Cytoskeletal dynamics of human erythrocyte

Ju Li*, George Lykotrafitis†, Ming Dao†, and Subra Suresh†‡§

*Department of Materials Science and Engineering, Ohio State University, Columbus, OH 43210; and †Department of Materials Science and Engineering and
‡Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

Communicated by L. B. Freund, Brown University, Providence, RI, January 10, 2007 (received for review November 15, 2006)

The human erythrocyte (red blood cell, RBC) demonstrates extraor-

dinary ability to undergo reversible large deformation and fluidity.
Such mechanical response cannot be consistently rationalized on
the basis of fixed connectivity of the cell cytoskeleton that com-
prises the spectrin molecular network tethered to phospholipid
membrane. Active topological remodeling of spectrin network has
been postulated, although detailed models of such dynamic reor-
ganization are presently unavailable. Here we present a coarse-
grained cytoskeletal dynamics simulation with breakable protein
associations to elucidate the roles of shear stress, specific chemical
agents, and thermal fluctuations in cytoskeleton remodeling. We
demonstrate a clear solid-to-fluid transition depending on the
metabolic energy influx. The solid network’s plastic deformation
also manifests creep and yield regimes depending on the strain
rate. This cytoskeletal dynamics model offers a means to resolve
long-standing questions regarding the reference state used in RBC
elasticity theory for determining the equilibrium shape and defor-
mation response. In addition, the simulations offer mechanistic
insights into the onset of plasticity and void percolation in cy-
toskeleton. These phenomena may have implication for RBC mem- Fig. 1. An experimental in vitro demonstration of the ‘‘fluidization’’ of a
brane loss and shape change in the context of hereditary hemolytic healthy human RBC through a microfluidic channel at room temperature. The
disorders such as spherocytosis and elliptocytosis. series of images show the shape of an RBC as it is squeezed through a 4 ␮m ⫻
4 ␮m channel made of polydimethyloxysilane (PDMS), under a pressure dif-

BIOPHYSICS
cytoskeleton remodeling 兩 computer simulation 兩 fluidization 兩 plasticity 兩 ferential of 1.5 mm of water. Note the recovery of shape upon egress from the
spectrin–actin dissociation channel. Images a, b, c, and d were taken at relative times of 0, 0.4, 0.8, and
1.4 s, respectively.

D uring its 120-day life span, a red blood cell (RBC) circulates
a million times in human body, often squeezing through
narrow capillaries. The erythrocyte’s remarkable mechanical
work. Therefore, the confluence of mechanical strain energy,
nonspecific thermal fluctuation energy, and specific biochemical
properties originate from the unique architecture of its cell wall, activation energy fundamentally influences the behavior of the
which is the main load bearing component as there are no stress cytoskeleton and the membrane tethered to it.
fibers inside the cell. The cell wall comprises a spectrin tetramer Optical tweezers (OT) experiments (13, 14) have shown that the
network tethered to a phospholipid bilayer (1, 2). It is very membrane with cytoskeletal attachment can sustain significant
flexible, and yet resilient enough to recover the biconcave shape shear, with an estimated shear modulus ␮ ⫽ 4–10 ␮N/m. Indeed,
whenever the cell is quiescent (see Fig. 1). It has been proposed the in-plane shear energy that an RBC can apparently store in these
that the spectrin network connectivity is not fixed and can OT experiments is ⬇200 pN ⫻ 8 ␮m/2 ⫽ 5,000 eV, which far
experience extensive remodeling as the RBC undergoes large exceeds the total bending energy it stores (⬇8␲␬ ⫽ 50 eV), for
deformation. Experiments show that, under physiological con- bending modulus ␬ ⬇ 2 ⫻ 10⫺19 J (15). Classic Canham–Helfrich
ditions, the spectrin tetramers in an unstressed intact cell wall explanation of the biconcave equilibrium shape of erythrocyte (16,
exist in rapid dynamic equilibrium with the dimers, and that 17) requires minimization of total bending energy only, without
shear-induced cell wall deformation displaces the balance in considering the much larger in-plane shear energy. Equilibrium
favor of the dimers (3). Here, the cell wall behaves as a weak shape calculations with inclusion of in-plane energies, at both
elastic solid at low strain levels, whereas it can be fluidized continuum (14, 18) and discrete spectrin network (14, 19) levels,
beyond a certain level of shear deformation, similar to soft thus exhibit a seemingly paradoxical behavior. At the experimental
matter such as emulsions and colloidal pastes (4, 5). Upon range of ␮ and ␬ values, the biconcave equilibrium shape is rarely
unloading, the dynamic stability is shifted toward tetramers and obtained in numerical simulations without careful tuning of the
the stiffness of the cell wall increases. Experiments also suggest stress-free reference state for the in-plane energy.
that cytoskeleton remodeling can be regulated by biochemical Based on extensive parametric studies, the following mechanistic
factors such as Ca2⫹ and ATP (adenosine 5⬘-triphosphate) hypothesis was proposed (14) to resolve the paradox. In an ideal
concentrations (6, 7). It has been postulated that phosphoryla-
tion affects spectrin–actin binding and produces changes in the
shape and deformability of human RBC (8–11); this is equivalent Author contributions: J.L., G.L., M.D., and S.S. designed research, performed research,
to the injection of metabolic energy specifically to spectrin–actin analyzed data, and wrote the paper.

junctions, thereby inducing local rupture between actin and The authors declare no conflict of interest.
spectrin. Prior analytical modeling addressed the impact of Abbreviations: OT, optical tweezer; CD, cytoskeletal dynamics.
ATP-induced cytoskeletal defects arising from spectrin–actin §To whom correspondence should be addressed. E-mail: [email protected]
dissociation on RBC shape and membrane fluctuation (12). This article contains supporting information online at www.pnas.org/cgi/content/full/
Activation of these biochemical processes can result in lower 0700257104/DC1.
connectivity and, consequently, a softer or even fluidized net- © 2007 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0700257104 PNAS 兩 March 20, 2007 兩 vol. 104 兩 no. 12 兩 4937– 4942
Fig. 2. Coarse-grained cytoskeletal dynamics computer simulation. (a) Schematic of cytoskeleton of the human RBC. (b) A minimal physically realistic model
with breakable actin–spectrin interaction. A (red sphere) represents an actin protofilament, and B (green and gray spheres) represents a spectrin segment. Only
the two ending spectrin units (green spheres) of a spectrin chain can bind to A. (c) Interaction potentials between A–A (steric repulsion), A–Bend (Lennard–Jones
potential), A–Bmid (steric repulsion), B–B[1] (spring potential between nearest neighbor spectrin units), and B–B[2] (steric repulsion between any other spectrin
units). Bmid represents a nonending spectrin unit, and Bend represents an ending spectrin unit. Note that A–Bend forms breakable linkage. (d) Lipid membrane
introduces additional effective repulsion between A–A (12), modeled by a linear potential with cutoff.

limit, for any RBC shape, the cytoskeleton always undergoes any dynamic cytoskeletal remodeling due to mechanical, thermal,
remodeling in topological connectivity at a certain rate to relax its or chemical driving forces.
in-plane shear elastic energy to zero. Therefore in considering the
equilibrium shape of an RBC, one does not need to account for the Computational Model
in-plane shear energy, even though in OT experiments it can be The erythrocyte cell wall consists of a lipid bilayer, cytoskeleton
injected temporarily into the cytoskeleton. The remodeling process network, and integral membrane proteins. The intramembrane
has been studied heuristically in (14) by simulating simple liquids proteins band-3 and glycophorin tether the cytoskeleton net-
confined on a surface and interacting via the Lennard–Jones or work to the bilayer via additional binding proteins (e.g., ankyrin
Stillinger–Weber potentials (20). This produces cytoskeleton that and 4.1). A schematic representation is shown in Fig. 2a. Spectrin
appears similar to atomic force microscopy scans (21–23), akin to is a protein tetramer formed by head-to-head association of two
reverse Monte Carlo (24) modeling. Despite these considerations, identical heterodimers (31). Each heterodimer has an ␣-chain,
detailed simulations of spectrin network dynamics promoting plas- which has 22 triple-helical segments, and a ␤-chain, which has 17
ticity and fluidization of RBC are presently unavailable. triple-helical segments. A minimalistic representation of spectrin
is therefore chosen to be S ⫽ 39 spherical beads (B in Fig. 2b)
In this paper, a cytoskeletal dynamics (CD) simulation frame-
connected by S ⫺ 1 unbreakable springs. The spring potential,
work is developed to study the RBC spectrin network remodeling
VBB(r) ⫽ k0(r ⫺ r0)2/2, is plotted as the magenta curve in Fig. 2c,
process and fluidization. The framework comprises the following
with equilibrium distance r0 ⫽ Lmax/(S ⫺ 1), where Lmax is the
key ingredients: (i) a minimal representation of the cytoskeleton contour length of the spectrin (chosen to be 200 nm). Thus, r0 ⫽
geometry that can self-organize and dynamically evolve, and (ii) 5.26 nm. The spring constant k0 is chosen later. Beads that are
mechanisms for nonthermal energies such as the strain energy or not topologically connected on the chain, or are on different
specific biochemical energy to influence and regulate structural chains, repel each other sterically. Their interaction potential is
evolution. By abstracting out many details, such a cytoskeletal taken to be simply VBB(r)H(r0 ⫺ r), shown as the black dash curve
dynamics simulation provides detailed mechanistic insights into the in Fig. 2c, where H(x) is the Heaviside step function.
mechanics and physics of RBC deformation. This model could then Among the S beads of a spectrin chain, there are two special
be used in studying RBCs with cytoskeletal defects which are typical beads colored in green in Fig. 2b that represent the free ends. They
in blood disorders such as hereditary spherocytosis and elliptocy- can bind noncovalently to junction complexes (2), composed of a
tosis (25, 26). The present work thus seeks to provide a significant short actin proto-filament and protein band 4.1. Experiments
advance over previous models that were either formulated at the indicate that, without protein 4.1, spectrin only binds weakly to
continuum scale (18), thereby ignoring the discrete cytoskeletal actin, with equilibrium association constant K1 ⬇ 5 ⫻ 103 M⫺1. With
structure, or that assumed a fixed cytoskeleton connectivity (14, 19, 4.1, however, strong binding occurs, forming ternary complexes
27–30), thereby useful only to study elastic cell deformation without with equilibrium constant K2 ⬇ 1 ⫻ 1012 M⫺1 (32). A separate

4938 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0700257104 Li et al.

measurement indicated that the equilibrium association constant of
protein 4.1 with actin itself is K3 ⬇ 1 ⫻ 107 M⫺1 (33). In the
schematic of the simulation model, the large red bead (A in Fig. 2b)
denotes the junction complex. The terminal green beads of spectrin
are attracted to A via the Lennard–Jones potential, VAB(r) ⫽
4␧((␴/r)12 ⫺ (␴/r)6), plotted as the green curve in Fig. 2c. The A–B
association energy ␧ is chosen to be 14 kcal/mol or 0.6 eV. The
characteristic interaction length scale ␴, on the other hand, is
chosen so that the equilibrium A–B distance 21/6␴ is precisely 2r0.
The ‘‘capture radius’’ of A–B attraction is ⬇20 nm (Fig. 2c). The
capture diameter is therefore ⬇40 nm, which matches the actual
actin proto-filament length of ⬇35 nm. To eliminate a free param-
eter, k0 is chosen to be identical to the curvature of VAB(r) at its well
bottom, k0 ⫽ 36 (2)2/3␧␴⫺2. The molecular mass of a spectrin
tetramer is almost 1,000 kDa (1). Thus, the B beads are chosen to
have a mass of 1,000/S kDa each. The mass of A bead is chosen to
be 121 kDa, as the sum of the masses of actin proto-filament and
band 4.1 (1).
The novelty of this model over prior spectrin-based simulations
(14, 19, 27–30) is that the A–B association is breakable, as well as
Fig. 3. CD simulations with only mechanical energy input. (a) Snapshot of
reformable, with the network topology changing dynamically. Un-
the equilibrium network structure at 300 K, top view and side view. (b)
der increasing tensile force, the A–B association breaks when their Snapshot of the sheared (␥ ⫽ 1) network structure at 300 K, top view. (c) Shear
distance reaches the inflexion point of VAB(r), rinflexion ⫽ (26/7)1/6␴, stress-strain response at strain rate ␥˙ ⫽ 3 ⫻ 105 per s.
indicated as the red circle in Fig. 2c. This corresponds to 10.9%
bond strain and 24.9 pN force. A saturation effect exists for the A–B side only (Vconfine(z)H(z)). In other words, the spectrin chain can
affinity because most of the actin proto-filaments have, at most, six freely swing down, but will be hampered when swinging up. The
slots to hook up with spectrin ends (via band 4.1) (30). This is stiffness of both confinement potentials are quite arbitrarily
implemented in the simulation quite naturally as follows. The chosen to be kconfine ⫽ 0.1k0.
attractive part of the Lennard–Jones potential, ⫺4␧(␴/r)6, is turned We perform 3D coarse-grained molecular dynamics simulations
on for an ending B, if and only if there are less than six other ending governed by the aforementioned potentials. Its fundamental time
Bs already within the inflexion radius rinflexion. In other words, when scale (chain oscillations) is at the nanosecond level, and it is typical
six ending Bs are already associated with A (judged by their

BIOPHYSICS
for a whole CD simulation run to cover a few microseconds. The
distances to that A), all other ending Bs only see the repulsive part Berendsen thermostat (35) is used to control the system’s temper-
of the potential 4␧(␴/r)12. ature, and the instantaneous stress tensor ␶ij is calculated by using
Nonending Bs have only steric repulsion with A, taken to be the Virial formula (36). Hydrodynamic interactions (37) are ig-
simply VBB(r⫺r0)H(2r0⫺r), shown as the red dash curve in Fig. 2c. nored [see supporting information (SI) Text]. We begin with the
Similarly, any two As would repel each other at short range with configuration in Fig. 2b that is a perfect triangular network in a
VBB(r ⫺ 2r0)H(3r0 ⫺ r), shown as the blue dash curve. This periodic supercell, containing 7 ⫻ 8 ⫽ 56 junction complexes, 168
cytoskeletal interaction potential model has the maximum simplic- chains, and 6,552 spectrin beads, spanning an area ⬇1.5 ␮m ⫻ 1.5
ity in terms of choice of parameters. Essentially, once the A–B ␮m at T ⫽ 0 K. Although discrete spectrin-based whole-cell
association energy ␧ is known, there are no free parameters to simulations have been performed before (14), and it is not out of
choose. the question even for the present cytoskeletal dynamics model, it is
The lipid bilayer exerts additional forces on cytoskeleton. The much more appealing to begin with a study of some unit processes
fluid-like lipid bilayer is slightly crumpled at equilibrium (12) (see in a representative area element.
Fig. 2d) pinned by the junction complexes (34), and it exerts an
effective repulsion between two neighboring junction complexes. It Results
can be shown that the repulsive force due to a bent elastic cylindrical Chemically Induced Spectrin–Actin Dissociation. Starting with perfect
shell is constant with respect to end-to-end distance, until the shell triangular network at T ⫽ 0 K, where A–A nearest-neighbor
is fully straightened out. To model this effect, we add an additional distance is Lmax ⫹ 27/6␴ ⫽ 221 nm, the network is gradually heated
repulsive potential between pairs of As that are nearest neighbors while maintaining the internal pressure near zero. As expected, the
4␲␬ bare membrane(Rcut⫺r) system shrinks because of entropic elasticity, to an average nearest-
VnearestA-A (r) ⫽ ␣ H共R cut ⫺ r兲, neighbor A–A distance of 88 nm at T ⫽ 300 K and total area ⬇0.6
3Rcut ␮m ⫻ 0.6 ␮m (Fig. 3a). The vertical fluctuations of spectrin beads
[1] are ⬇40 nm. The topology of network remains nearly perfect at T ⫽
300 K. The mean degree of the network 具d典 is taken to be the
where Rcut is the distance at which the membrane becomes flat. average number of spectrin ends attached to a junction complex.
We take Rcut ⫽ 106.1 nm, which is 20% larger than the thermally 具d典 ⫽ 6 if the network topology is perfect. Upon further heating,
averaged A–A distance, chosen to be R0 ⫽ 88.4 nm. With the broken-link, degree-5 defects are spontaneously created (12).
estimated bare membrane bending modulus ␬bare membrane ⫽ 2 ⫻ Fixing T ⫽ 300 K, we then apply simple shear to the supercell
10⫺20 J (12), ␣ ⫽ 0.36, so that the pressure of the entire system with strain rate ␥
˙ ⫽ 3 ⫻ 105 per s, where ␥ is the engineering shear
is nearly zero at T ⫽ 300 K and R0 ⫽ 88.4 nm. strain. Final sheared network configuration is shown in Fig. 3b, and
A and B are also confined vertically by the lipid bilayer. Soft the shear stress versus shear strain curve is plotted in Fig. 3c. Linear
harmonic confinement potentials Vconfine(z) ⫽ kconfinez2/2 are elastic shear modulus ␮0 ⬇ 10 ␮N/m agrees well with literature
used in the z direction to mimic this effect without explicitly values from OT and micropipette aspiration experiments (13, 14).
simulating the fluid bilayer. Because the junction complex is Using the worm-like chain model for the spectrin molecule (14),
attached to the bilayer, A’s confinement potential is in both ⫹z this ␮0 corresponds to a persistence length of p ⬇ 6 nm, which is
and –z directions (Vconfine(z)), whereas B is confined on the ⫹z close to the B bead diameter (5.26 nm). This numerical agreement

Li et al. PNAS 兩 March 20, 2007 兩 vol. 104 兩 no. 12 兩 4939

Fig. 4. Shear stress-strain response of network at strain rate ␥˙ ⫽ 3 ⫻ 105 per s. (a) Energy hit rate 10 per ␮s and final network structure at ␥ ⫽ 1 (Inset). (b) Energy
hit rate 2.5 per ␮s and final network structure at ␥ ⫽ 1 (Inset).

in ␮0 is perhaps fortuitous without any intentional fitting to the response (not shown) is similar as well. To further confirm this
shear response. finding, we plot the potential energy U of reformed network (SI Fig.
At large shear strains, ␥ ⫽ 80%, the tangent shear modulus 6b Inset), which approaches that of the perfect network reference
stiffens to ␮f ⬇ 25 ␮N/m. This value then amounts to a prediction value over time. Thus the cytoskeleton ‘‘re-gels’’ from fluidized
of the simulation. Even at ␥ ⫽ 1, few topological defects appear in state after biochemical energy influx is turned off, with a time
the simulation. The stress–strain response is nearly perfectly re- constant ␶g ⬇ 0.1 ␮s, taken from the half life of 具d典 and U recoveries
versible and elastic. Again, the agreement is good with OT exper- in SI Fig. 6b.
iments (13, 38). To match the experiments up to 100% RBC The characteristic recovery time ␶g in present simulation is
elongation with a finite-element model, a third-order hyperelastic- perhaps much faster than that in reality. A–B association is repre-
ity constitutive relation is needed for cell wall material with sented by a smooth potential VAB(r) with no activation energy
stiffening behavior (13, 38). The value of ␮f estimated from finite barrier (Fig. 2c). Thus, once A loses one B association and its d is
element simulations is 20 ␮N/m at ␥ ⫽ 1. ⬍6, it turns on a smooth attractive force field with capture diameter
Until now, the cytoskeleton behaves just like an ordinary inert ⬇40 nm for all free spectrin ends. In reality, both association and
material. Room-temperature thermal fluctuations do not over- dissociation reactions have activation barriers (42), catalyzed by
come A–B binding to effect topology change. Because of homeosta- protein kinases (7) as well. We now nondimensionalize all time and
sis (nearly constant body temperature), cytoskeletal changes cannot rate labels by ␶g. The fluidization at H ⫽ 100 per ␮s is thus attributed
arise from significant temperature changes. However, the CD could to H␶g ⬎⬎ 1. In other words, the rate of biochemical activation and
be driven by chemical energy flux, pushing the system out of induced dissociation far exceeds that of intrinsic recovery, whereby
equilibrium (39). To explore this effect, we mimic biochemically a disconnected spectrin chain reconnects somewhere to reduce the
activated actin–spectrin dissociation by an instantaneous kinetic free energy. The recovery rate depends on applied stress and
energy transfer of X ⫽ 0.7 eV to an ending B bead. The magnitude temperature, which change driving force and kinetics of reattach-
of X matches roughly the energy release from ATP hydrolysis ment (forming a stretched chain with two constrained ends versus
reaction, which is ⬇20–25kBT per molecule (40). The direction of a coiled chain with one or two free ends).
kinetic energy transfer is random in the 4␲ spherical angle. Because We next choose the borderline case of H ⫽ 10 per ␮s, H␶g ⫽ 1.
the binding energy between A–B is ␧ ⫽ 0.6 eV, it is highly likely that The cytoskeleton shear stress vs. shear strain is plotted in Fig. 4a,
such a hit would lead to dissociation of the particular AB junction. at the same strain rate ␥ ˙ ⫽ 3 ⫻ 105 per s or ␥
˙␶g ⫽ 0.03. The linear
The schedule of kinetic energy hits conforms to a random Poisson modulus ␮0 is softened from 10 to 5.3 ␮N/m. More dramatically, the
process, with equal probability for all spectrin ends, irrespective of shear stress vanishes above ␥ ⫽ 60%. A look at the structure
their hit histories. The center-of-mass of the cytoskeleton is held indicates that the cytoskeleton has indeed fluidized (41), with large
fixed at all times. holes percolating through the simulation supercell (Fig. 4a Inset).
Initially, we assign a hit rate of H ⫽ 100 per ␮s per spectrin end This behavior is in stark contrast to Fig. 3 with no biochemical
without simultaneously shearing the cytoskeleton. The network activation, where the network deforms elastically, and even stiffens
quickly becomes proliferated with topological defects, as indicated to ␮f ⬇ 25 ␮N/m. Thus, at hit rate H␶g ⬇ 1, a softened, but still solid,
by the 具d典 statistics (see SI Fig. 6a). Larger and larger holes appear, cytoskeleton is obtained at small strain, which transforms into fluid
which eventually percolate through the supercell (41). The network at critical strain ␥fluidize ⬇ 50%. RBCs commonly encounter such
then loses shear-bearing capability; that is, the solid is completely strains in circulation (43).
fluidized at H ⫽ 100 per ␮s without mechanical stress (gel-to-sol The mechanism for this fluidization transition could be summa-
transition). The fluidization process reflects a dynamic percolation rized as follows. Under shear strain, some spectrin chains are
of broken bonds across the network (41), which could lead to mass elongated and some shortened, depending on their alignment to the
and momentum transport even below the static percolation limit. principal axes of stress. The elongated chains exert an effective
The importance of the percolation limit of broken bonds to shear tensile force FWLC at the A–B junction roughly according to
elasticity was pointed out in ref. 12. worm-like chain entropic force (14). At small ␥, the maximum
To see the ‘‘annealing’’ or ‘‘recovery’’ behavior (sol-to-gel tran- elongation is small, so even after dissociation the probability of
sition), we then turn off the kinetic energy hits (H ⫽ 0), and the reattachment is large. However, at large ␥, the initial elongation is
cytoskeleton reconstitutes readily, as indicated by the 具d典 statistics too large (see Fig. 4b Inset). Therefore, once a spectrin link is
(see SI Fig. 6b), within the CD simulation time scale. The network broken, the chain recoils and becomes exponentially unlikely for the
that reforms from fluidized state does not have a perfect triangular- spectrin chain to fluctuate back to the original length to reestablish
net topology, it contains topological ‘‘mistakes,’’ but structural the connection.
similarity to a triangular network is strong. The shear stress–strain The response seen in the above CD simulation comes from a

4940 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0700257104 Li et al.

nonequilibrium system, which cannot be made similar to an inert
system by simple scaling (law of corresponding states). This is
because one essential element, FWLC, is proportional to kBT, where
T is the thermodynamic temperature; but the other element, the
rate of A–B association/dissociation, is related to the rate of
phosphorylation. This is analogous to the concept of two temper-
atures (39), one for general degrees of freedom including chain
vibrations and bending (thermodynamic temperature) and one just
for topological change (effective or ‘‘structural evolution’’ temper-
ature refs. 5, 44, and 45). When the thermodynamic temperature is
raised, due to equi-partition theorem, all degrees of freedom tend
to be equally excited. In contrast, biochemical activation is highly
targeted due to the specific biochemical machinery required, e.g.,
for phosphorylation; this is reflected in our model, because when
H is raised, only the A–B associations (strategic nodes of the
network) are strongly excited. The effective temperature concept is
generic in soft glassy matter (45), irradiated materials (46), and
generally nonequilibrium materials (39) under mechanical, chem-
ical, or radiative energy influxes. However, it is perhaps in the
biological context that the concept is put to use in a very sophis-
ticated manner (47). Biochemical activation is specific and local.
Only certain cytoskeletal proteins can be activated to elicit certain
structural response. Fig. 5. Refined CD simulation. (a) A model with breakable actin–spectrin
We then examine the other limit, H␶g ⬍⬍ 1, with H ⫽ 2.5 per ␮s. and/or ␣-spectrin–␤-spectrin interaction. Red spheres represent actin proto-
The shear stress–strain response is plotted in Fig. 4b, with final filaments, and green, gray, blue, and yellow spheres represent a spectrin
configuration at ␥ ⫽ 100% (Fig. 4b Inset). The cytoskeleton is a segment. Only the two ending spectrin units (green spheres) of a spectrin
solid throughout the entire range of strain, because although voids chain can bind to red sphere. (b) Shear stress-strain response of network at ␥˙ ⫽
exist, they never percolate through the network. Its linear modulus 3 ⫻ 105 per s and dimer– dimer association energy of 0.6 eV. Corresponding
shear stress-strain response of network at 3 ⫻ 105 per s and 0.56 eV (c) and 3 ⫻
␮0 (8.5 ␮N/m) is very close to that with no energy hits. The
105 per s and 0.47 eV (d) are shown.
difference with the zero chemical energy influx situation (Fig. 3) is
that there is a plastic displacement burst at ␥ ⫽ 35–50%, similar to

BIOPHYSICS
incipient plasticity events in metals (48). However, the system is still Dimer–Dimer Dissociation Due To Shearing. In Fig. 2b, two features
essentially a solid. Therefore, we conclude that, at H␶g ⬍⬍ 1, the of cytoskeleton are ignored: the confinements due to ankyrins and
cytoskeleton behaves like a plastically deforming solid, with the rate band 3 proteins (2) that also bind the spectrin to the membrane, and
of plastic deformation governed by H and stress. the possibility that a spectrin tetramer can dissociate into two
The strain ␥F at the first plastic displacement burst depends on heterodimers (3). These two aspects are considered here (Fig. 5a),
the energy hit-rate H, the energy per hit X, and the spectrin–actin where interest is shifted from spectrin–actin dissociation (due to
association energy ␧. SI Table 1 shows that ␥F and the shear moduli locally delivered chemical energy) to the effects of dimer–dimer
(at the initial elastic response) decrease as the energy hit-rate dissociation during shearing of the network. The strength of dimer–
increases from 0 to 10 per ␮s per spectrin. The constant strain rate dimer association is known to be weaker than the actin–4.1–spectrin
used in all of the deformation simulations, ␥ ˙ ⫽ 3 ⫻ 105 per s, is very association, with equilibrium constant K4 ⬇ 1.5 ⫻ 106 M⫺1 (42). In
high in terms of absolute value. However, if scaled by ␶g, the Fig. 5a, the confinement due to ankyrin and band 3 protein is
nondimensionalized strain rate ␥ ˙␶g is actually quite reasonable with modeled by applying the same confinement potential Vconfine(z) to
a clear physical meaning. Take the last case of H␶g ⫽ 0.1: combined the blue beads, which were randomly placed two beads ahead of or
with ␥˙␶g ⫽ 0.03, this would mean that during the course of straining behind the center of each chain. Rupturing of the chain (symbol-
the cytoskeleton to 100%, each spectrin end would receive 3.3 izing dissociation of a spectrin tetramer into dimers) was allowed to
biochemical activation hits on average. We have also investigated occur in the middle between the two yellow beads. The original
the effect of strain rate on ␥F (SI Table 2). When H ⫽ 0, ␥F is large harmonic potential k0(r ⫺ r0)2/2 between the yellow beads B⬘ was
(⬎100%) and nearly independent of strain rate. At finite H, we substituted by a Lennard–Jones potential VB⬘B⬘ ⫽ 4␧B⬘B⬘ ((␴/(r ⫹
discovered two regimes of behavior. Take the case of H␶g ⫽ 0.1. r0))12 ⫺ (␴/(r ⫹ r0))6), which has similar characteristics as VAB. The
When ␥ ˙␶g ⬍ 0.014, ␥F is sensitive to ␥˙ in almost linear relationship; only difference is that the minimum of VB⬘B⬘ is shifted to r0, which
but when ␥ ˙␶g ⬎ 0.014, ␥F becomes much less sensitive to ␥ ˙. The is the equilibrium distance between all spectrin beads.
reason is that, at small ␥ ˙, the first plastic displacement burst is As above, the perfect triangular network was initially heated
governed more by damage accumulation caused by H than by from T ⫽ 0 to 300 K while the internal pressure was kept close to
mechanical stress ␶. The time tD to accumulate enough damage to zero. ␧B⬘B⬘ was varied from 0.6 eV to 0.47 eV (that is, from 100%
facilitate a displacement burst depends more on H, and less on ␶ or to 78% of the actin–spectrin association energy ␧). This choice
␥, so ␥F ⫽ ␥ ˙tD can be approximated by ␥F ⬇ ␥ ˙tD(H), and thus the resulted in an equilibrated network of reduced integrity. In partic-
linear relationship. But when ␥ ˙ is large, stress ramping is fast ular, although all of the actin–spectrin connections were unbroken
compared with damage by H, and the high stress begins to play a (具d典 ⫽ 6), the integrity at T ⫽ 300 K of the dimer–dimer associations
much more substantial role in inducing displacement burst, and the varies from 100% to ⬇90%. Next, the same simple shear defor-
strain threshold for this to happen becomes more constant. These mation was applied to model networks of various integrities at a
two plastic deformation regimes are analogous to the creep and strain rate of ␥
˙⫽ 3 ⫻ 105 per s. No specific biochemical energy hits
yield regimes of engineering materials, which are sensitive (much were injected at the network associations, and only mechanical
less sensitive) to the strain rate at small (large) strain rates, energy was supplied.
respectively, with the latter regime possessing a much more clear- In the first simulation (Fig. 5b), ␧B⬘B⬘ ⫽ 0.6 eV, whereas the
cut yield strain threshold behavior. integrity of dimer–dimer connections at T ⫽ 300 K was 100%. The

Li et al. PNAS 兩 March 20, 2007 兩 vol. 104 兩 no. 12 兩 4941

response of the network was initially similar to the response shown show a progressive decrease of the dimer–dimer association integ-
in Fig. 3c, where no dimer–dimer dissociation was allowed. At small rity during shearing (SI Fig. 7).
strains, it exhibited an elastic shear modulus of ⬇9 ␮N/m. Then,
at ⬇ 45% strain, it became stiffer with a shear modulus of 20 ␮N/m. Discussion and Concluding Remarks
At 90% strain, where the shear stress reached the value of ⬇12.5 We have developed a cytoskeletal dynamics simulation frame-
␮N/m, a plastic type displacement burst occurred. This burst was work that allows active remodeling of the 3D cytoskeleton via
caused by dimer–dimer dissociations, because the integrity of breakable as well as reformable associations of the junction
actin–spectrin associations was 100% throughout the numerical complex and spectrin tetramer. Possible roles of external shear
experiment. At ␧B⬘B⬘ ⫽ 0.56 eV (7% lower than ␧) the response of stress (related to deformability of healthy RBC at large strains),
the network is shown in Fig. 5c. It initially exhibited a shear modulus specific chemical energy (i.e., ATP, as a possible means to
of ⬇8 ␮N/m, slightly lower than in the previous case. At 25% strain, actively adjust RBC deformability in circulation), and protein
the network shows a larger tangent modulus of 13 ␮N/m. At ⬇45% association strength (possibly related to RBC disease states with
strain, a plastic displacement burst occurred. Then the response was weakened/broken cytoskeletal connectivity) versus temperature
elastic with a tangent shear modulus of 13 ␮N/m, until 80% shear and strain rate can be studied parametrically by using the
strain where another plastic displacement burst started. proposed model. These results offer mechanistic rationale for
At ␧B⬘B⬘ ⫽ 0.47eV (22% lower than ␧), which corresponds to 91% the erythrocyte cytoskeleton mechanical function under loading.
integrity of the dimer–dimer connections, the response of the The model demonstrates that cytoskeletal remodeling through
spectrin network to shear deformation changed dramatically (Fig. specific biochemical activation is a possible means for RBC
5d). Initially, the spectrin network showed a linear elastic behavior deformability to be actively ‘‘tuned’’ during its circulation in
with a reduced shear modulus of ⬇4 ␮N/m. From 20% until 50%, microvasculature. On one hand, structural integrity of the cell
the shear stress was constant. Finally, at 60% strain, the shear stress demands certain minimal properties of the cytoskeleton. For
response almost vanished and the cytoskeleton fluidized. instance, the largest flaw in the RBC cytoskeleton cannot exceed
In the case of dimer–dimer dissociation due to shearing, the yield a critical size, otherwise lipid membrane loss will occur via the
strain depends on the association energy between the dimers. The vesiculation instability (49). On the other hand, RBC also
spectrin network was first heated from 0 to 300 K and then encounters different environments (artery, capillary, splenic
equilibrated. SI Table 3 shows that, during the above procedure, the sinus, etc.) and mechanical conditions in circulation, and its
mechanical properties may need to change ‘‘on the fly’’ (50). The
resulting spectrin network integrity at the equilibrium reduced from
relative influence of mechanical stress during constrained shear
almost 100% at ␧B⬘B⬘ ⫽ 0.6 eV to 91% at ␧B⬘B⬘ ⫽ 0.47 eV. The yield
flow, energy exchange and biochemical modification of dimer–
strain was also reduced from 90% to 20%, respectively. The
dimer or actin–spectrin binding energy is largely unknown at this
‘‘thermal equilibrium’’ dimer content depends on kBT/␧B⬘B⬘. The
time, and could depend on the natural variations in the cytoskel-
plastic strain threshold is very sensitive to the above dimer content,
etal structures in a given population of cells and the local
because when kBT/␧B⬘B⬘ is raised, not only do we have more dimers,
biochemical environment. Our parametric CD model is a first
but even the existing tetramers are severely weakened by entropic step toward unraveling possible mechanistic underpinnings of
elasticity. The trend of our results matches very well with the these complex, intertwined pathways.
experimental data presented in ref. 3. There, it was shown that the
cell wall stability decreases when the dimer content of the RBC We thank David J. Quinn for preparing the images in Fig. 1 from
increases. The most striking outcome of the above work (3) is the microfluidics experiments conducted in the laboratory of S.S. This work
observation that shear deformation induces dissociation of tetram- was supported by National Institutes of Health/National Institute of
ers into dimers. This finding matches our numerical results, which General Medical Sciences Grant 1-R01-GM076689-01.

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4942 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0700257104 Li et al.