometimes they differ for the same protein expressed in different cell lines (4, 5). Deviations from pure diffusion are more readily apparent when the trajectories 1

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2 1

Figure 1 Modes of diffusion of individual membrane proteins as revealed by single-molecule tracking techniques. The hypothetical trajectory of an individual plasma membrane protein as traced by single-particle tracking techniques is shown. An individual protein can switch between several different modes of over time, which include conned diffusion (region 1), free diffusion (region 2), and immobilization (region 3).

of single molecules are analyzed (Fig. 1). Individual molecules exhibit a range of diffusive behaviors, characterized as immobilization, transient connement, free diffusion, and in some cases directed motion (6). Features of cell membranes that contribute to this complex diffusional behavior include the presence of membrane domains, interactions of proteins with the cytoskeleton and extracellular matrix, and molecular crowding because of the high protein concentrations found in cellular membranes (4, 5). The diffusional mobility of proteins and lipids can also be modulated actively. For example, crosslinking of certain cell surface molecules causes dramatic changes in their mobility, which reects interactions with other cellular components. A well-studied example of such a protein is FcR1, the high afnity IgE receptor, which undergoes a substantial loss of mobility on the formation of small aggregates of IgE-FcR1 (7). Using an advanced technique known as FCS cross correlation analysis, it is now possible to detect transient interactions of membrane proteins that occur during cell signaling by virtue of their codiffusion (8). Until recently, experiments that probe protein diffusion relied on uorescent antibody-based probes, and thus were limited to plasma membrane proteins with extracellular epitopes. In contrast, the mechanisms that regulate diffusion of intracellular membrane proteins remained unexplored because of their inaccessibility to labeling. With the development of genetically encoded uorescent probes, such studies have become tractable because proteins targeted to a particular organelle can be uorescently labeled directly with GFP. In addition, improved technology has now made it possible to monitor the movement of multiple types of proteins or lipids, tagged with different markers simultaneously. Some examples of the types of questions it is now possible to address include:

Is the mobility of proteins inuenced by how they are attached to membranes?

Lateral diffusion is predicted to be relatively insensitive to the size of the transmembrane domain of the diffusing species. This prediction suggests that membrane anchorage should not strongly inuence diffusion. However, membrane anchorage would be expected to inuence the types of interactions that occur between a given protein and the complex environment of the cell. For example, the diffusion of peripheral membrane proteins localized to the inner leaet of the plasma membrane by lipid anchors has been shown to be much faster than transmembrane proteins and nearly as fast as that of lipid probes in some instances (11). This nding suggests that because of their lipid anchors, these molecules do not experience the same barriers to diffusion as transmembrane proteins. It also likely reects the ability of some proteins to undergo rapidly reversible binding to membranes (12, 13).

Do cholesterol-enriched lipid domains organize proteins into functional complexes?

Interest in the role of membrane domains in regulating protein and lipid diffusion has recently been revitalized by the lipid raft model, which proposes that cell membranes are divided into cholesterol and sphingolipid-enriched and -depleted microdomains. As a result, much effort has been made recently to relate lipid and protein diffusion in cells and model membranes (14) and to determine the effect of cholesterol levels on protein and lipid diffusion by both FRAP and FCS (11, 15). The role of cholesterol-dependent domains in regulating events such as T-cell signaling has also been investigated by examining closely the mechanisms of diffusional trapping of proteins at the single molecule level by TIRF microscopy; unexpectedly, proteinprotein interactions seem to play a more important role than cholesterol-dependent domains in this process (16).

How is protein diffusional mobility regulated in intracellular compartments?

Some of the rst investigations of diffusion in organelle membranes such as the Golgi complex and endoplasmic reticulum suggest that unlike the plasma membrane, protein diffusion in intracellular membranes approaches theoretic limits (9). These ndings suggest that intracellular membranes contain considerably fewer barriers to free diffusion than the cell surface. Diffusion studies can also be used to test whether protein immobilization is responsible for retaining proteins within a specic organelle, the effect of unfolding on protein mobility, and how various perturbations inuence both the luminal and membrane environment experienced by proteins (10). 2

Membrane trafcking
Although integral membrane proteins and lipids are laterally mobile within the plane of a given cell membrane, they cannot exchange between different membrane compartments unaided because of the high energetic barriers to exposing their hydrophobic portions to water. The movement of transmembrane proteins between cellular compartments is accomplished by the formation of closed bilayer structures known as transport vesicles that bud off from one compartment and fuse with another. The term membrane trafcking refers to the movement of

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these vesicular structures between organelles and/or the plasma membrane, and includes the processes of endocytosis and exocytosis. Membrane trafcking involves many highly regulated events, which include cargo selection, recruitment of coat and accessory proteins, vesicle budding, intra-organelle transport, targeting to and fusion with the target membrane, and recycling of cellular machinery for another cycle of transport (17). Recent work has now begun to focus on the temporal and spatial regulation of these events in living cells (1820). Such approaches are especially powerful when coupled with cell biologic manipulations such as drug treatments, the introduction of mutant proteins into cells, or knockdown of protein expression to probe the cellular mechanisms that underlie membrane trafcking (20). Examples of the types of questions being addressed are listed in the following sections.

are sorted to various intracellular destinations. Sorting requires the pH-dependent release of cholera toxin from caveolar domains in endosomes; SV40 remains immobilized under these conditions (24).

Cycling of proteins on and off membranes

Although integral membrane proteins must rely on membrane trafcking as their sole mechanism for traversing the cell, many peripheral membrane proteins can move on and off membranes in a reversible manner by shifting between a membrane-bound and a soluble state. This reversible binding is critical for the function of coat proteins involved in the formation of transport vesicles (clathrin, COPI, and COPII), proteins that play roles in organelle identity and membrane trafcking (Rabs and Arf proteins), and a wide variety of signaling proteins (Ras, Raf, and protein kinase C) (2628). Biochemically, such exchange can be detected by fractionating cells into membrane and soluble fractions, and quantifying the amount of protein found in each. Using live cell imaging approaches, it is possible to begin to monitor these events in real time to address questions such as the following.

What are the kinetics of intracellular transport through the secretory pathway?
The transmembrane protein VSVG is a classic tool used to study protein transport through the secretory pathway. Some early studies of vesicular transport in living cells visualized the movement of a GFP-tagged version of VSVG by time-lapse confocal microscopy (21, 22). From these data, it was possible to derive a kinetic model of the movement of this well-studied protein between intracellular compartments, while at the same time providing information about the nature of the vesicular structures involved in this process (21, 22).

On what cellular membranes does exchange occur?

One major advantage to studying membrane/cytosol exchange of proteins in living cells is that it is possible to compare the recruitment of proteins with different organelles simultaneously. For example, such studies have been instrumental in showing that Ras activation, as reported by the membrane recruitment of the FP-tagged Ras binding domain of Raf from the cytosol, occurs on Golgi membranes as well as the cell surface, and that the kinetics of recruitment are different for the two compartments (29).

What pathways do pathogens use to enter cells?

Certain viruses and bacterial toxins are endocytosed by a specialized pathway that involves invaginations of the plasma membrane known as caveolae. Recent studies have traced out this caveolar endocytic pathway by jointly tagging the cellular machinery with GFP and labeling viruses or bacterial toxins with uorescent dyes, which enables their dual visualization in real time (23, 24). Such work has revealed several unanticipated properties of this pathway, such as the ability of viruses to induce formation of actin comets that propel virus-containing vesicles (23) and the stable, immobile nature of the caveolar coat that encases these vesicles (24).

How rapidly do proteins cycle on and off membranes?

Compared with membrane trafcking, which occurs with characteristic rates of 3% per minute for the case of VSVG movement from the endoplasmic reticulum to the Golgi complex (22), the cycling of proteins on and off membranes can occur over very rapidly. In our own work (30), we have observed in photobleaching studies of GFP fusion proteins that a mutant form of HRas that lacks both palmitoylation sites is able to constitutively cycle on and off membranes of the Golgi complex with halftimes of 5 s (Fig. 2).

How do cargo molecules and trafcking machinery progress through the endocytic pathway?
Movement of cargo through the endocytic pathway could occur by vesicular transport between stable compartments. Alternatively, it could involve progressive maturation of endosomal membrane. Recent work has tested these models by using fast, live cell imaging in combination with tools to quantify dynamic changes in the levels of the small GTPases Rab5 and Rab7 on endosomal structures during transport of uorescently labeled cargo (25). These data suggest a model in which conversion in Rab proteins is a mechanism by which cargo progresses between early and late endosomes. Other studies have focused on how specic cargo molecules that enter the cell via identical pathways, such as cholera toxin and SV40, ultimately

Is exchange constitutive or regulated?

Membrane/cytosol exchange is sometimes constitutive, but more often it occurs in a tightly regulated manner. Many proteins are recruited to membranes in response to the transient generation of protein or lipid binding sites on the membrane, which are recognized by modular protein interacting or lipid interacting domains contained within the recruited protein. Alternatively, membrane binding can be regulated by loss of membrane binding sites or perturbation of binding motifs within 3

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IgG
~7 0

GFP 30

Prebleach

t=0 (a)

50 s

~3

50 (b) 140 Biarsenicaltetracysteine 6 amino acids ~2030 (d)

Fluorescence intensity (% initial)

100

0
80 60 40 0 20 40 60 Time (s) (b)
Figure 2 Visualization of reversible protein binding to the Golgi complex. (a) COS-7 cells that express a GFP-tagged HRas palmitoylation mutant were imaged over time before and after photobleaching uorescent molecules localized to the Golgi complex (circle). Fluorescence recovers rapidly and completely within 1 min. Bar, 10 m. (b) Kinetics of recovery for the GFP-HRas palmitolyation mutant after photobleaching the entire Golgi complex. Data are from a representative experiment similar to that shown in panel A. The rapid recovery kinetics are highly suggestive of reversible membrane binding. (Reproduced from Reference 30. Copyright 2005 Rockefeller University Press.)

140

80
80 100

(a) DsRed Tetramer ~60 ~80 (c)

proteins. For example, recent evidence suggests that KRas, which contains a cluster of basic amino acids that facilitates its binding to the plasma membrane, can be released via phosphorylation of residues in the polybasic domain. This release in turn allows for the regulated redistribution of KRas to mitochondria, where it promotes apoptosis (31).

Figure 3 Size of typical probes used in studies of membrane dynamics. Relative sizes of (a) Immunoglobulin G, (b) green uorescent protein, (c) the Discoma red uorescent protein (DsRed) tetramer, and (d) biarsenical tetracysteine. (Reprinted with permission from Reference 61.)

Small organic dyes

Small organic dyes are available with a wide range of spectral properties encompassing the entire visible range, including the near UV and far red (32). Prior to the advent of FPs, small organic dyes were the workhorses for uorescent labeling of plasma membrane proteins, either by using dye-labeled antibodies (Fig. 3a) or by labeling proteins directly that bind to plasma membrane proteins or lipids such as bacterial toxins or growth factors. Indeed, most early work that denes the diffusional mobility of proteins on the plasma membrane relied on immunodetection of cell surface proteins with antibody fragments labeled covalently with uorescent dyes (5). The use of small organic dyes to label proteins uorescently remains a valuable tool to study the endocytic itineraries of ligands, viruses, and bacterial toxins (24). However, dye-labeled proteins have historically been much less useful in studies of intracellular proteins in living cells, because this task requires their introduction by either microinjection or permeabilization of the plasma membrane. The susceptibility of many uorescent dyes to photobleaching also limits their use in live cell imaging applications.

Chemical Tools and Techniques to Study Cell Membrane Dynamics

Probes
Fluorescent probes of membrane dynamics fall into two general classes: exogenous and genetically encoded. Exogenous probes, which include small organic dyes and quantum dots, usually are targeted to the protein of interest via immunolabeling approaches. They are used commonly to label cell surface proteins, although they can be introduced into cells by microinjection or by permeabilizing cells. Genetic approaches make use of intrinsically uorescent proteins (FP) or genetic tags that generate binding sites for small dyes. Here, molecular biology tools are used to engineer DNA constructs that contain fusions of the protein of interest and the genetic tag. The DNA is then introduced into cells by transfection or transduction (Fig. 3). 4

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Probes of lipid dynamics

Synthetic lipid analogs such as DiI and BODIPY-labeled lipids are classic reporters of lipid dynamics (32, 33). Fluorescent glycolipid analogs have also been used for many years, especially in studies of endocytosis and lipid trafcking (34). However, it is important to note the limitations of these probes. Many make use of short-chain analogs to enable their delivery into cells. In addition, some uorophores used to label these lipids are used to replace a fatty acid chain. As a result, these lipids may not exhibit behavior similar to that of their native counterparts. To address these limitations, polyene-lipids, which have a structure similar to that of natural lipids, have been developed recently as an alternative to classic uorescent lipid analogs (35). Fluorescent cholesterol analogs have joined the growing list of uorescent lipid probes (36). Of these, dihydroergosterol is most likely to be a useful structural analog of cholesterol. An alternative approach to the use of uorescent lipid analogs to study lipid dynamics is the use of uorescent reporter proteins that bind specic lipids. Such experiments are also of interest from the biologic standpoint of protein function. For example, plekstrin homolog (PH)-domain-containing proteins bind phosphoinositide lipids (26, 27), whereas cholera toxin binds ganglioside GM1 (20). It should be noted however that protein reporters that interact with a specic lipid might not be neutral from a biologic point of view, as they may compete for binding with endogenous molecules.

it is linked (Fig. 3b). Second, the expression of fusion proteins typically relies on transfection, which can be problematic in some primary cell lines and can lead to overexpression artifacts. Third, certain uorescent proteins form obligate oligomers (Fig. 3c). Finally, spectral overlap limits the number of different uorescent proteins that can be used in the same experiment, although this problem is diminishing as new variants are developed.

Chemical labeling of fusion proteins

Recently, methods have been developed to label proteins site-specically with small molecules for live cell imaging studies as an alternative to the use of FP fusion proteins (4244). The strategy of these approaches relies on genetically incorporating a receptor domain that can serve as a specic binding site for a small molecule to the protein of interest. After expressing the fusion protein in cells, it can then be labeled using cell-permeant small molecule probes. This general scheme offers several major advantages over FP fusion proteins, which include 1) the potential to label proteins with relatively small, and thus in principle, minimally perturbing tags; 2) the possibility of taking advantage of small molecule probes with a wide range of chemical properties; and 3) the ability to control the time at which the proteins of interest are tagged, which allows for temporal regulation of labeling. The tetracysteine-biarsenical system is one of the rst examples of such technology (45). Here, a 12-residue sequence that includes four cysteines is incorporated into the protein of interest to enable binding of membrane permeant biarsenic dyes (FlAsH and ReAsH) (42). In addition to the small size of the tetracysteine motif (Fig. 3d), advantages of this approach include the possibility to perform correlative electron microscopy analysis and pulse-chase labeling (42). Furthermore, the uorescence intensity of the bisarsenic dyes increases substantially on binding, which decreases background uorescence. However, the biarsenical dyes exhibit nonspecic binding to cysteine-rich proteins and require a reducing environment for labeling. Compared with the FP, this and other methods that combine genetic tags with small molecules are still in their infancy, but with additional iterations of renement, they are likely to become useful in the future.

GFP and its derivatives

Many recent advances in the study of cell membrane dynamics (and indeed in much of cell biology) have been driven by the discovery of green uorescent protein from Aequorea victoria (37). Importantly, expression of GFP as part of a fusion protein results in visible green uorescence, and despite its relatively large size (27 kDa), GFP tagging often does not interfere with normal protein targeting or function. Since the initial discovery and cloning of GFP, mutational analysis has been performed to increase its brightness, photostability, and speed of folding as well as to generate a range of spectral variants. Of particular importance to the study of membrane proteins was the discovery of a point mutation that prevents the weak tendency of GFP to dimerize (38). Similar proteins were isolated from reef corals and sea anemones, which expanded the spectrum of available colors. Photoactivatable uorescent proteins undergo a substantial change in their spectral properties (switching either to a dark state or to another color) in response to irradiation with light of a particular wavelength, intensity, and duration. This effect can be either reversible or irreversible depending on the particular protein (39). The recently discovered KEIMA protein (40) is a very promising uorescent protein that provides an easy way to analyze dynamics of cellular membranes by simultaneously following two uorescent-tagged subsets of proteins. Additional properties of the uorescent proteins are described in Reference 41. Despite the many attractive features of uorescent fusion proteins, they are not without their limitations. First, GFP is relatively large (27 kDa) and thus has the potential to disrupt the structure, function, and/or localization of the protein to which

Quantum dots
Quantum dots are uorescent semiconductor nanocrystals that have been incorporated recently into the toolbox of uorescent labeling techniques (42). When coated appropriately, quantum dots can be conjugated with streptavidin or antibodies for protein labeling applications in cells (46). Quantum dots are exceptionally bright and photostable, -have a broad absorbance spectrum, and can be tuned to emit at specic wavelengths depending on their size. Because of their brightness and photostability, they can be very useful for detecting low abundance proteins and are attractive probes for long-term single particle tracking studies. However, their applications in live cells are hampered somewhat by their large size (10 to 30 nm) and blinking behavior. For example, the size of quantum dots has limited their use primarily to studies of extracellularly localized plasma membrane proteins because their introduction into cells 5

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requires membrane permeabilization. The potential for multivalent binding of quantum dots is also a potential concern in single-molecule tracking studies.

Fluorescence microscopy techniques for live cell imaging

Many dynamic processes, such as membrane trafcking and regulated cycling of proteins on and off membranes, can be visualized by capturing sequential images of living cells by uorescence microscopy. The forms of uorescence microscopy used most commonly for live cell imaging are wide eld and confocal microscopy. In wide eld microscopy, uorescence is typically excited with an arc lamp and emission is collected using a CCD camera. Such systems can be congured readily for imaging live cells over time. An advantage of this approach is its relative simplicity compared with other imaging modalities. However, it collects light emitted from the entire depth of the specimen including out-of-plane uorescence. In confocal microscopy, out of plane uorescence is eliminated by the incorporation of a pinhole in the light path, which enables the collection of three-dimensionally resolved images. This task is accomplished by using a single pinhole for the case of laser scanning confocal microscopy or with a series of rotating pinholes in spinning disk confocal microscopy. Because the intensity and position of the laser used to excite samples can be modulated rapidly in many laser scanning confocal microscopes, it is possible to use them for specialized applications such as photobleaching and photoactivation (see below). Spinning disk confocal microscopes are not well suited for photobleaching or photoactivation applications, but they have a greater image acquisition rate than laser scanning confocals. In addition to confocal microscopy, a technique known as multiphoton microscopy can also be used to generate three-dimensionally resolved uorescence images. In this case, excitation is limited to those uorophores that are present within the small region where the laser is focused and thus are of sufciently high power to enable a single uorophore to absorb two or more photons simultaneously (47). Other advantages of multiphoton microscopy include reduced photobleaching outside of the focal plane as well as increased penetration into samples because of the use of longer wavelength excitation, which makes this a technique of choice for tissue imaging in vivo . In some instances, it is of interest to focus exclusively on events that occur at the plasma membrane. Here, a technique known as TIRF microscopy is particularly valuable (48). TIRF uses an evanescent wave generated by a process referred to as total internal reection to excite the sample. Total internal reection occurs when light traveling from a medium of high refractive index arrives at an interface with a medium of lower refractive index above a so-called critical angle. Under these conditions, the light is reected back into the high refractive index material, and an evanescent eld is generated in the lower refractive index medium. The eld decays exponentially away from the interface, with a typical depth between 50 to 150 nm. Because experimentally, this interface represents the surface at which cells attach to a coverslip, it is possible to selectively excite and visualize only those uorescent molecules found at this 6

surface. TIRF can also be combined with the advanced methods described in more detail below, which include single molecule imaging (16), FCS (49), and FRAP (50). Thus, TIRF offers an attractive imaging modality for probing plasma membrane dynamics using a variety of techniques. For a more in depth discussion of the pros and cons of each of these approaches in live cell imaging studies, we refer the reader to several recent reviews (51, 52).

Advanced techniques used to study membrane dynamics

FRAP
FRAP (also known as uorescence photobleaching recovery or FPR) has been used for many years to study lateral diffusion of plasma membrane proteins as characterized by their diffusion coefcient and mobile fraction (5, 53). In these experiments, a population of uorescence molecules is bleached irreversibly by exciting with an intense, focused laser spot. Recovery of uorescence in the bleached region is then monitored over time to determine to what extent and how rapidly the bleached molecules are replaced by unbleached molecules from other regions within the cell (Fig. 4a). Two fundamental assumptions that underlie these experiments are that the bleaching event does not damage the labeled protein or surrounding region of the cell and that the bleaching is irreversible, in other words, that recovery occurs because of diffusional exchange and not by recovery of uorescence of an individual uorophore. FRAP curves are typically t by equations for free diffusion plus an immobile fraction (53). A variation on this approach is to measure FRAP as a function of spot size, which is a technique that is sensitive to the cycling of proteins on and off membranes (13). Until the development of the uorescent proteins, FRAP measurements were conned to measurements of protein or lipid diffusion in the plasma membrane using uorescently labeled Fabs or uorescent lipid analogs that could be added to cells exogenously. For such experiments, little need existed for spatially resolved measurements because the uorescence signal was localized to the plasma membrane. Thus, most classic FRAP studies made use of a spot photobleaching apparatus that consisted of an epiuorescence microscope, computer-controlled shutter, laser used both for bleaching and for low-level excitation of the sample, and detector to collect uorescence emission coupled with electronics to record the measured uorescence intensity (54). In recent years, commercially available confocal microscopes have incorporated FRAP protocols, which allows for imaging-based FRAP measurements of intracellular uorescent proteins. These two developments have brought FRAP into the mainstream of cell biologic techniques, especially those related to questions of membrane dynamics. Examples of useful applications of confocal FRAP include selective photobleaching and uorescence loss in photobleaching (FLIP) (18, 55). In contrast with conventional spot photobleaching FRAP, in confocal FRAP it possible to visualize both the bleach region and the surrounding area of the cell. In addition, confocal FRAP techniques typically use

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Photobleach Recovery (a)

and off membranes (28) as well as to assess the kinetics of vesicular and nonvesicular transport in the secretory and endocytic pathways (18) (Fig. 2). In FLIP, a single region of interest is bleached repetitively, which allows for recovery of uorescent material to occur in between each repetition. This technique causes a gradual depletion of uorescence material in regions of the cell that are in communication with the bleach region (Fig. 4c). Thus, the rate and the extent of loss of uorescent material from the area outside of the bleach region depends on the degree of connectivity between compartments (18).

Photoactivation
Photoactivate Redistribution (b)

Bleach 1

Bleach 2 (c)

Bleach 3

Although simple time-lapse imaging is often sufcient to monitor vesicular trafcking and transient membrane binding events, for other experiments it is advantageous to mark a particular group of uorescent molecules and watch their redistribution over time. The generation of photoactivatable uorescent proteins (39) has now made it possible to highlight a population of molecules for optical tracking experiments (55). As a result, movement of the photoactivated molecules can be visualized directly in a pulse-chase experiment (Fig. 4b). Many of the same concepts described above for FRAP experiments can also be applied to photoactivation.

Single particle tracking

Photobleach Recovery (d)

Photobleach No recovery (e)

Figure 4 Principles of photoactivation and photobleaching experiments. For purposes of illustration, regions of bright uorescence are shaded gray, and areas that contain little or no uorescence are shown in white. (a) FRAP. Here, molecules in a region of interest (box) are photobleached, and their exchange with uorescent molecules from the surrounding region is monitored over time. (b) Photoactivation. Similar to FRAP in principle except here the molecules in the region of interest (box) are converted to a different state. The redistribution of photoactivated molecules can then be monitored selectively. (c) FLIP. Repeated photobleaching of a region of interest (square) is performed, while monitoring the loss of uorescence from other regions of the cell. (d, e) Selective photobleaching. Fluorescent molecules in an individual compartment (as illustrated here for the Golgi complex, circled) are photobleached. The sample is then monitored over time to determine whether uorescence can recover (d) or not recover (e) from other regions of the cell. (Adapted from Reference 62 with permission from Elsevier.)

large and/or complex regions of interest and may incorporate repetitive bleaching protocols instead of a single bleaching event. For example, in selective photobleaching experiments, an entire subcellular compartment such as the Golgi complex is photobleached to examine the mechanism and kinetics of recovery from elsewhere in the cell (Fig. 4d-e). Using this approach, it is possible to monitor the kinetics of coat protein cycling on

Single particle tracking is a technique that visualizes directly the movements of individual molecules, small groups of molecules, or even viruses by either uorescence microscopy or light microscopy (54, 56). For such experiments, individual proteins can be expressed as GFP fusion proteins, labeled with uorescent antibodies, or labeled with quantum dots. Alternatively, they can be immunolabeled with probes that can be detected by light microscopy such as 3040 nm gold particles or latex beads. In the limit of sparse labeling, the trajectory of individual tagged molecules can then be tracked with high temporal and spatial accuracy using highly sensitive cameras (54). Although the resolution of uorescence microscopy is 250 nm, the centroid of a single molecule can be determined with an accuracy of 10 nm (54). Typical rates of image acquisition are 30 frames/sec, although much faster acquisition rates can be obtained with specialized cameras. The resulting trajectories can be plotted directly to show the movements of each molecule, or can be analyzed even more to determine the mean squared displacement as a function of time (Fig. 1). For example, during free diffusion, the molecule moves randomly, which results in a characteristic linear relationship between mean squared displacement and time. A hallmark of conned diffusion is that the mean squared displacement of the particle is at rst linear, then plateaus, which reects the limited movement of the molecule within a conned region of the membrane. A given molecule may shift between several modes of motion during the observation period. Thus, the percentage of molecules that undergo each type of motion and/or the fraction of time they exhibit a particular behavior is often reported. An obvious advantage of single molecule tracking is that it allows for highly detailed analyses of the movements of individual molecules that are obscured in population-based measurements such as FRAP and photoactivation experiments. However, SPT 7

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experiments are not trivial to perform and are subject to several potential artifacts. The rst challenge is to demonstrate that single molecules are being studied. Crosslinking can occur in studies that use antibody-labeled gold beads or quantum dots as probes, and lead to changes in mobility. In addition, large probes can potentially interact with the extracellular matrix. Photobleaching of organic dyes or FP-fusions can be rapid, which limits visualization times. However, photobleaching can also be used to conrm that single molecules are being visualized, because they will undergo a single-step photobleach. Finally, because of the intrinsic variability in trajectories, careful analysis is required to distinguish between motions that arise from free diffusion and conned diffusion.

images collected as a function of time. This spatial-temporal version of ICS can be used to monitor slow protein dynamics, and even be used to generate vector maps of directed protein movements in living cells (60).

Acknowledgments
I apologize to those whose original research could not be cited because of space limitations. I thank Maria Kiskowski, Lynne Lapierre, Erik Snapp, and Michael Edidin for their comments on the manuscript and Erik Snapp for providing Figure 2. Supported by R01GM-073846.

FCS and ICS

FCS is not an imaging technique per se, although it is often performed using a microscope-based system. Instead, FCS measures the movement of individual uorescent molecules through a dened observation volume, recorded as uctuations in uorescence over time (57). Such measurements require a sensitive photodetector, a dilute sample (nM) and a sampling volume with femptoliter dimensions. The observation volume can be generated using a laser focused to a diffraction-limited spot with a confocal pinhole placed in front of the detector, multiphoton illumination, or TIRF excitation in conjunctions with a pinhole. A notable strength of FCS is its sensitivity to uorescence uctuations over a wide range of timescales, from fast kinetics corresponding to photophysical properties of uorescent proteins to the diffusion of proteins in cell membranes (58). In addition, FCS measurements can be collected within user-dened regions in an individual cell or articial membrane vesicle, which allows for comparison of protein or lipid dynamics in distinct membrane environments (15). FCS can also be used to measure kinetic rate constants, for example, the association and dissociation of uorescently labeled molecules from the plasma membrane (49). The uorescence uctuations measured by FCS can be analyzed in several ways. The most common technique, autocorrelation analysis, provides information about characteristic diffusion time of uorescent molecules through the observation volume. It also reports on the average number of molecules present in the observation volume, and thus the concentration of uorescent moleculesn (14, 49, 56, 57). Other types of FCS analysis can be used to analyze molecular brightness and the oligomeric state of the uorescent molecule. Finally, cross-correlation FCS monitors uctuations jointly from molecules labeled with two or more different uorophores. This technique provides a powerful approach to assay for intermolecular interactions, because molecules that are bound either directly or indirectly to one another should diffuse as a single unit (8, 59). Whereas FCS measures uorescence uctuations over time, a related technique, ICS, measures uorescence uctuations over space, in particular from images collected using a laser-scanning microscope (56). ICS analysis of pixels within a single image provides information about protein clustering and density. A variation of ICS known as image cross-correlation spectroscopy evaluates the interactions of molecules labeled with different uorescent probes. ICS can also be performed on stacks of 8

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See Also
Imaging Techniques: Overview of Applications in Chemical Biology Lipid Bilayers, Properties of Membrane Assembly in Living Systems Membranes, Fluidity of Membrane Trafcking

Further Reading
Edidin M. Lipids on the frontier: a century of cell-membrane bilayers. Nature Revi. Mol. Cell. Biol. 4:414418. Gennis RB. Biomembranes. 1989. Springer-Verlag, New York. Jans DA. The Mobile Receptor Hypothesis. 1997. Chapman & Hall, New York.

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