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doi: 10.1242/10.1242/jcs.00195

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Myosin II contributes to the posterior contraction and the anterior extension during the retraction phase in migrating Dictyostelium cells

Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan

View larger version (32K): [in a new window]   Fig. 1. Mechanical properties of the silicone substrate. The bead displacement in response to the force applied by a microneedle was plotted. The slope of the line represents the stiffness of silicone substrate. (a) The stiffness of the softest substrate is 5.74 nN/µm. (b) The stiffness of the hardest substrate is 8.59 nN/µm. (c,d) Percent recovery of the beads toward their original position of the softest (c) and the hardest (d) substrates. The average of percent recovery was 87% in both cases.

View larger version (89K): [in a new window]   Fig. 2. Differential interference contrast images of a Dictyostelium cell on the silicone substrate. (a) A single image of a cell surrounded with beads. (b) Five successive images acquired at 6 second intervals were superimposed, which clearly shows the movement of beads around the cell. Bar, 5 µm. Movie 1 showing cell migration on the silicone surface is available at jcs.biologists.org/supplemental.

View larger version (35K): [in a new window]   Fig. 3. A typical movement of beads around a migrating wild-type cell. The gray and black lines represent a superimposed image of two cell contours at 0 seconds and 21 seconds in a, at 21 seconds and 42 seconds in b, at 42 seconds and 63 seconds in c, at 63 seconds and 84 seconds in d, respectively. Large closed arrows represent the direction of cell migration. The gray and black dots represent positions of the beads at 0 seconds and 21 seconds in a, at 21 seconds and 42 seconds in b, at 42 seconds and 63 seconds in c, at 63 seconds and 84 seconds in d, respectively. The crosses represent the original position of beads before the cells were placed on the silicone substrate. Small open arrows of red, green and blue represent the vectors of bead movements around anterior, side and posterior regions, respectively. Black open arrows represent the bead movement underneath the cell body. The length of the open arrows is twice as long as the displacement of the beads. Between 0 and 42 seconds (a,b), the beads around the anterior and posterior regions moved toward the cell body, and those around the side region moved away from the cell. Subsequently, between 42 and 84 seconds (c,d), the bead movements were reversed. Insets in b and d, schematic representations of the directions of the movement of beads surrounding the cell are shown. Large black arrows represent the direction of the cell migration. Closed arrows of red, green and blue represent the directions of bead movements around anterior, side and posterior regions, respectively. The pattern of bead movement between 0 and 42 seconds is referred to as pattern 1 (inset of 3b) and the pattern between 42 and 84 seconds as pattern 2 (inset of 3d). The beads underneath the cell body occasionally moved in a different manner and direction. In many cases, this feature could not be expressed in these figures because the directional change occurred in a short time. The stiffness of this silicone substrate is 5.97 nN/µm. Bar, 5 µm.

View larger version (33K): [in a new window]   Fig. 4. Cyclic behavior of cells corresponds with specific patterns of bead movement. (a) A representative time course of the gained area of a migrating wild-type cell. Two successive contours of a cell were superimposed, and the gained area was calculated by subtraction of the retraction area from the extension area. Note that extension and retraction phases alternated. The average time of a cycle was 70.4±28.3 seconds (n=22). Two patterns of bead movement (pattern 1, circled number 1; pattern 2, circled number 2) were well correlated with the extension and the retraction phases, respectively. The vertical lines are drawn on the basis of the transition points of the two patterns. (b) The displacement of the anterior (squares) and posterior (triangles) edges of the cell was plotted over time. The definition of the anterior and the posterior edges is described in Materials and Methods. When the speed of the advance of the posterior edge reached its maximum value, the bead movement switched from pattern 1 to pattern 2 (arrows in b). Note that the anterior edge advanced significantly after the peak of the forward movement of the posterior edge (arrowheads in b).

View larger version (27K): [in a new window]   Fig. 5. Alternate movements of beads did not occur in MHC-null cells. Representative movement of the beads around a migrating MHC-null cell (a-d). The gray and black lines represent a superimposed image of two cell contours at 0 seconds and 120 seconds in a, at 120 seconds and 240 seconds in b, at 240 seconds and 360 seconds in c, at 360 seconds and 480 seconds in d, respectively. Large closed arrows represent the direction of cell migration. The gray and black dots represent positions of the beads at 0 seconds and 120 seconds in a, at 120 seconds and 240 seconds in b, at 240 seconds and 360 seconds in c, at 360 seconds and 480 seconds in d, respectively. The bead movement did not reverse. Insets in c and d, schematic images of the direction of movement of beads surrounding the cell. The bead movements showed only the pattern 1 observed in wild-type cells. (e) A representative time course of the gained area of a migrating MHC-null cell. Note that MHC-null cells also repeated two phases, extension and retraction phases. The average time of a single cycle was 151.7±38.0 seconds (n=15). (f) The displacement of the posterior and the anterior edges of the cell was plotted over time. Note that the position of the posterior edge moved roughly at a constant rate when compared with the case of wild-type cells (see Fig. 4b), whereas the rate of the displacement of the anterior edge had obvious peaks (arrows in f). The cell shown in e and f was different from the cell in a-d. The stiffness of silicone substrate was 5.97 nN/µm. Bar, 5 µm.

View larger version (30K): [in a new window]   Fig. 6. Correlation between bead displacement and phase transition of cell locomotory behavior. Solid squares on the line graphs represent the bead displacement around three regions, anterior (a,d), side (b,e) and posterior (c,f) of a wild-type (a-c) and a MHC-null (d-f) cell, respectively. The method for the measurement of bead displacements was described in Materials and Methods. Zero at the y axis indicates the original position of the beads before the cells were placed on the substrate. Negative values indicate displacement toward the cell, and positive values indicate displacement away from the cell. In wild-type cells, the bead displacement around the anterior region reversed and increased to positive values during the retraction phase (a), representing a pushing force in this region. The bead displacement around the side and the posterior regions reached zero during the retraction phase (b,c), suggesting that the adhesion of the cell body to the substrate in both regions was lost. Note that the bead displacement in MHC-null cells was not reversed but enhanced throughout the extension and the retraction phases. All graphs include results of several beads (multiple plotted lines).

View larger version (201K): [in a new window]   Fig. 7. Cell migration under the pressure of overlaid thin agar sheet. Wild-type (a) and MHC-null (b) cells were compressed between a thin agar sheet and a glass coverslip. Arrows represent the direction of the anterior extension. The wild-type cell was able to migrate under the pressure of the agar sheet. This cell showed both the posterior retraction and the anterior extension. However, the MHC-null cell was not able to migrate. Asterisks represent the position of the nucleus of this cell. The MHC-null cell did not show the posterior retraction, although it showed anterior extensions. Bar, 10 µm. Movies 2 and 3 showing cell migration are available at jcs.biologists.org/supplemental.

View larger version (26K): [in a new window]   Fig. 8. A schematic model of how the cell exerts the traction force against the substrate during migration. The flexible silicone substrate is shown as straight or wavy lines. The wavy lines indicate the distortion of the silicone surface. The gray large arrows (a,f) show the direction of the cell movement. The circles and crosses indicate the present and the original position of beads, respectively. The displacement of beads is shown by thin black arrows parallel to the substrate. The shaded part of the cell represents the putative region generating motive force for migration. The cell transmits the traction force or the motive force (thick black arrows) to the substrate through putative feet (black vertical bars). (a-e) In the wild-type cell, during the extension phase (a-c), the extension force of the anterior region drags the posterior cell body and causes the pulling traction force at the anterior feet, and the posterior feet are passively dragged and pull the beads forward. During this phase, formation of new feet may occur in the newly extended anterior region (c,d). During the retraction phase (d,e), the posterior region of the cell contracts (two small arrows at the rear) mediated by the myosin II, which localizes at this region. It causes the detachment of the adhesion sites in the posterior region, the substrate is relaxed, and the beads return to their original positions (the circle overlaps with the cross) in d. (e) Then the posterior contraction generates a pushing force of the anterior cell body and also causes anterior extension. At this time, the posterior feet exert the traction force backward to the substrate and the anterior feet push the beads forward. (f-i) In the case of MHC-null cells, the beads always move toward the cell body since the contraction of posterior regions is deficient. The posterior edge moves at a constant rate whereas the extension progresses cyclically at the anterior region. Figs j and k explain the difference between the `pulling force' and the `pushing force' at the anterior region. (j) The pulling force is caused by the reaction to the anterior motive force generated by actin polymerization or myosin Is. (k) The pushing force is generated by the posterior contraction mediated by myosin II.

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