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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.021576

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Actin-based propulsive forces and myosin-II-based contractile forces in migrating Dictyostelium cells

Department of Functional Molecular Biology, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8512, Japan

View larger version (39K): [in this window] [in a new window]   Fig. 1. Typical stress map under a migrating Dictyostelium cell. (A) Wild-type Dictyostelium cells (strain AX2) or myosin-II-null cells (strain HS1) transformed with GFP-myosin-II, E476K mutant myosin II or ABD120k constructs were placed on elastic silicone or gelatin substrata embedded with orange- or red-fluorescent marker beads (200-nm or 20-nm diameter). The fluorescence of GFP and the marker beads was imaged simultaneously under TIRF or confocal microscopy. Migrating cells cause strains in the elastic substratum and displacements of the beads in the substratum (arrows). (B) A typical image of 200 nm marker beads under confocal microscopy. The outline of a migrating cell is superimposed as a white line. Displacements of 300 marker beads under and surrounding a migrating cell were measured. (C) The coordinates of each bead and their displacements were transformed to those of each node of a triangle mesh. The length of the sides of each triangle is 400 nm (=8 pixels). The stresses in the surface of the substratum were calculated using original software based on the triangle finite element method (see Materials and Methods). (D) Stress map for a migrating wild-type cell on a gelatin substratum. The distributions of stresses in the substratum were visualized by pseudocolor. The direction of the strain in the substratum at each small white circle is indicated by a white bar. The length of the white bars is three times as long as the strain. The yellow allow indicates the direction of cell migration. Large rearward traction stresses emerged as `stress spots', indicated by the two white arrows. Forward stresses emerged at the posterior edge, as indicated by the white arrowhead. Numerical values in the kPa scale are indicated near each arrow and arrowhead. Bars, 4 µm (B-D).

View larger version (70K): [in this window] [in a new window]   Fig. 2. Colocalization of rearward traction stress spots and F-actin accumulation sites, and close contact of the cell surface to the substratum in wild-type cells. (A) Simultaneous recording of fluorescence of GFP-ABD120k (left) and traction stresses (right) of a migrating wild-type cell transformed with GFP-ABD120k under confocal microscopy. Stress spots (a'-c') appeared at locations where F-actin accumulated (a-c) in the anterior and middle regions of the cell. The typical sequential stress map shown was made from three migrating cells randomly selected from 11 cells. (B) Simultaneous recording of fluorescence of GFP-ABD120k (left) and IRM imaging (right) of a migrating wild-type cell transformed with a GFP-ABD120k construct under confocal microscopy. Dark spots surrounded by white rings (d'-g') appeared at locations where F-actin accumulated (d-g). The white ring surrounding a dark spot is a feature of focal contacts in Dictyostelium (Uchida and Yumura, 2004). The time-courses are indicated for each picture of GFP-ABD120k. Yellow arrows at 0 seconds in panels A and B indicate the direction of cell migration. Bars, 4 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 3. Rearward traction stresses in myosin-II-null cells are identical to those in wild-type cells. (A) Simultaneous recording of the fluorescence of GFP-ABD120k (left) and traction stresses (right) of a migrating myosin-II-null cell transformed with a GFP-ABD120k construct under confocal microscopy. Stress spots (a'-c') appeared at locations where F-actin accumulated (a-c) in the anterior and middle regions of the cell. The typical sequential stress map shown was generated from three migrating cells randomly selected from 13 cells. The time-courses are indicated for each picture of traction stresses. The yellow arrows at 0 seconds indicate the direction of cell migration. (B) Simultaneous recording of the fluorescence of GFP-ABD120k (left) and IRM imaging (right) of a migrating myosin-II-null cell transformed with a GFP-ABD120k construct under confocal microscopy. Dark spots surrounded by white rings (d'-f') appear at the locations where F-actin had accumulated beforehand (d-f). Bars, 4 µm. (C) Average distance between the centers of each F-actin accumulation and corresponding stress spot in wild-type and myosin-II-null cells. (D) Average interval between the time at which the fluorescence of GFP-ABD120k at the center of each F-actin accumulation reached its maximum value and the time at which the stress value at the corresponding stress spot reached its maximum. (E) Average maximum values of rearward traction stresses for each stress spot in wild-type and myosin-II-null cells. (F) Average stress spot duration times for wild-type and myosin-II-null cells. There was no significant difference between wild-type cells and myosin-II-null cells in the maximum magnitude or duration of stress at each stress spot.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Myosin-II-null cells migrate in a direction counter to the sum of the strain vectors at each spot. (A) The trajectory of a migrating myosin-II-null cell and strain vectors at each point for a period of 400 seconds. The black line is the trajectory of the centroid of a migrating myosin-II-null cell. Each blue line emerging from the trajectory indicates the average strain vector under the cell. The length of the blue lines is 50 times the real strain in the substratum. Blue lines were drawn every 17.5 seconds. The black arrow indicates the direction of migration and represents a length of 1 µm. The trajectory was made from one of the three stress map sequences of myosin-II-null cells. (a-f) Stress maps when the cell was positioned at points a-f in A, respectively. Bar, 3 µm (a). (B) The sequential images of the cell outline at the moment when the cell was at each position marked with asterisks of the same color in panel A. The trajectory in panel B is reduced to one-third in comparison with panel A. Myosin-II-null cells continued to migrate in a direction opposite to the direction of the traction stresses.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Myosin II contributes to forward traction stress at the posterior edge. (A) Consecutive maps of traction stresses under a migrating wild-type cell. The time-course is indicated in each picture. The yellow arrows indicate the direction of cell migration. Posterior cell edges are indicated by a white arrow (b at 27 seconds). The cell retracted its posterior edges quickly at 0-84 seconds after forward traction stress reached a maximum. Three sequential stress maps were made from 11 migrating cells, and a typical sequence is shown. Bar, 4 µm. (B) Time-courses of the values of traction stresses (red) and the length of the posterior region (the length of the perpendicular lines from the posterior edge to the white lines at 0 seconds in panel A). The magnitudes of traction stresses at the posterior edges (red lines) strongly increased at the beginning of posterior edge retraction. After the increase in traction stress, the posterior edges retracted quickly. (C) Average values of forward traction stresses at the posterior edges (n=12 in five cells) and rearward traction stresses at the stress spots in the anterior and middle regions (n=16 in five cells) in wild-type cells when the forward traction stress reached a maximum. The magnitude of the forward traction stress at the posterior edge significantly exceeds that of the rearward stresses at stress spots in the anterior and middle regions (P<0.001). (D) Average values of forward traction stresses at the posterior edges (n=5 in three cells) and that of rearward traction stresses at the stress spots in the anterior and middle regions (n=8 in three cells) in myosin-II-null cells when the forward traction stresses at the posterior edges reached a maximum. There was no significant difference between rearward and forward stresses (P>0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Myosin II contributes to traction stresses under the tips of retracting pseudopodia in wild-type cells. (A,B) Fluorescence of GFP-ABD120k (left) and stress maps (right) under two typical pseudopodia from five migrating wild-type cells. Large traction stresses were observed under the tips of retracting pseudopodia where F-actin accumulated. (A',B') Time-courses of traction stresses (red lines) under the tips of each pseudopod (A,B) and pseudopod lengths (blue lines). (C-F) Fluorescence of GFP-ABD120k (left) and stress maps (right) under four typical pseudopodia from three migrating myosin-II-null cells. Large traction stresses were not observed under the tips of retracting pseudopodia where F-actin accumulated. (C'-F') The time-courses of pseudopod lengths and traction stresses just under the tip of each pseudopod. Blue and red lines indicate pseudopod lengths and stresses, respectively. Although a small rise in stress took place under the tip of a large pseudopod (arrow in F'), stresses under the pseudopod tips were unchanged typically between the elongation and retraction. (G) The average periods of elongation and retraction of pseudopodia in wild-type (n=5 in three cells) and myosin-II-null cells (n=7 in three cells). Although there was no significant difference in elongation time between wild-type and myosin-II-null cells, the retraction time of pseudopodia in wild-type cells was significantly shorter than in myosin-II-null cells. Bars, 1.5 µm. The look-up table (LUT) of A and B is indicated at the right side of B, and the LUT of C-F is at the right side of D.

View larger version (43K): [in this window] [in a new window]   Fig. 7. Traction stresses under retracting pseudopodia mediated by myosin II. (A) Simultaneous recording of GFP-myosin-II fluorescence and traction stresses using a combination of TIRF and force microscopy: the dynamics of myosin II filaments (upper images) and traction stresses (lower images) under a pseudopod of a migrating myosin-II-null cell are shown. The stress map was made from three migrating cells randomly selected from nine cells. The time elapsed is indicated in each GFP-myosin-II image. Myosin II filaments begin to accumulate in the pseudopod during elongation (0-40 seconds), followed by increased traction stress in the silicone substratum just under the pseudopod (40-80 seconds). Retraction began at the same time as the beginning of the increase in traction stress (40 seconds). Bar, 1 µm. (B) Time-courses of pseudopod area (blue) and fluorescence intensity of GFP-myosin-II (red). (C) Time-courses of fluorescence intensity of GFP-myosin-II (red) and average traction stress in the pseudopod (blue). The increase in pseudopod area was followed by accumulation of myosin II (B). Traction stress in the pseudopod subsequently reached a maximum during retraction (C).

View larger version (44K): [in this window] [in a new window]   Fig. 8. The motor activity of myosin II is required for the generation of traction forces by retracting pseudopodia. (A) Dynamics of E476K myosin II filaments (upper images) and traction stresses (lower images) under a pseudopod of a migrating myosin-II-null cell expressing GFP-E476K myosin II. The stress map was made from three migrating cells randomly selected from six cells. E476K myosin II filaments began to accumulate in the pseudopod during elongation of the pseudopod (0-24 seconds), similar to wild-type myosin II. After elongation, traction stress slightly increased only under the tip of the pseudopod (arrows). Bar, 1.5 µm. (B) Time-courses of pseudopod area (blue) and fluorescence intensity of GFP-myosin-II (red). (C) Time-courses of fluorescence intensity of GFP-myosin-II (red) and average traction stress in the pseudopod (blue). The increase in pseudopod area was followed by the accumulation of E476K myosin II in the pseudopod, as was the case for normal myosin II (Fig. 7B). However, the traction stress increased only at the tip of the retracting pseudopod.

View larger version (31K): [in this window] [in a new window]   Fig. 9. A model for the migration of fast-moving irregularly shaped cells. (A) The migrating cell comprises a frontal towing zone (a) and a rear trailing and contractile zone (p). These two zones are connected by an elastic transition zone (e) that includes the cytoskeleton. Polymerization of actin (red-filled circles) produces a force (F) for pseudopod elongation in the frontal towing zone and a rearward force (rf) at stress points (s) in the anterior or middle of the cell. Part of F' is transmitted to the rear zone through the elastic transition zone, and this passively generates a forward force (ff) at the stress points in the rear trailing and contractile zones. F' increases as the elastic transition zone extends. (B) When the passive force F' reaches a threshold level, an active strong force (Fm) is exerted by accumulated myosin II at the rear trailing and contractile zones. After contraction of the rear zone by myosin II, a new cycle of the cell migration process begins (panel A).

View larger version (18K): [in this window] [in a new window]   Fig. 10. The method of calculating the traction stress. (A) Each triangle element has three nodes, A1, A2 and A3, whose original positions are (x1, y1), (x2, y2), (x3, y3) and displacement vectors are (u1, v1), (u2, v2), (u3, v3), respectively. The strains at each point in one element are assumed to be distributed linearly. P is an arbitrary point whose original position is (x, y) and the displacement vector is (u, v). (B) The longitudinal and shearing strains, x, y and xy, at the minute square in the element. (C) The normal and shearing stresses, x, y and xy, at the minute square in the element.

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