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From the
Received for publication, November 13, 2001, and in revised form, February 26, 2002
Rapid sol-gel transitions of the actin
cytoskeleton are required for many key cellular processes, including
cell spreading and cell locomotion. Actin monomers assemble into
semiflexible polymers that rapidly intertwine into a network, a process
that in vitro takes ~1 min for an actin concentration of
1 mg/ml. The same actin filament network, however, takes ~1 h to
exhibit a steady-state elasticity. We hypothesize that the slow
gelation of F-actin is due to the slow establishment of a homogeneous
meshwork. Using a novel method, time-resolved multiple particle
tracking, which monitors the range of thermally excited displacements
of microspheres imbedded in the network, we show that the increase in
elasticity in a polymerizing solution of actin parallels the progressive decline of the network microheterogeneity. The rates of
gelation and network homogenization slightly decrease with actin
concentration and in the presence of the F-actin cross-linking proteins
Many key cellular processes, including cell spreading and cell
locomotion, require rapid sol-gel transitions of the actin cytoskeleton
(1, 2). Under physiological conditions,
G-actin1 monomers assemble
into semiflexible F-actin polymers, which rapidly overlap into a
network, a process that in vitro takes ~1 min for an actin
concentration of 1 mg/ml (3). The same actin filament network, however,
takes ~1 h to exhibit a steady-state stiffness, a direct measure of
the extent of gelation (see "Results" below). We also observe that
increasing actin concentrations decelerate gelation but accelerate
polymerization (see "Results"). Moreover, the gelation of F-actin
in the presence of auxiliary proteins is much slower than required to
produce morphological changes at the edge of adherent cells (4).
Cellular protrusions, including filopodia and lamellipodia, typically
appear and vanish in minutes even in slow-locomoting fibroblasts (1).
We hypothesize that the large discrepancy in time scales between actin
polymerization and actin network gelation in vitro is due to
the slow establishment of a homogeneous network.
No existing method can quantitatively probe the evolution of
the organization of a cytoskeletal network in real-time. Current methods either ensemble-average physical properties of the network yielding an average mesh size or average correlation length
(e.g. neutron and light scattering) or have not been made
quantitative (e.g. cryo-electron microscopy). We introduce a
new method, time-resolved multiple particle tracking, which makes use
of the distributions of Brownian displacements of microspheres imbedded
in the network. This approach monitors the spatio-temporal organization
of actin filaments in solution and quantifies the
time-dependent degree of heterogeneity in F-actin solution
undergoing gelation.
Preparation of Actin and F-actin-binding Proteins--
Unless
specified, all reagents were purchased from Sigma Chemical Co. (St.
Louis, MO). Actin was prepared from chicken breast using Sephacryl
S-300 for gel filtration (5, 6). Purified actin was stored as
Ca2+-actin in continuous dialysis at 4 °C against buffer
G (0.2 mM ATP, 0.5 mM dithiothreitol, 0.2 mM CaCl2, 1 mM sodium azide, and 2 mM Tris-HCl, pH 8.0). Mg2+-actin filaments were
generated by adding 0.1 volume of 10× KMEI (500 mM KCl, 10 mM MgCl2, 10 mM EGTA, 100 mM imidazole, pH 7.0) polymerizing salt to 0.9 volume of
G-actin in buffer G. Time-dependent Microheterogeneity via
Time-dependent Multiple Particle Tracking--
The
trajectories of fluorescent carboxylated microspheres imbedded in actin
solutions undergoing gelation were recorded using a silicon-intensified
target camera (VE-1000, Dage-MTI, Michigan City, IN) mounted on an
inverted epifluorescence microscope equipped with a 100× oil-immersion
objective (numerical aperture 1.3) (Nikon, Inc., Melville, NY) (9). The
centroid of each microsphere, defined as the intensity-weighted
center of gravity of the microsphere, was tracked with a resolution of
~5 nm, as determined by immobilizing microspheres on a glass
microslide using a strong adhesive and tracking their apparent
displacements (10). MPT tracks the movements of the probe microspheres
in the plane of focus of the microscope objective (i.e.
two-dimensional tracking). Two-dimensional tracking assumes that the
probed material is isotropic (11) but not necessarily homogenous.
Microspheres that are slightly out of focus may appear larger than they
are, but this is inconsequential, because we do not measure the (known)
size of the particles only the displacements of their centroids. Fields
of view were selected at random at each probed time during the course
of gelation. Despite the fact that MPT can track hundreds of particles
simultaneously, the number of tracked particles per field of view was
chosen to be low (between 5 and 20) to avoid particle-particle
interactions mediated by the filaments. For each tested actin
concentration, the movements of a total of 110-140 microspheres were
tracked, which necessitated 5-7 specimens per tested concentration
and gelation time. Images of the microspheres were captured at a
frequency of 30 Hz for 20 s, initially every 8 min for ~1.5 h
followed by 15 min for ~2.5 h, using a custom routine incorporated to
the software Metamorph (Universal Imaging Corp., West Chester, PA).
Mean squared displacements of individual particles were computed from
the microsphere trajectories (12) then transformed into local
compliance as described previously (13) (see "Results"). Surface
effects between the probe microsphere and the filaments may cause the
local network moduli to be underestimated by particle tracking (14)
without affecting measurements of the network heterogeneity. The
compliance distributions were statistically analyzed by their moments
and bin distributions as described previously (15, 16). We verified
that our MPT measurements of network compliance distribution were
independent of the size of the microspheres for diameters > 0.2 µm (data now shown). We observed little sedimentation of the 1-µm
microspheres primarily used in our experiments. All MPT measurements
were conducted at room temperature.
Gelation Kinetics via Time-resolved Mechanical
Rheometry--
The rate and extent of gelation of F-actin networks
were probed using a strain-controlled cone-and-plate rheometer
(ARES-100, Rheometrics, Piscataway, NJ) as described (17, 18). The
lower plate of the rheometer is coupled to a computer-controlled motor, which applies small oscillatory shear deformations of controlled frequency and amplitude. The upper cone is connected to a torque transducer, which measures the stress induced by shear deformations within the F-actin specimen, maintained here at room temperature. We
report the time-dependent in-phase component of the stress divided by the amplitude of the applied oscillatory deformation of
fixed frequency, i.e. the storage (or elastic) modulus,
G'(t), where t is the elapsed time
after initiation of actin polymerization. Each G-actin solution was
mixed with a one-tenth volume of 10× KMEI and immediately loaded onto
the lower plate of the rheometer using a micropipette with a cut-off
tip to limit filament breakage. The dead time between the time of
protein mixing and data collection was constant and equal to 30 s
for all experiments. For each specimen, F-actin solution gelation was
measured by recording G' by application of two cycles of
(small) shear deformation of 1% amplitude and fixed frequency of 1 rad/s, every 30 s for 6 h. We verified that these repeated
deformations did not affect the kinetics of gelation by applying
1%-amplitude shear deformations every 10 min for 6 h (data not
shown). Evaporation of the specimen was avoided by using a vapor trap
placed around the lower and upper tools; no apparent evaporation was
observed compared with the control without the trap, for which about
one-fourth of the sample had evaporated after 6 h. Using the
strain-controlled rheometer, we also measured the
frequency-dependent elastic modulus G'( Actin Polymerization via Time-resolved Fluorescence
Spectroscopy--
To monitor actin assembly, actin was labeled with
N-(1-pyrenyl)iodoacetamide as described (19, 20), which
produced 80 mol% pyrene-labeled actin. For each fluorometric
measurement, a 0.7-ml mixture containing 90% unlabeled actin and 10%
pyrene actin was used. The actin polymerization assay was conducted in an LS-50S luminance spectrophotometer (PerkinElmer Life Sciences, Shelton, CT). The excitation wavelength was set at 365 nm; the emission was monitored at 407 nm for 2 h at room temperature
after initiation of actin polymerization.
Homogenization of F-actin Solutions as Measured by Time-resolved
Multiple Particle Tracking--
We hypothesized that the slow increase
in elasticity displayed by an F-actin solution undergoing gelation is
due to the slow establishment of a homogeneous network (i.e.
constant mesh size), not to the rate of monomer assembly into
filaments. To test this model of actin gelation, we monitored the rate
and extent of microheterogeneity in F-actin solutions using
time-resolved multiple particle tracking. Fluorescently labeled, 1-µm
diameter microspheres were suspended in 3 µM, 10 µM, and 24 µM G-actin solutions (Fig.
1A), where polymerization was
initiated by addition of KMEI salt (see "Materials and Methods"). The Brownian trajectories of the centroids of a total ~130 particles per actin concentration were recorded with 5-nm spatial resolution and
30-Hz temporal resolution, for 20 s every 8 min for 1.5 h, then every 15 min for 2.5 h (Fig. 1). Typical trajectories of microspheres imbedded in actin solutions of increasing concentration and in an F-actin solution at different times during the gelation process are shown in Fig. 1. As expected, the range of the
displacements was reduced by increasing actin concentration (Fig.
1C), i.e. the local compliance of the network
decreased for increasing actin concentration. The extent of these
movements decreased (slightly) with gelation time (Fig. 1D).
As described next, these trajectories serve as raw data to analyze the
degree of heterogeneity of actin filament networks undergoing
gelation.
To quantify the microheterogeneity of F-actin networks, individual mean
squared displacements (MSD),
Distributions of the local network compliance at different time scales
were generated from measured MSDs (Fig. 2, C and
D) and statistically analyzed in terms of bin distributions
(see below). We found that the local compliance of a 10 µM actin solution showed a relatively wide distribution
during a short time after initiation of actin polymerization (Fig.
2C). At long elapsed times, the distribution in compliance
became progressively narrower and more symmetric (Fig. 2D),
a qualitative effect that can be quantified using bin distributions
(see below). This change of the compliance distribution was
particularly pronounced at long time scales (Fig. 2, compare
C and D with their respective insets), presumably due to the decrease in the extent of heterogeneity in
F-actin gels after a long equilibration time (see more in
"Discussion").
To determine the degree of heterogeneity in the physical properties of
F-actin solutions undergoing gelation, compliance distributions were
statistically analyzed by computing time-dependent means and bin distributions. As expected, the network stiffened during gelation, as detected by the decrease of the ensemble-averaged compliance, which is obtained by averaging all (~130) values of local
compliance at a given gelation time for each tested actin solution
(Fig. 3, A and B).
Directly comparing statistical parameters of distributions
(i.e. standard deviation, skewness, kurtosis) that encompass
different values can be somewhat misleading, we therefore opted to
report the "bin partition" of compliance values, which is
insensitive to the actual values in the distribution. Bin partitions of
the compliance distributions were obtained by comparing the
contributions of the 10%, 25%, and 50% highest values of the local
compliance to the mean compliance for each tested actin concentration
and at different points in time during gelation. The fractional
contributions of the highest 10%, 25%, and 50% local compliance to
the mean compliance should be close to unity for a highly heterogeneous
solution and close to 0.1, 0.25, and 0.50 for a perfectly homogeneous
solution; these parameters therefore describe the degree of
heterogeneity in compliance of the F-actin network. We verified that
these parameters were indeed close to their homogenous values for
perfectly homogenous glycerol solutions (Fig.
4A) and were close to unity
for a highly heterogeneous solution (as qualitatively assessed by
confocal microscopy) containing both fascin-mediated actin bundles and
Actin Polymerization and Gelation Kinetics: Effect of Actin
Polymerization and F-actin Cross-linking Proteins
It is often assumed that the rate of gelation, which describes the
kinetics at which a steady-state elasticity is reached, can be enhanced
by the presence of filament cross-linkers. We tested this hypothesis by
polymerizing actin in the presence of the prototypical F-actin
cross-linker chicken Network Homogenization Controls the Rate of Gelation of F-actin
Networks--
The sol-gel transition of the actin cytoskeleton is
believed to be one of the necessary steps in cell locomotion (1, 2, 23). Yet the fundamental mechanism of F-actin network gelation is not
well understood because rates of gelation and actin polymerization occur at completely different time scales. This large time scale discrepancy means that the rate at which the elasticity increases in an
F-actin network undergoing gelation is not governed by the onset of
topological overlaps between filaments, which occurs as soon as actin
filaments have polymerized, at least at the tested actin
concentrations. Indeed, as soon as filaments are formed, they will
overlap even at concentrations as low as 3 µM, due to the
fact that F-actin is a semiflexible polymer (24). To the best of our
knowledge, the concentration dependence of the rate of gelation has not
been previously reported. We note, however, that the rate of gelation
measured at 24 µM was slightly lower than that measured
by Janmey et al. (25), but the actin used in those
experiments is now known to display thiol oxidation, which artificially
enhances the elasticity of the F-actin network. Our measured rates of
actin polymerization were in quantitative agreement with results
obtained by many groups (20, 26). We also note that precautions were
taken to eliminate possible artifacts due to presence of capping
proteins (which would accelerate actin polymerization) (27) and
enhanced interactions between filaments due to thiol oxidation (25). In
particular, the actin used in our experiments was doubly gel-filtered,
was continuously dialyzed against buffer G for storage, was used within
2 days of purification, and was never frozen (28). Therefore, gelation
kinetics does not seem to correspond to the onset of topological
overlaps in the network during actin polymerization and after actin
polymerization has occurred.
Based on our results, our working model to explain the slow rate of
F-actin network gelation (despite rapid actin polymerization) is that
filaments take a long time to form a homogeneous network. The large
pores initially formed at random in the network greatly reduce the
overall elasticity of the network during the early phase of gelation.
We speculate that actin filaments progressively move into those large
network pores to gain conformational entropy, a process that is slow
because actin filaments are long and semiflexible. Note that the
commonly used falling-ball assay, which measures the viscous friction
generated by a heavy bead moving down a tube of polymerizing actin,
cannot be used to monitor actin polymerization, because it effectively
probes F-actin network gelation. To investigate the degree of network
heterogeneity during F-actin gelation, we introduced a novel method,
time-resolved multiple particle tracking, that monitors the dispersion
of displacements of microspheres imbedded in the network. These new
measurements, which were complemented with traditional rheological and
fluorescence measurements, suggest that the rate of network
homogenization, not the extent of polymerization, controls the kinetics
of F-actin network gelation. In support of this model of gelation, we
found that the rate of network homogenization and the rate of gelation
decrease both with actin concentration while the rate of polymerization
increases with concentration and the degree of heterogeneity increases
with actin concentration. We also found that the rate of gelation and
rate of homogenization are comparable.
We suggest that the rate of homogenization decreases with actin
concentration, because filaments, which are quickly formed, take an
increasing time to diffuse toward large pores. That time increases
partly because the mesh size of the network is smaller at high actin
concentration (29) and because polymer transport becomes slow and
sub-diffusive in a progressively more congested network (30, 31). We
note that the characteristic time for network homogenization to set in
(Fig. 4C) is of the same magnitude as the relaxation time of
filaments in an entangled solution (32). This relaxation time is the
diffusion time required for a filament to move approximately its own
length (33). We shall test our hypothesis that polymer diffusion is a
mechanism of network homogenization in future studies. Can other slow
processes, including filament annealing and fragmentation and ATP
hydrolysis, be responsible for the slow homogenization of F-actin
networks (34, 35)? We think that filament fragmentation and annealing
are unlikely to play a major role in setting the pace for F-actin
gelation, because polymer length has no effect on the overall
elasticity of a semidilute solution of semiflexible polymers according
to well-accepted models of polymer physics (36, 37). This is due to the
fact that polymer length does not affect the mesh size and, therefore,
the modulus of polymer networks (30, 33). Moreover, the rate of
filament annealing increases with actin concentration, whereas the rate
of gelation decreases with actin concentration. Finally, ATP hydrolysis
of F-actin has been shown not to affect the mechanical properties and
ultrastructure of actin filaments (38).
Why F-actin Cross-linking/Bundling Proteins Do Not Enhance the Rate
of Gelation of F-actin Solutions--
One may speculate that the rate
of gelation could be enhanced by gelation factors such as F-actin
cross-linking and bundling proteins. Using mechanical rheometry, we
measured the rate of gelation of F-actin solutions in the presence of
different types of ubiquitous F-actin binding proteins. In the presence
of the prototypical F-actin cross-linking protein Implications for the Cell--
The physical state of
the actin network is clearly more complex in vivo than that
of reconstituted actin network systems. Nevertheless, insight into
possible mechanisms of actin gelation in vivo can be derived
from our results in vitro. The observed slow rate of actin
gelation in the presence/absence of actin cross-linking proteins
implies that either the cell requires levels of elasticity that are
lower than those obtained at steady state with cross-linking/bundling proteins or that F-actin networks are homogenized by another activity of F-actin binding proteins. A good candidate to rapidly form homogeneous and stiff F-actin structures, when and where needed (for
instance, during cell migration at the leading edge), is the protein
complex Arp2/3. The complex Arp2/3 nucleates dendritic actin structures
at the leading edge of migratory cells (40). Cryo-electron microscopy
of detergent-extracted lamella shows that the Arp2/3-rich cytoskeleton
at the leading edge of locomotive keratocytes is indeed remarkably
uniform (41, 42). The cytoskeleton becomes, however, more heterogeneous
in the perinuclear region and in the myosin/
Our rheological measurements show that increasing concentrations of
actin slightly slow down gelation kinetics. This results suggests that
passive entropy-driven diffusion of filaments into the large pores of
the meshwork or other passive effects such as annealing and breaking of
filaments that may influence the rate of homogenization are too slow to
sustain fast morphological changes of the cell. The homogenization of
the actin cytoskeleton by passive filament diffusion could to be
complemented by an activated process. This process could be initiated
by motor proteins such as myosin, which would bias and enhance the
transport of filaments into large meshwork pores (44). Large pores
could also be "filled in" by localized polymerization of actin via
nucleators such as the Arp2/3 complex and/or by the activation of
F-actin severing/nucleating proteins such as gelsolin. Using the method
of time-resolved multiple particle tracking introduced in this report,
we are currently measuring the rate of gelation and ultrastructural
homogenization of F-actin networks in the presence of myosin, the
Arp2/3 complex, and gelsolin.
We believe that the versatility of the novel method presented in this
report, time-resolved multiple particle tracking, will help shed new
light on multiple problems in the biological sciences, including the
determination of the local micromechanical properties of cells during
cell locomotion and chemotactic migration, and materials science,
including the local microstructure of complex fluids undergoing a glass
transition or gelation.
We thank David Sept for helpful discussions.
*
This work was supported by National Science Foundation Grant
CTS007227.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, March 11, 2002, DOI 10.1074/jbc.M110868200
The abbreviations used are:
G-actin, globular
actin;
F-actin, filamentous actin;
MPT, multiple particle tracking;
MSD, mean-squared displacement.
Microheterogeneity Controls the Rate of Gelation of Actin
Filament Networks*
§,, and§¶
Department of Chemical Engineering,
§ Program in Molecular Biophysics, ¶ Department of
Materials Science and Engineering, The Johns Hopkins University,
Baltimore, Maryland 21218
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actinin and fascin, whereas the rate of actin polymerization increases dramatically with actin concentration. Our measurements show
that the slow spatial homogenization of the actin filament network, not
actin polymerization or the formation of polymer overlaps, is the
rate-limiting step in the establishment of an elastic actin network and
suggest that a new activity of F-actin binding proteins may be required
for the rapid formation of a homogeneous stiff gel.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Actinin was purified from chicken gizzard (7,
8). Human fascin was expressed as a glutathione
S-transferase fusion in Escherichia coli; the
fusion protein was dialyzed against phosphate-buffered saline/dithiothreitol and liberated from glutathione
S-transferase by cleavage with thrombin, followed by
glutathione-Sepharose chromatography (6). Fascin and -actinin were
used within 5 days of purification.
) over
the frequency range of 0.5 rad/s-100 rad/s. The collection of each
frequency spectrum took about 2 min, a time much shorter than
characteristic times of gelation. Such a spectrum was collected every
10 min until a steady-state frequency profile was reached (see
"Results").
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Principle of multiple particle tracking
(MPT) measurements in F-actin solutions. A, randomly
selected frame of a movie of fluorescently labeled carboxylated
1-µm diameter polystyrene microspheres dispersed in a 3 µM actin solution in the presence of the polymerizing
salt KMEI (see "Materials and Methods"). The concentric squares
around each microsphere allow the MPT software to continuously identify
the particles during movie capture. Their sizes are initially set
manually. The centroid of each microsphere is video-tracked with 5 nm
of spatial resolution; a field of view of 120 µm × 120 µm is
monitored for 20 s every 8 min for 1.5 h and then every 15 min for 2.5 h. B, 20-s trajectories of microspheres
(shown in A) collected 30 min after initiation of
polymerization. C, detail of a typical trajectory of a
microsphere placed in a 3, 10, and 24 µM F-actin
solution, 194 min after initiation of polymerization. D and
E, detail of a 20-s displacement of a microsphere dispersed
in a 10 µM F-actin solution collected 6 and 240 min after
onset of polymerization.
r2(
)
,
of microspheres distributed throughout the solution were computed from
the microspheres' time-dependent two-dimensional coordinates, which were then transformed into local
time-dependent compliance values, 
(). We have indeed
previously proved that the MSD is directly proportional to the (local)
compliance of the network produced by the small random forces of order
kBT/a created by the
thermally driven fluctuations of the particles (13). The
time-dependent compliance, (), is related to the measured MSD by () = (
a/kBT)r2(),
where kBT is the thermal energy and
a is the radius of the microsphere (13). A liquid of shear
viscosity 
subjected to a constant (small) stress displays a creep
compliance that increases linearly with time, () = /;
a Hookean solid of modulus G0 under stress
displays a time-independent compliance = 1/G0; the time-dependent compliance
of a viscoelastic system such as an F-actin network shows an
intermediate behavior (13). The magnitude and time dependence of the
compliance traces (Fig. 2, A
and B) show that the actin network at early times is softer (i.e. more compliant, more deformable) and more viscous-like
(i.e. the slope of is higher) than at later times during
the gelation process.
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Fig. 2.
Time-resolved MPT measurements in actin
filament networks undergoing gelation. Typical compliance traces
( ) of the network in the vicinity of microspheres dispersed in a 10 µM F-actin solution. Compliance traces obtained
(A) 6 min and (B) 240 min after addition of
polymerizing salt to the G-actin solutions. The vertical
lines in A and B show where distributions in
C and D were collected. C and
D, compliance distributions (n ~ 130)
collected at a time lag of 0.1 s. Insets: associated
distributions at a time lag of 1.0 s.
-actinin-cross-linked filaments (15). During gelation, contributions
from the highest compliances to the ensemble-averaged compliance
decreased toward values close to the theoretical values for homogeneous
networks (Fig. 4, A-D). Higher actin concentrations
typically produced values of these homogeneity parameters that were
higher than obtained at low concentrations (compare Fig. 4,
B and D), i.e. concentrated F-actin
solutions were more heterogeneous than dilute solutions. Moreover,
these parameters took longer to decrease toward their homogeneous
values in concentrated actin solutions, i.e. the rate of
network homogenization decreased with actin concentration. Therefore,
the degree of heterogeneity decreases during gelation, a homogenization
process that is slowed down by high actin concentrations.
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Fig. 3.
Ensemble-averaged network compliance.
A, time lag-dependent mean compliance
(ensemble-averaged ) in 3 and 24 µM actin solutions
measured 6 min (upper curve), 1 h (intermediate
curve), and 4 h (lower curve) after initiation of
actin polymerization. The last two curves are superimposed.
B, evolution of the ensemble-averaged evaluated at a
time lag of 0.1 s during solution gelation.
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Fig. 4.
Statistical analysis of network compliance
( ) distributions. A, fractional contributions of the
highest 10%, 25%, and 50% values of local compliance to the mean
compliance in a glycerol solution measured at a time scale of 0.1 s (first columns) and 1.0 s (second
columns). These contributions are close to the expected values for
a perfectly homogeneous solution, i.e. 0.10, 0.25, and 0.50. B-D, time-dependent contributions
(defined as fractions of unity) of the highest 10, 25, and 50%
compliance values measured at a time scale of 1 s to the mean
compliance in B, 3 µM; C, 10 µM; and D, 24 µM actin
solutions.
-Actinin and
Fascin--
Time-resolved multiple particle tracking measurements were
complemented by traditional fluorescence and rheological measurements to directly compare the rates of homogenization of actin solution to
the rates of actin polymerization and rates of gelation. Gelation kinetics, as detected by the time-dependent elasticity
G'(t), where t is the elapsed time
after initiation of actin polymerization and which is measured at a
fixed frequency of 1 rad/s, did not follow a simple exponential
behavior (Fig. 5A). Therefore,
the rate of gelation was conveniently defined as the inverse of the time necessary to reach 90% of the steady-state value of G'
(averaged here over 10 min). The rate of gelation measured by rheology
slightly decreased with actin concentration (Fig. 5B). We
monitored the propensity of actin filaments to diffuse in solutions
during gelation by probing the frequency dependence of the elastic
modulus, G'() every 10 min for 6 h. Each frequency
sweep took ~2 min, a time much shorter than characteristic times of
gelation (as shown in Fig. 5B). Moreover, we verified that
the gelation process (i.e. extent and rate of gelation) were
independent of the rate of data acquisition, i.e. subjecting
the network to (small) deformations did not affect its gelation process
(see "Materials and Methods"). At the beginning of gelation,
G'() values were low at low frequencies and elevated at
high frequencies, which reflects a relatively high degree of mobility
of the filaments in the network (Fig. 5C), presumably due to
the presence of large heterogeneities at early times. This steep
dependence on frequency declined progressively before reaching a
steady-state frequency-dependent profile (Fig. 5C). The elastic modulus remained relatively unchanged at
high frequencies, but greatly increased at low frequencies (Fig.
5C). These measurements therefore corroborate the MPT
measurements, which showed that compliance profiles displayed a steep
time scale dependence at early times during gelation and less time
scale dependence at later times (Fig. 2, A and
B).
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Fig. 5.
F-actin polymerization and solution gelation:
Effect of F-actin cross-linking proteins. A,
time-dependent elasticity G'(t)
measured at a fixed frequency of 1 rad/s and deformation amplitude of
1%. Closed arrows indicate when actin polymerization has
reached a steady state (times taken from E); open
arrows indicate when G' has reached 90% of its
steady-state value. Note the increasing time distance between gelation
times and polymerization times for increasing actin concentration.
B, concentration-dependent gelation rate of
F-actin solution as probed by rheometry. These rates correspond to the
inverse of the time required to reach 90% of the steady-state
elasticity (see "Materials and Methods"). Error bars
correspond to variations around the mean time required to reach
steady-state values. C, gelation time-dependent,
frequency-dependent elasticity, G'( ), of a 24 µM actin solution undergoing gelation. G'()
was measured at a fixed deformation amplitude of 1% at the gelation
times indicated in the figure. D, rate of gelation of 24 µM actin solutions in the presence of the F-actin
bundling protein fascin and the F-actin cross-linking/bundling protein
-actinin divided by the rate of gelation of a 24 µM
F-actin solution (= 7.7 × 10
2 min1).
E, fluorescence intensity increase due to F-actin assembly
using the pyrene assay (see "Materials and Methods"). F,
concentration dependence of the rate of fluorescence intensity
increase.
-actinin (15, 21, 22) and the F-actin bundling
protein human fascin (6, 10). We found that the rate of gelation as
probed by rheology changed little or slightly decreased in the presence
of fascin and -actinin (Fig. 5D). In contrast, the pyrene
assay showed that the rate of actin assembly was relatively insensitive
to the presence of -actinin (data not shown), but greatly increased with actin concentration (Fig. 5, E and F). We
observed that the polymerization rates were much higher than the rates
of gelation (Fig. 5B) and the rates of network
homogenization (Fig. 4). The difference between the rate of gelation
and the rate of network homogenization was enhanced at high actin
concentrations as indicated by the increasing time distance between
arrows in Fig. 5a, which show the times at which
actin polymerization and actin gelation have reached 90% of their
steady-state values.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actinin, we found that the rate of gelation was, somewhat surprisingly, decreased at low
molar ratios of -actinin to actin and became constant at high molar
ratios (see also Refs. 6, 15). Although -actinin greatly increased
the final steady-state values of the elasticity of F-actin, it did not
enhance the rate to reach that steady-state value (39). A similar
effect was observed in the presence of the prototypical F-actin
bundling protein fascin (6), which promotes the parallel arrangement of
actin filaments: The rate of gelation of F-actin slightly decreased at
low fascin concentrations and became constant at high concentrations.
These results suggest that actin cross-linking/bundling proteins are
not necessarily good candidates for gelation factors. This result is
readily explained by recent steady-state multiple particle tracking
measurements. Both fascin and -actinin were shown to greatly enhance
the degree of heterogeneity of F-actin networks (10, 15) even a long time (~6 h) after initiation of actin polymerization. We think that
two conflicting effects are therefore at work when F-actin cross-linking/bundling proteins are present. Actin
cross-linking/bundling proteins decrease the propensity for actin
filaments to move in solution, which increases the overall network
stiffness, but also slows down network homogenization.
-actinin-rich lamella.
Our recent local micromechanical measurements in situ also
show that the elasticity at the edge of the cell is much higher than in
the perinuclear region (43). Arp2/3 would therefore not only help
provide the cell with some of the propulsive forces for its locomotion
as suggested by earlier work (3) but also structurally support new
protrusions more effectively and more rapidly than conventional F-actin
cross-linkers such as -actinin.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed. Tel.:
410-516-7006; Fax: 410-516-5510; E-mail: wirtz@jhu.edu.
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