Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA
* Author for correspondence (e-mail: Knecht{at}UCONN.edu)
Accepted 27 May 2003
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Summary |
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Key words: Chemotaxis, Myosin, Force, Deformation, Under-agarose assay, Folate, Dictyostelium, Actin cross-linking
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Introduction |
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The organization of these actin filaments into functional arrays and the
dynamics of these arrays is also not well understood. To date, more than
twenty actin-binding proteins have been discovered in D. discoideum.
Among these are a number of actin filament cross-linking proteins, including
ABP120 (Condeelis et al.,
1981),
-actinin
(Fechheimer et al., 1982
),
fimbrin (Prassler et al.,
1997
), cortexillins I and II
(Faix et al., 1996
), and a 34
kDa protein (Fechheimer and Taylor,
1984
) that presumably provide rigidity to the cortex. Why the cell
needs so many different actin cross-linking proteins, and what specific roles
each plays in processes that involve rearrangements of the actin cytoskeleton
such as chemotaxis, cytokinesis, endocytosis and phagocytosis, is unclear.
The myosin motors also play a major role in cytoskeletal function.
Non-muscle myosin II assembles into minifilaments, and these filaments are
able to apply force to move actin filaments relative to each other
(Clarke and Spudich, 1974;
Hynes et al., 1987
;
Sheetz et al., 1986
). Myosin
II minifilament assembly is regulated by the phosphorylation state of the
heavy chain tail (Egelhoff et al.,
1993
) and the motor activity is stimulated by phosphorylation of
the regulatory light chains (Griffith et
al., 1987
). The essential light chain appears necessary for myosin
motor function since myosin lacking this protein assembles minifilaments and
binds actin, but has no measurable actin activated ATPase activity
(Chen et al., 1995
;
Xu et al., 1996
). While not
generally thought of as an actin cross-linking protein, myosin II
minifilaments presumably also have this capability
(Wachsstock et al., 1994
;
Humphrey et al., 2002
).
Mutants lacking myosin II (mhcA-) are able to accomplish
both random and chemotactic motility; however, they move slowly and have
defects in pseudopod extension (Peters et
al., 1988
; Wessels et al.,
1988
). Although mhcA- cells are able to
aggregate, they are unable to complete the developmental program
(Knecht and Loomis, 1988
).
The developmental defect of mhcA- cells appears to be
due to their inability to move in a restrictive environment
(Shelden and Knecht, 1995).
During early development, cells acquire surface adhesion proteins and so
movement occurs while cells are continually making and breaking adhesive
contacts with their neighbors as well as the substratum. Unlike movement on a
planar substratum, this form of motility is analogous to the movement of
metastatic cancer cells away from a primary tumor, or the extravasation of
immune cells through capillaries and into a wound site. It requires cells to
overcome a barrier of resistance to their movement. Movement in restrictive
conditions is also important for Dictyostelium development. Ponte et
al. showed that while development of actin-binding protein mutants on agar
plates is normal, development on soil plates is defective
(Ponte et al., 2000
). Soil is
presumably a more restrictive environment for cell motility than a planar agar
surface. Myosin II seems to be essential for this multidimensional process of
migration, apparently by providing cortical integrity, since the
mhcA- cells became stretched and distorted when attempting
to move in aggregation streams (Shelden
and Knecht, 1995
).
Surprisingly, cells lacking the essential light chain
(mlcE-) behave normally in this environment indicating
that the motor activity of myosin is not required for motility in restrictive
conditions (Xu et al., 2001).
Since the environment of aggregation streams is so complex, we sought to
develop a simpler and more versatile means by which cell motility in a
restrictive environment could be investigated. An under-agarose folate
chemotaxis assay has been developed in which cells are induced to move between
a planar substratum (glass or plastic) and a layer of deformable agarose of
varying stiffness (Laevsky and Knecht,
2001
). Using this system, we have investigated the movement of
cells lacking specific cytoskeletal proteins. Consistent with our previous
results, it appears that the actin binding activity of myosin II, and not the
motor activity, is required for movement and cortical stability in this
restrictive environment. None of the other actin cross-linkers tested have as
major a role in this process as myosin II.
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Materials and Methods |
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Under-agarose assay
The under-agarose assay was performed as described previously with minor
modifications (Laevsky and Knecht,
2001). 14 ml of SeaKem® GTG agarose (BMA, Rockland, ME, USA),
made with SM medium (Sussman and Sussman,
1967
), was poured into 100 mm plastic Petri dishes. The agarose
was allowed to solidify for 1 hour at 22°C. Three 2 mm wide troughs were
cut 5 mm apart with a standard razor blade (4 cm length) using a template
(Fig. 1). 100 µl of 0.1 mM
folic acid (Research Organics, Cleveland, OH, USA) was added to the center
trough and allowed to form a gradient for 1 hour at room temperature. Cells
were harvested, adjusted to 1x106 cells/ml for individual
analysis and 1x107 cells/ml for population analysis. 100
µl of cell suspension was then added to the peripheral troughs.
|
Analysis of cell movement
Images were taken of the cell populations using a Zeiss® IM inverted
microscope (Carl Zeiss, Oberkochen, Germany), Paultek Imaging Inc. CCD camera
(Advanced Imaging Concepts, Princeton, NJ, USA), Scion Inc. LG3 frame grabber
(MVI, Avon, MA, USA) and NIH Image software (developed at the US National
Institutes of Health and available on the Internet at
http://rsb.info.nih.gov/nih-image/).
Overall distance traveled by cells under-agarose was determined by measuring
the average distance the ten front most cells in a field of view were from the
trough edge, approximately 3 hours after the cells were applied to the trough.
The analysis of the movement of mhcA- cells was done near
the trough edge as soon as they could be seen to have moved underneath the
agarose in order to examine cells prior to stretching and fragmentation.
Individual cell speed and direction change was determined using DIAS®
software (Solltech, Oakdale, IA, USA). Speed was calculated using the
displacement of the centroid from frame to frame during 1-minute intervals.
Direction change was measured as the absolute value of the difference in the
direction of movement of the centroid from frame to frame, measured in
degrees. Cross sectional area measurements were made using NIH Image software.
The cross sectional area is measured as the area of the image of a cell seen
using phase contrast microscopy.
Fluorescence imaging
For fluorescence imaging experiments, 0.75 ml of agarose was added to a
Rose chamber (Rose et al.,
1958), or 4 ml to a 60 mm glass bottom Petri dish (Willco Wells,
Amsterdam, Netherlands) so that cells could be imaged through a 0.17 mm thick
glass coverslip. Two troughs were cut in the Rose chamber with a 10 mm long
razor blade, and the amount of cells and folate was decreased proportionally.
Confocal imaging of GFP-labeled cells was performed using a Leica TCS SP2
confocal microscope system (Leica Microsystems, Heidelberg, Germany) and an
MRC 600 (Bio-Rad Laboratories, Hercules, CA, USA) equipped with a 25 mW
krypton-argon laser and COMOS software.
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Results |
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Cells lacking myosin II (mhcA-) are able to move on a
liquid covered planar surface at rates about one third of wild-type cells
(Wessels et al., 1988).
However, these mutants are unable to penetrate aggregation streams, which are
presumed to be a viscous restrictive environment
(Clow and McNally, 1999
;
Shelden and Knecht, 1995
). In
order to examine more directly whether this defect is the result of their
inability to move in a restrictive environment, the under-agarose chemotaxis
assay was used to examine the motility of the mhcA- cells.
In 0.5% agarose, wild-type cells moved relatively freely out of the troughs
within 1 hour and continued to do so over the next 9 hours reaching a distance
of about 3000 µm from the trough
(Laevsky and Knecht, 2001
)
(Fig. 2). In contrast, few
mhcA- moved out of the trough, and those that did never
migrated more than 500 µm from the trough
(Fig. 2). Because of this, the
movement of individual mhcA- cells was measured near the
trough edge soon after exit. The speed of these cells was about two thirds of
wild-type cell speed and their movement was directed toward the folate trough
(Table 1). At concentrations of
agarose 1% or above, the mhcA- cells did not move out of
the troughs at all (Figs 2 and
3). In order to confirm that
the inhibition of mhcA- movement was due to the stiffness
of the agarose and not the adherence of the agarose to the plastic dish, the
same experiment was performed except that the agarose layer was either rotated
180° or lifted out and placed in a fresh dish prior to cutting the
troughs. The same results were obtained when the agarose was freed from the
surface in this way (data not shown) indicating that it is the local
deformation of the agarose and not the adhesion of the agarose to the surface
that inhibits the movement of mhcA- cells.
|
|
Effects of agarose overlay on mhcA-
morphology
When moving under 0.5% agarose, the mhcA- cells did not
move as far or as fast as the wild-type cells, however, the most unusual
aspect of their behavior was the dramatic elongation of the cells as they
attempted to move (Figs 3,
4,
5). This behavior is
reminiscent of mhcA- cells moving in wild-type aggregation
streams using the chimeric aggregation assay
(Shelden and Knecht, 1995;
Xu et al., 1996
) (see
Discussion). However, it was difficult in that assay to pinpoint the precise
cause of the stretching. In the under-agarose assay, the stretched appearance
of cells was found to be due to a failure of retraction of the rear of the
cell body. Dictyostelium cells do not normally have a well-defined
uropod, but this process generated a structure resembling the uropod of
mammalian cells. Time-lapse analysis of cell movement indicated that the rear
of the cell would often become stuck to the surface while the cell body
continued to move. This uropod would eventually only be connected by a thin
bridge of cytoplasm and this bridge sometimes broke as the cell body moved
away (Fig. 4). Even cells that
did not fragment ceased moving about 500 µm from the trough edge
(Fig. 2). The posterior of
wild-type cells is enriched in F-actin as shown by the bright fluorescence of
the GFP-ABD120 probe in this region
(Laevsky and Knecht, 2001
).
The posterior of mhcA- cells is also enriched in F-actin
and the probe is concentrated in the cytoplasts released from
mhcA- cells (Fig.
5). This loss of cellular actin may account for the eventual
cessation of movement by these cells (Fig.
5). However, it is interesting to note that cells that do not
fragment also eventually stop moving about 500 µm from the trough edge.
|
|
Previously, we showed that increasing the concentration of agarose results
in an increased surface area of wild-type cells, indicating that the cell is
less able to deform the agarose upward and becomes more compressed and
flattened as a result (Laevsky and Knecht,
2001). If the cortex of the mhcA- cells were
flaccid, one would expect that they would have a greater surface area than
wild-type cells at the same agarose concentration. The elongated appearance of
the mhcA- cells makes this comparison more complicated,
however, the mhcA- cells that were able to move under 0.5%
agarose did have a significantly greater surface area than wild-type cells
(Table 1).
There are two possible reasons why the mhcA- cells might not be able to exit the troughs at high agarose concentrations. The first possibility relates to their behavior when moving under 0.5% agarose. The stretching and fragmentation indicates that the cells have trouble releasing and retracting their uropods. While this is seen to some extent when mhcA- cells are moving in liquid media without an agarose overlay (D.A.K., unpublished observations), it is far more dramatic under agarose. Thus it is possible that at 1% and higher agarose concentrations, this problem is magnified. In this scenario, the cells would not move beyond the trough edge because once they move the cell body underneath the agarose at the edge, they become trapped because they cannot retract their uropods and move any farther. If this were the case, we would see stretched cells at the very edge of the trough. The other possibility is that the cells are unable to deform the agarose upward and bring the midbody (the thickest part of the cell) underneath, indicating a weakness in the ability of the general cell cortex to deform the agarose. In order to distinguish between these possibilities, mhcA- cells were examined at the edge of the trough as they tried to move out under the agarose (Fig. 6). The cells moved to the edge of the trough, and then frequently extended pseudopods under the agarose sheet, but the cell body was never able to move underneath. However, the cells were able to withdraw the pseudopod and continue moving along the agarose interface. Stretched cells under the agarose at the edge were not observed indicating that uropod retraction was not the cause of the defect. This data indicates that the defect in mhcA- cells is in creating the force necessary to push the stiffer agarose out of the way and move the cell body underneath.
|
Essential light chain mutants move normally under agarose
Myosin II from cells lacking the essential light chain of myosin
(mlcE-) has actin-binding activity, but lacks ATPase motor
function (Chen et al., 1995;
Xu et al., 2001
). In the
chimeric aggregation assay, mlcE- cells moved normally and
did not become elongated like mhcA- cells
(Xu et al., 2001
).
MlcE- cells were tested in the under-agarose chemotaxis
assay to see if the motor function of myosin was necessary for movement in
this restrictive environment. Under all agarose concentrations tested,
mlcE- cells moved the same distance and at the same speed
as the control rescued cells in which the essential light chain was
reintroduced into the cells (Table
1). DIAS analysis of individual cell behavior indicated that the
rate of direction change was consistent with cells undergoing positive
chemotaxis, as opposed to cells moving randomly
(Table 1)
(Laevsky and Knecht, 2001
).
The mlcE- mutants maintained a surface area of about 198
µm2, similar to that of the control cells
(Fig. 2,
Table 1) and became comparably
flatter under 2.5% agarose. Morphologically, no obvious difference was seen
between the two cell lines when viewed under agarose
(Fig. 3).
Localization of F-actin and myosin II during under-agarose
chemotaxis
In order to determine if the localization of F-actin in
mlcE- cells was altered, the GFP-ABD120 probe was
introduced into the cells (Pang et al.,
1998). This probe dynamically associates with F-actin filaments in
live cells allowing visualization of the actin cortex. In both wild-type and
mlcE- cells moving under agarose, the probe localized to
an arc around the posterior and rear edge of the cell and transiently to new
protrusions at the leading edge (Fig.
7A,B). No significant difference in the localization of this probe
was observed in mlcE- cells.
|
Previous work has shown that myosin is distributed throughout the cortex in
cells in buffer or media, but when placed under agarose, it rapidly
relocalizes to the rear of the cell
(Neujahr et al., 1997;
Yumura et al., 1984
). In order
to examine the localization of myosin II during under-agarose chemotaxis,
wild-type and mlcE- cells, expressing myosin IIGFP were
examined. Confocal optical slices about 0.5 µm thick were acquired every 5
seconds at a point just above the surface of the coverslip. In both cell
types, myosin II is concentrated in an arc at the rear of cells undergoing
under-agarose chemotaxis (Fig.
7C,D). In addition to its prevalent localization in the rear,
myosin II is also found to transiently localize to small patches of the cortex
at the front of the cell. No significant differences in myosin-GFP
localization were observed between the wild-type and the
mlcE- cells during under-agarose motility.
In order to visualize the three-dimensional localization of myosin through the volume of the cell, 0.2 µm thick z-sections were acquired with the confocal microscope. The cells are about 4-5 µm thick under this condition, and actin (not shown) and myosin II are present in an arc or ring at the edge of the cell throughout much of this volume (Fig. 8). There is much less myosin near the dorsal surface of the cell. This conical wall of myosin (and actin) sometimes extends all the way around the cell (Fig. 8), but is frequently just in the rear half of the cortex as in the cells shown in Fig. 7. The only significant difference between wild-type and mlcE- cells was that the later frequently had small round dots of fluorescence in the rear of the cell, which is probably results from a disassembly defect in myosin lacking the essential light chain. We hypothesize that the cortical rim of acto-myosin is the structural element that allows the cell to resist the downward pressure of the agarose and move in this environment.
|
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Discussion |
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Our results indicate that myosin II is a surprisingly important player in
the maintenance of cortical integrity, especially when a cell is challenged to
move in a restrictive environment. Even more surprising is the finding that
this action of myosin II does not appear to require the normal contractile
activity. The most likely interpretation of this result is that ELC-myosin II
retains the ability to bind and cross-link actin filaments and thereby the
cortex, in addition to its ability to rearrange those filaments when called
upon to contract. This result is consistent with rheological measurements that
show that mixing myosin II with actin filaments in the presence of ADP can
dramatically stiffen the matrix (Humphrey
et al., 2002). How the cell might regulate this aspect of myosin
function is unknown, but a precedent exists in the latch state of smooth
muscle myosin where force production is not always directly linked to actin
binding (Sweeney, 1998
).
We, and others have previously shown that cells lacking myosin II are
unable to accomplish morphogenetic movements
(Clow and McNally, 1999;
Shelden and Knecht, 1995
). In
aggregation streams the mhcA- cells were unable to move
amidst the mass of adhered cells and became dramatically stretched as they
tried to make and break contacts with neighboring cells in this environment
(Shelden and Knecht, 1995
).
The defect was interpreted as a failure in cortical integrity, allowing cells
to be stretched abnormally by externally applied forces. Surprisingly, cells
lacking the essential light chains of myosin II behaved normally in this
chimeric aggregation assay (Xu et al.,
1996
). In the absence of the essential light chain, myosin is
found associated with the actin cortex, so presumably can bind actin, but
there is minimal actin-activated ATPase motor activity
(Chen et al., 1995
;
Xu et al., 1996
). This result
indicated that the motor activity of myosin II is not required for the
maintenance of cortical integrity.
We envision at least three distinct force-generating steps in movement
under agarose. First is the protrusive force at the leading lamella or
pseudopod causing forward movement of the leading edge. Because this part of
the cell is relatively thin, and there is a small space between the agarose
and the planar surface, the agarose concentrations we are using are probably
not especially inhibitory to this protrusion process. The second step is the
upward deformation of the agarose necessary to allow the thickest part of the
cell (the nuclear region) to squeeze underneath. This localized upward
deformation as the cell crawls was shown to occur by tracking the movement of
fluorescent beads embedded in the agarose
(Laevsky and Knecht, 2001). As
the agarose concentration is increased, it becomes more and more difficult for
the cells to deform the agarose, and so movement slows down and eventually
ceases around 3% agarose. The third step is the retraction of the rear of the
cell. Dictyostelium cells do not have well defined uropods, and
therefore it was not obvious that this would be separate from the
translocation of the cell body. However, the finding that cells lacking myosin
II have long trailing extensions of cytoplasm when moving under 0.5% agarose
indicates that the detachment of the rear of the cell is indeed a separate and
important issue in translocation. However, the stretching is not simply a
matter of increased surface adhesion. Jay et al. examined the movement of
mhcA- cells on surfaces of varying adhesiveness and did
not observe the uropods being left behind
(Jay et al., 1995
). Instead
the mhcA- cells were unable to move at all on sticky
surfaces that the wild-type could still crawl on.
The inability of the mhcA- mutant cells to move under concentrations of agarose above 0.5% is likely to be a different problem. In this situation, the cells still make protrusions under the agarose at the trough edge, but the cell body never flattens and continues up the gradient. Thus this is not a problem of rear retraction, since the cells never get the uropod underneath the agarose. A clue to understanding this phenotype, comes from visualization of the dynamic localization of GFP-myosin in these cells. Actin and myosin are not prominent in the ventral or dorsal cortex of cells under agarose, but are enriched in the peripheral cortex, either in the rear portion as an arc, or surrounding the cell (Figs 7 and 8). This vertical ridge of actomyosin is likely to be responsible for deforming the agarose upward and allowing the nucleus to fit underneath. In the absence of myosin II, we presume that the cortex does not have the stiffness to deform higher agarose concentrations, and so the nucleus cannot fit underneath and the cells are trapped at the trough edge.
The model in Fig. 9 explains
the events proposed to occur during protrusion and retraction events. The
crosshatching indicates the orthogonal network of actin filaments that lie
beneath the membrane. This network would be held together by actin binding
proteins, such as ABP-120, -actinin, cortexillin, talin and myosin II.
The wild-type, mlcE- and mhcA- cells
are able to extend protrusions under the agarose
(Fig. 9A,C). The linkage
between the pseudopod and the cell body in wild-type and
mlcE- cells is retained and the cell moves as an integral
unit under the agarose (Fig.
9B). The mhcA- cell
(Fig. 9D) is able to retract
the nuclear region under 0.5% agarose, but not under 2.5% agarose. At the
higher agarose concentrations, the cell apparently cannot produce sufficient
force to make further progress.
|
The implication of these results is that the cell cortex acts to integrate
the cell as a whole, and myosin II is crucial to integrating the actin cortex.
The surprising result is that normal contractile activity is not needed for
myosin II to carry out this function. It is possible that some contractile
activity below the limit of our assays is present in ELC-myosin, and this is
sufficient to allow myosin II to integrate the cortex. It has been determined
that Aspergillus nidulans myosin I mutants with less than 1% of
wild-type actin-activated MgATPase activity retain essential in vivo functions
(Liu et al., 2001). However,
we have shown that mlcE- cells cannot undergo contraction
of detergent extracted cortices, which would be a direct test of contractile
activity of myosin in situ (Xu et al.,
2001
). Another possibility is that because the actin-activated
ATPase activity is lost in the mlcE- mutant, this mutant
myosin has become a permanent actin cross-linker and this cross-linking
activity replaces the normal contractile activity of myosin. This is possible,
but it seems unlikely that such a dramatic change in function could allow
cells to behave so normally or would allow normal organization of the actin
filament network. We favor a third hypothesis, that as in smooth muscle,
non-muscle myosin II is not constitutively applying force to the actin
cytoskeleton, but can enter a state in which it is bound to actin like a
cross-linker, while not actively engaged in the ATPase cycle. Myosin II may
only be called upon to contract when the cell changes shape, as happens in
cytokinesis. The mlcE- mutation would allow the myosin II
to function in its cross-linking state, but not enter a contractile mode.
Our data indirectly indicates that the cortex of mhcA-
is less stiff than wild-type cells. Attempts have also been made to directly
measure the cortical integrity of cells using biophysical techniques. The
results are contradictory and confusing. Pasternak
(Pasternak et al., 1989)
showed only a slight decrease (32%) in the cortical stiffness of
mhcA- cells using a `cell poker' that measured the
resistance of the cell to inward deformation. Egelhoff
(Egelhoff et al., 1996
), using
a vibrating glass rod, measured a 50% decrease in cortical stiffness in myosin
II mutant cells. However, Merkel et al.
(Merkel et al., 2000
) used a
pipette aspiration system and found a dramatic increase in the resistance of
mhcA- cells to outward deformation from a suction pipette,
indicating a stiffer cortex in the myosin II mutants. Feneberg et al.
(Feneberg et al., 2001
) used a
microrheology technique based on colloidal magnetic tweezers to measure the
viscoelastic forces within the cytoplasm. They found the apparent viscosity of
myosin II null mutants was higher, also implying a stiffer cortex. Some of the
discrepancies may be the result of the methodologies used. Live cell imaging
of the actin cortex in cells containing the GFP-ABD120 probe shows that any
time a cell makes contact with an object (another cell, a bead or an
obstacle), there is a rapid accumulation of F-actin in the contact region
(D.A.K., unpublished observations). Thus, application of a pipette or poker
may lead to an actin polymerization response that will interfere with the
measurements. By using a biological assay, we have directly evaluated the
functionality of the cortex in what is to the cells, a relatively normal
environment.
ABP-120 and -actinin are, by mass, the two major actin cross-linking
proteins in the cell (Condeelis et al.,
1981
; Condeelis and Vahey,
1982
). Thus it is surprising that mutants lacking these proteins
had no altered phenotype in this assay. This result indicates that not only is
myosin II important for cortical integrity, but that so far, it is the single
most important protein providing this function. Clearly, cells lacking myosin
II have some cortical integrity or they would not be able to move at all. This
residual cortical integrity is presumably supplied by the myriad of other
actin cross-linkers or the rheological properties of actin filaments
themselves.
Our model is not intended to suggest that cells do not require the motor
activity of myosin II. Mutants lacking the essential light chain (and thus
motor activity) are unable to divide in suspension and have defects in
multicellular development (Chen et al.,
1995). In addition, we have previously shown that myosin II
contractile activity is needed for cells to generate shape in suspension or to
elongate vertically off a surface (Shelden
and Knecht, 1996
) and the essential light chain mutants are
defective in both functions (Xu et al.,
2001
). Our model, therefore, proposes that myosin plays a major
role in maintaining the physical integrity of the actin cortex, and that its
function can be separated into contractile and actin-binding activities.
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Acknowledgments |
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Footnotes |
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References |
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