Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan
* Author for correspondence (e-mail: yumura{at}po.cc.yamaguchi-u.ac.jp)
Accepted 27 September 2002
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Summary |
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Key words: Traction force, Pseudopod, Amoeboid movement, Silicone substrate
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Introduction |
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The traction force against the substrate is necessary for animal cells to
migrate. Information on the magnitude and the direction of the traction force
against the substrate is important for understanding how the cell crawls on
the substrate. The traction force against the substrate was first examined in
fibroblasts, which are irregular in shape and slow-moving cultured cells.
Harris et al. discovered significant traction force generated by locomoting
fibroblasts using a thin film of silicone as a substrate
(Harris et al., 1980). The
traction force generated by fibroblasts is directed inwards, leading to
compression of the substrate and the formation of wrinkles perpendicular to
the direction of their lamella extension
(Harris et al., 1980
). Burton
and Taylor reported that the wrinkles appeared parallel to the equator of
dividing fibroblast (Burton and Taylor,
1997
).
The detailed vectors of the traction force can be examined by using a
flexible silicone substrate to which many small beads are embedded. The
traction force causes the deformation of the substrate and the displacement of
the beads without the formation of wrinkles. The elastic properties of the
silicone medium were calibrated by measuring the displacement of the beads
during the application of known force using flexible glass microneedles. This
calibration enabled us to calculate the force exerted by the cells on the
substrate (Lee et al., 1994;
Oliver et al., 1995
). This
approach showed that the fibroblasts exert centripetal force directed toward
the center of the cell (Roy et al.,
1997
; Pelham and Wang,
1999
). Lee et al. measured the vectors (both the direction and the
magnitude) of the force exerted by keratocytes, fast-moving cells with
constant shape (Lee et al.,
1994
). The keratocytes exert centripetal force directed toward the
center of the cell, and the largest force is located in the lateral regions
(Lee et al., 1994
;
Oliver et al., 1995
;
Burton et al., 1999
;
Galbraith and Sheetz,
1999
).
The traction force is considered to be generated by the interaction between
actin and myosin. The traction force disappears after the treatment with
cytochalasin D, an actin-depolymerizing drug, and butanedione monoxime, an
inhibitor of myosin ATPase (Oliver et al.,
1999; Pelham and Wang,
1999
; Riveline et al.,
2001
), but not after the application of nocodazole, a
microtubule-depolymerizing drug (Pelham
and Wang, 1999
). The force vectors are related to the distribution
of the actin cytoskeleton and the polarity of actin bundles in the cell
(Cramer et al., 1997
;
Svitkina et al., 1997
;
Burton et al., 1999
;
Oliver et al., 1999
;
Pelham and Wang, 1999
). These
findings suggest that the force generated by the actomyosin system is
transmitted to the substrate through the cell-substrate adhesion sites.
Previous studies of the traction force were limited to mammalian cultured
cells, especially to fibroblasts and keratocytes. The former are irregularly
shaped, slow-moving cells, whereas the latter are regularly shaped,
fast-moving cells. So, it is worthwhile to examine the traction force of
irregularly shaped, fast-moving cells. In this study, we examined the vectors
of the traction force exerted by Dictyostelium cells, irregularly
shaped, highly motile cells, using an improved flexible silicone prepared by a
newly modified method, in which cross-linking of silicone was performed by
heating instead of glow discharge, which was how the previous method worked
(Oliver et al., 1995), and as
a result elasticity was much improved, that is, the rate of recovery from
deformation was around 80 to 90% or more. We found that the direction of the
force reversed periodically in the anterior region of the cells during
migration. Furthermore, in order to clarify the role of myosin II in cell
migration, we examined the traction force exerted by MHC-null cells. We found
that myosin II is involved in the generation of `pushing force' responsible
for a part of the anterior extension and the cancellation of the traction
force in the side and the posterior regions of the cells.
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Materials and Methods |
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Preparation of silicone rubber film
The preparation of silicone substrate was performed by modifying the method
of Lee et al. (Lee et al.,
1994). About 4 µl of the silicone fluid (dimethylpolysiloxane;
1000 centistokes of viscosity; Sigma, St. Louis, MO) was spread in 10 square
mm with the thickness of 10-30 µm onto a cleaned coverslip (18x18 mm,
Matsunami Glass, Japan) by a handmade glass spreader. The coverslip was
settled on a tile plate and heated at 450°C using a custom-made hot plate,
which was retained 2 mm above the coverslip. In the preparation of silicone
substrate for the experiments of live cells, the silicone was heated for 20
minutes for wild-type cells and 25 minutes for MHC-null cells, respectively.
The stiffness of silicone substrate was controlled via the heating time.
Calibration of bead displacement
The mechanical properties of the film were characterized by modifying the
method of Lee et al. (Lee et al.,
1994). Highly flexible microneedles with sharply pointed tips were
made using a pipette puller (Narishige, Japan), and calibration was done by
plotting the amount of tip deflection generated by weights in the range of
15-100 µg made from strips of 15 µm thick aluminum sheets. The force
required to displace a bead on a silicone substrate was determined as the
force that reproduces the deflection of the tip of the highly flexible
microneedle. Video images of the calibration experiments were used to know the
extent of tip deflection for a given bead displacement. The percent recovery
of a bead toward its original position was measured 15 seconds after the
substrate was released from microneedle. The percentage recovery was defined
as [(pq)/p]x100, where p is the bead displacement and q is the
bead displacement after recovery.
Microscopy and acquisition of images
The observation chamber was made from a solid silicone sheet
(17x17x0.5 mm) with a hole of about 10x10 mm, which was
placed on a silicone film spread on the coverslip. About 200 µl BSS
containing 5% carboxylate latex beads (0.5 µm in diameter, Polyscience,
Warrington, PA) was carefully poured into the chamber and pipetted gently to
attach the beads to the silicone surface. After 20 minutes, the chamber was
washed with BSS to remove unattached beads. The silicone substrate was coated
with 500 µg/ml bovine serum albumin (BSA) because the surface was so sticky
without coating that cell migration became much slower.
The cell suspension was placed in the chamber filled with BSS, and the chamber was sealed with a coverslip. The cells were monitored by an inverted microscope equipped with a confocal system (LSM 510; Carl Zeiss, Germany) with x63C Apochromat objective lens (NA 1.2). Resolution of acquired images was 512x512 or 1024x1024 pixels. Sequential images were stored in a computer at intervals 2-6 seconds and analyzed by Scion Image software (Scion Corporation, Frederick, MD). Drift of silicone substrate was corrected in a series of images on the basis of the position of some beads that were far away from the cell and did not move. Each image was moved to adjust the position of these beads on the same software. All experiments were performed at 22°C.
Image analysis
To analyze cell movement, the gained area was calculated over time by
subtracting the retraction area from the extension area. Here, the extension
area is defined as the increase in area determined from cell contours taken
from two successive images, and the retraction area is defined as the decrease
in area between two successive images
(Weber et al., 1995;
Yumura and Fukui, 1998
).
The positions of the anterior and posterior edges of the migrating cell
were also measured over time. The anterior and posterior edges were defined as
both of the extreme edges perpendicular to the vector of instantaneous
migration of the cells, which is defined from the cyto-centers of three
continuous images (Uchida and Yumura,
1999). If the cell turns more than 30°, the data was not
counted. Image processing was performed by macro programs in Scion image
(Scion Corp.) and improved by Photoshop (Adobe Systems Inc., Japan).
Position of beads in an image was measured at the center of the beads. If displacement of a bead was less than 0.2 µm (less than 3 pixels in the case of 1024x1024 pixel image), the bead displacement was not quantified. The vector of bead displacement, derived from the present position and the original position of the bead, was divided into perpendicular and parallel vector components to the direction of cell migration. We took the parallel component of the vector of the bead displacement as the substantial bead displacement around the anterior and the posterior regions of the cell and the perpendicular component as that around the side regions.
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Results |
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Alternate movements of beads during cell migration
Dictyostelium cells were allowed to settle on the silicone
substrate, and the movement of beads around the cells was monitored and
analyzed. The movement of beads reflected the distortion of the silicone
substrate caused by the cell during its migration. The analysis was performed
on straight moving cells. Fig.
2a shows a differential interference contrast image of a cell on
the silicone substrate. The superimposed image of five successive images
acquired at 6 second intervals clearly shows the movement of beads around the
cell (Fig. 2b).
Fig. 3 shows a typical example
of two alternate patterns of bead movement around a migrating cell. The
direction of bead movement reversed repeatedly. In
Fig. 3a,b, the beads around the
anterior and the posterior regions of the cell moved toward the cell body, and
those around the side region moved away from the cell. Subsequently, the
direction of bead movement was reversed
(Fig. 3c,d). The beads around
the anterior and the posterior regions moved away from the cell, and those
around the side regions moved toward the cell. Interestingly, most of beads in
the side and the posterior regions returned to their original positions, and
the beads around the anterior region moved away from the cell over their
original positions. The beads moved in similar patterns repeatedly. In
addition, all 51 cells exhibited similar patterns. Hereafter the first pattern
of bead movement is referred to as pattern 1 (inset in
Fig. 3b) and the second as
pattern 2 (inset in Fig.
3d).
|
|
Correlation between specific patterns of bead movement and cyclic
behavior of cells during migration
The cyclic change in the pattern of bead movement observed in
Dictyostelium cells may be correlated with cyclic behavior of cells
during migration, which has been described previously (Soll et al., 1987;
Weber et al., 1995). Then,
contours of a cell taken at two successive times were superimposed, and the
difference between the extension and the retraction areas (gained area) was
plotted over time. If the difference is more than zero, the extension is more
prominent than the retraction (the extension phase). If the difference is less
than zero, the retraction is more prominent than the extension (the retraction
phase). The cell alternated between the extension and the retraction phases as
shown in Fig. 4a. The average
duration of a cycle was 70.4±28.3 seconds (n=22).
|
Interestingly, the two alternate patterns of bead movement (insets in Fig. 3b,d) were well correlated with the extension and the retraction phases of cell behavior during migration, respectively (circled numbers in Fig. 4a). During the extension phase, beads around the anterior and the posterior regions moved toward the cell body, and those around the side region moved away from the cell (pattern 1). During the retraction phase, beads around the anterior and the posterior regions moved away from the cell, and those around the side region moved toward the cell (pattern 2). All the beads around the cell simultaneously changed their direction of movement at the transition between the extension and the retraction phases.
The positions of the anterior and the posterior edges of the migrating cell were also monitored, and the displacement of both edges was plotted over time (squares and triangles in Fig. 4b). When the bead movement switched from `pattern 1' to `pattern 2', the peaks of forward movement of the posterior edge appeared at these transitions (arrows in Fig. 4b). After the peaks of the displacement of the posterior edge, the anterior edge began to move forward significantly (arrowheads in Fig. 4b).
Alternate movements of beads do not occur in MHC-null cells
Myosin II, conventional myosin, is considered to play an important role in
the force generation during cell movement. However, the Dictyostelium
MHC-null mutant cells can still migrate slowly on a substrate
(Wessels et al., 1988). To
investigate the role of myosin II in the generation of traction force, bead
movement caused by MHC-null cells was analyzed. The analysis was performed on
straight moving cells. Fig.
5a-d shows a typical bead movement during migration of a MHC-null
cell. Interestingly, these cells showed only `pattern 1' observed in wild-type
cells. Fig. 5e shows the gained
area in a MHC-null cell as a function of time. The MHC-null cells still
repeated the two phases, the extension and the retraction phases. The average
duration of a cycle was 151.7±38.0 seconds (n=15), which was
twice as long as that of wild-type cells. These results suggest that the
alternate biphasic patterns of bead movement observed in wild-type cells is
not directly related to the extension and the retraction phases of cell
locomotory behavior.
|
Posterior contraction mediated by myosin II generates a pushing force
of the anterior extension
Analysis of the displacement of the anterior and the posterior edges of the
migrating MHC-null cells explained the loss of the pattern 2 in their bead
movement. In Fig. 5f, the
posterior edge of the MHC-null cell moved forward almost at a constant rate,
in contrast to wild-type cells showing apparent peaks at the beginning of the
retraction phases (arrows in Fig.
4b). Only the rate of the movement of the anterior edge changed
cyclically in migrating MHC-null cells (arrows in
Fig. 5f), and this cyclic
anterior extension mainly contributed to the biphasic changes of their gained
area. At all time, beads around the anterior and the posterior regions moved
toward the cell body (insets of Fig.
5c,d). After the cell moved far enough away, the beads returned to
their original positions owing to the relaxation of the substrate.
Comparing the results in Fig.
4b and Fig. 5f, we
found that the considerable forward movement of the posterior edge during the
retraction phase in wild-type cells is caused by myosin-II-dependent
contraction. After the forward movement of the posterior edge, the anterior
edge also began to move forward significantly during the retraction phase
(arrowheads in Fig. 4b). This
anterior extension pushed the beads forward in wild-type cells
(Fig. 3c,d,
Fig. 6a). These observations
strongly suggest that during the retraction phase, the `pushing force' of the
anterior extension is generated by the posterior contraction mediated by
myosin II, which localizes at the rear cortex of the migrating cells
(Yumura et al., 1984). This is
the first convincing evidence to show the role of myosin II in the anterior
extension of migrating cells.
|
Myosin II cancels the traction force at the side and the posterior of
the cell
Where and how does the cell exert force against the substrate? The time
course of displacements of beads from their original positions was
investigated around three divided regions (anterior, side and posterior
regions) of the migrating cells (Fig.
6). The vector of bead displacement was divided into components
that were perpendicular and parallel to the direction of cell migration. The
bead displacement around the anterior and the posterior regions was
represented by the parallel component and that around the side region was
represented by the perpendicular component. The bead displacement around the
anterior region of a wild type cell changed from -1 µm during the extension
phase to +1 µm during the retraction phase
(Fig. 6a). Here, negative
values indicate the movement of beads toward the cell body, and positive
values indicate the movement away from the cell body. In the case of MHC-null
cells, the bead displacement around the anterior region decreased from -1
µm to -3 µm (Fig. 6d),
and these cells never pushed the beads forward during the retraction phase.
These results suggest that the `pushing force' for anterior extension depends
on myosin II during the retraction phase.
The bead displacement around the side region in wild-type cells was +1 µm during the extension phase, decreased to zero during the retraction phase, and it never turned negative (Fig. 6b). Here, zero indicates the original position of beads before the cells were placed on the substrate. The bead displacement around the posterior region was -1.5 µm during the extension phase and it reached zero during the retraction phase (Fig. 6c). Similar results were obtained in analyses of 50 different wild-type cells. The bead displacement around the side and the posterior regions returned to zero during the retraction phase, suggesting that the traction force of these regions was cancelled.
In the case of MHC-null cells, however, the bead displacements around all of three regions were enhanced both during the extension and the retraction phases (Fig. 6d-f). The bead displacement around the side region increased from +1 µm to +4 µm (Fig. 6e). The bead displacement around the posterior region decreased from -1 µm to -4 µm (Fig. 6f). Similar results were obtained in 20 different cells. Taken together with the results of wild-type cells, myosin II is required for the cancellation of the traction force, probably by detaching the adhesion of middle and posterior part of the cell body from the substrate.
Wild-type cells migrate more efficiently than MHC-null cells via the
posterior contraction
The magnitude of the traction force was calculated by multiplying the
stiffness of the substrate by the bead displacements
(Table 1), which showed that
the calculated magnitude of the traction force of MHC-null cells was larger
than that of wild-type cells. However, we could explain these contradictory
values by the lack of the cancellation process of the traction force in the
MHC-null cells. Furthermore, the comparison of the locomotory behavior between
a wild-type cell and a MHC-null cell under the pressure of overlaid agar sheet
(Yumura and Fukui, 1985;
Yumura, 2001
) showed that the
MHC-null cell extended long cell processes, but could not migrate
(Fig. 7b), whereas the
wild-type cell could easily migrate. These observations suggested that the
posterior contraction mediated by myosin II is required for the cells to
migrate under the pressure of the agar, and the wild-type cells could utilize
the traction force more efficiently than the MHC-null cells, with the
contraction of the posterior region.
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Discussion |
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Fibroblasts, slowly moving cells with an irregular shape, mainly exert
inward force at the lamella and the tail regions
(Galbraith and Sheetz, 1997;
Roy et al., 1997
;
Pelham and Wang, 1999
).
Keratocytes, rapidly moving cells with regular shape, mainly exert lateral
force directed toward the center of the cell
(Lee et al., 1994
; Oilver et
al., 1995; Burton et al., 1999
;
Galbraith and Sheetz, 1999
).
These cells do not change the direction of the traction force during
migration, suggesting a single mechanism. In the present study, we found that
Dictyostelium cells, fast moving cells with an irregular shape,
periodically changed the direction of the traction force with two alternate
mechanisms of cell migration. This is the first report of the novel mechanism
of cell migration.
Analysis of the displacement of the anterior and the posterior edges of
migrating cells helped us to explain the difference between wild-type and
MHC-null cells. Wild-type cells showed apparent peaks in the rate of
displacement of their posterior end, whereas MHC-null cells did not. It is
most likely that myosin II, which localizes at the posterior region
(Yumura et al., 1984),
mediates displacement via the contraction of the posterior cortex. In
wild-type cells, considerable anterior extension occurred after these peaks of
the posterior displacement during the retraction phase. This anterior
extension generated a `pushing force' as revealed by the analysis of bead
movement. This observation provides convincing evidence that the posterior
contraction mediated by myosin II contributes to a part of the anterior
extension. Recent work showed that MHC-null cells could not extend
quinine-induced protrusions (quinine is a kind of plant alkaloid), also
supporting the contribution of myosin II to cellular extensions
(Yoshida and Inouye, 2001
).
There was a time lag between the peak of the displacement of the posterior and
the following peak of the anterior edge. Probably, the energy of the
retraction is stored as in a spring by the viscoelastic properties of the cell
body.
MHC-null cells exerted traction force against the substrate continuously
without changing the direction of the force during migration
(Fig. 5). On the other hand, in
wild-type cells, the traction force was reversed in the anterior and relaxed
in the side and the posterior regions during the retraction phase. Though we
do not have any direct evidence at present, it is highly conceivable that
these regions are detached from the substrate in wild-type cells and that
MHC-null cells probably could not perform effective detachment. This idea is
supported by the observation that MHC-null cells could not migrate when they
were placed on a highly sticky surface
(Jay et al., 1995). So, myosin
II must play an important role in the detachment of the cell body from the
substrate via the posterior contraction.
What is the mechanism of the advance of the posterior edge of MHC-null cells? Since the cell body itself is elastic, the posterior part may be dragged passively by the anterior part.
The estimated magnitude of the traction force of MHC-null cells
(Table 1) was continuously at a
high level compared with that of wild-type cells. If myosin II performs the
detachment of the cell body from the substrate efficiently, the wild-type
cells do not need to keep such continuous high levels of the traction force
observed in the MHC-null cells. Since the MHC-null cells cannot detach their
attachments from the substrate effectively, they must migrate by dragging
their body, resulting in a continuous high level of traction force. In other
words, the wild-type cells could migrate faster and more efficiently, saving
power and energy. We think that the motive force of migration of the MHC-null
cells is mainly localized in the anterior region, probably powered by actin
polymerization or myosin Is or other myosins that are distributed in the
anterior edge (Fukui et al.,
1989; Jung et al.,
1993
; Peterson et al., 1995).
Information about the feet of cells is crucial for understanding how the
cells exert the traction force. Total internal reflection aqueous fluorescence
(TIRAF) shows that the cell-substrate gap is relatively uniform beneath the
entire ventral surface of Dictyostelium cell
(Todd et al., 1988),
indicating that there are not any apparently differentiated feet. Eupodia,
which are one of the candidates for feet of Dictyostelium cells,
appear only under the application of some pressure in agar-overlaid conditions
(Fukui and Inoue, 1997
). As the
other candidates for the feet of Dictyostelium cells, we previously
found the several actin foci, from which numerous actin filaments radiate,
associated with the ventral membrane of the cell
(Yumura and Kitanishi-Yumura,
1990
). Furthermore, the migrating cells shed the small dots, which
are immunostained with anti-actin antibodies, on the substrate
(Uchida and Yumura, 1999
), and
these dots are derived from actin foci, which remain attached to substrate in
the trail of a migrating cell (K.S.K.U. and S.Y., unpublished). So, it is
highly probable that the actin foci might be the feet, where the traction
force might be transmitted to the substrate.
Fig. 8 shows a schematic model of how the cell exerts force against the substrate during migration. For simplification, only the parallel components of the traction force to the direction of cell migration are considered, and the feet or the attachment sites of the cell to the substrate are represented as one or two imaginary points of the action of the traction force in each region of the cell. During the extension phase (Fig. 8a-c), extension force of the anterior cell body, probably generated by actin polymerization or myosin Is, results in the `pulling force' at the feet in the anterior as the reaction against the substrate, and the posterior part is passively dragged, resulting in the `pulling force' at the posterior feet (Fig. 8a-c). Fig. 8j explains the presumptive model of the vector of 'pulling force' during the anterior extension. During the retraction phase (Fig. 8d,e), in accordance with the posterior contraction mediated by myosin II, the posterior attachment sites are detached from the substrate resulting in the cancellation of the traction force. At this time, the beads return to their original position (Fig. 8d). Subsequently, the contraction of the posterior region pushes the endoplasm forward, resulting in the `pushing force' in the anterior region (Fig. 8e), and as a result the beads around the anterior region move forward, which is never observed in MHC-null cells. In the case of MHC-null cells, because the contraction of the posterior region is deficient, the beads are always dragged toward the cell body both in the anterior and the posterior regions. Nevertheless, the alternate extension and retraction phases occur in the MHC-null cells, resulting in the cyclic extension of pseudopods (Fig. 8f-i). Fig. 8k explains the vector of the `pushing force' in the anterior extension. Since the `pushing force' in the anterior region was observed only in wild-type cells, it is highly conceivable that it could be generated by myosin II via the posterior contraction.
|
In conclusion, the locomotory behavior of Dictyostelium cells is biphasic, and in accordance with these phases, the direction of the traction force changed repeatedly, representing the highly coordinated biphasic process of cell migration. From the comparison between wild-type and MHC-null cells, myosin II plays an important role, especially during the retraction phase, in the generation and the cancellation of the traction force via the posterior contraction. This is the first convincing evidence of the involvement of myosin II in the anterior extension.
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Acknowledgments |
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Footnotes |
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