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Phosphorylation of the myosin regulatory light chain plays a role in motility and polarity during Dictyostelium chemotaxis

¹ Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242, USA
² Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA

View larger version (49K): [in a new window]   Fig. 1. (A) The behavioral responses of wild-type cells to the spatial, temporal and concentration components of the different phases of the natural cAMP wave, derived from results obtained in prior studies (Varnum-Finney et al., 1987a; Varnum-Finney et al., 1987b; Wessels et al., 1992; Wessels et al., 2000b). (B) Protocols used in this study to determine the behavioral defects of S13A mutant cells.

View larger version (17K): [in a new window]   Fig. 2. Motility is developmentally regulated in the myosin regulatory light chain phosphorylation mutant S13A. Cells were removed from developing JH10, S13A-1 and WT-res cultures at noted times, dispersed on the wall of a perfusion chamber and analyzed for cell velocity over a 10 minute period while perfused with buffer. The mean instantaneous velocity (Inst. Vel.) was computed at each time point from the average instantaneous velocity of 20 to 30 amoebae selected at random without a velocity threshold. Results similar to those for S13A-1 cells were obtained for cells of the independent mutant strains S13A-2 and S13A-3.

View larger version (64K): [in a new window]   Fig. 3. Mutant S13A-1 cells retract anterior pseudopods and extend lateral pseudopods in a manner similar to that of wild-type JH10 cells. Retraction of the original anterior pseudopod and extension of a new lateral pseudopod over a 24 second period for a representative JH10 (A) and S13A (B) cell imaged through differential interference contrast optics. a, original anterior pseudopod, at time zero, and new anterior pseudopod at 24 seconds; u, uropod. Arrows indicate direction of retraction of original anterior pseudopod and direction of expansion of new lateral pseudopod for both cell types. Time is indicated in seconds in upper left hand corners of panels. Similar results were obtained for cells of strains WT-res, S13A-2 and S13A-3.

View larger version (32K): [in a new window]   Fig. 4. Mutant S13A-1 cells chemotax more efficiently than JH10 cells in a spatial gradient of cAMP. A histogram of chemotactic indices indicates that S13A cells attain high CIs (>0.8-1.0) more frequently than JH10 cells or WT-res cells. The number of JH10, S13A-1 and WT-res cells analyzed was 20, 28 and 29, respectively. Results similar to those for S13A-1 cells were obtained for S13A-2 and S13A-3 cells.

View larger version (29K): [in a new window]   Fig. 5. Mutant S13A-1 cells migrate faster and with fewer turns in a spatial gradient of cAMP. Computer-generated tracks are presented of the three JH10 (A), S13A-1 (B) and WT-res (C) cells with the highest chemotactic indices. Cells were selected from 20, 28 and 29 analyzed cells, respectively. Cell perimeters are drawn every 4 seconds. Results similar to those for S13A-1 cells were obtained for strains S13A-2 and S13A-3 cells.

View larger version (27K): [in a new window]   Fig. 6. Mutant S13A cells respond to a sequence of simulated temporal waves of cAMP generated in the absence of spatial gradients by increasing and decreasing velocity in a manner similar to wild-type JH10 cells. The instantaneous velocity is plotted as a function of time for a representative JH10 cell (A) and a representative S13A-1 cell (B) during four simulated waves. The estimated cAMP concentration, measured in dye experiments (Wessels et al., 2000b), is presented as a function of time through the four waves. Note that the velocity of neither JH10 nor S13A-1 cells increases in the front of the first simulated wave, a result previously reported for wild-type cells (Varnum et al., 1985). Instantaneous velocity was measured at 5 second intervals. Instantaneous velocity plots were smoothed 10 times with Tukey windows of 10, 20, 40, 20 and 10. Results similar to those for the representative JH10 and S13A-1 cells were obtained for nine additional cells of each respective cell line. Results similar to those for S13A-1 cells were obtained with S13A-2 cells.

View larger version (88K): [in a new window]   Fig. 7. Cell morphology and myosin II localization during the different phases of a simulated temporal wave of cAMP generated in the absence of a spatial gradient. A to C, D to F, and G to I represent differential interference contrast (DIC) microscopy images of representative JH10 cells fixed in the front, peak and back, respectively, of a simulated temporal wave of cAMP. A' to C', D' to F' and G' to I' are DIC images of representative S13A-1 cells fixed in the front, peak and back, respectively, of a simulated temporal wave of cAMP. J and K are representative JH10 cells in the front and the back, respectively, of a simulated temporal wave of cAMP stained with anti-myosin II antibody (first panel in each set) and scanned along the white line shown in the first panel for staining (pixel) intensity (second panel in each set). J' and K' are representative S13A-1 cells in the front and the back, respectively, of a simulated temporal wave of cAMP stained and analyzed in a fashion similar to the JH10 cells in panels J and K. Over 100 JH10 and 100 S13A-1 cells were analyzed for morphology in the front, peak and back of simulated waves, and found to exhibit the morphologies of the representative cells in this figure. Nine additional JH10 and S13A-1 cells in the front and back of simulated temporal waves were found to exhibit the distribution of myosin demonstrated for the representative cells in the figure.

View larger version (29K): [in a new window]   Fig. 8. S13A cells abnormally fail to lose polarity and abnormally continue to translocate, albeit at diminished velocity, at the peak and in the back of a simulated temporal wave of cAMP and after the rapid addition of 10-6 M cAMP. (A,B) Perimeter tracks of representative JH10 and S13A cells, respectively, at the peak and in the back of the second and third waves in a series of four simulated temporal waves generated in the absence of a spatial gradient. (C,D) Perimeter tracks of representative JH10 and S13A cells, respectively, after the rapid addition of 10-6 M cAMP.

View larger version (12K): [in a new window]   Fig. 9. Centroid tracks of representative JH10 cells (A) and S13A cells (B) prior to (-10 to 0 minutes) and after (0 to + 10 minutes) the rapid addition of 10-6 M cAMP. Time interval between centroids is 10 seconds. Similar results were obtained for 17 additional JH10 and S13A cells analyzed in the same fashion. Results similar to those of JH10 were obtained for WT-res cells analyzed in the same fashion and results similar to those for S13A-1 cells were obtained for S13A-2 and S13A-3 cells analyzed in the same fashion.

View larger version (33K): [in a new window]   Fig. 10. S13A cells remain abnormally elongate and continue to translocate, albeit at reduced velocity, at the deduced peak and in the deduced back of a self-generated natural wave of cAMP. (A,B) Velocity plots of a representative JH10 cell and a representative S13A-1 cell in respective homogeneous aggregation territories responding to three natural sequential waves of cAMP. The phases of the wave (A+B, C+D) are deduced from the velocity plots described previously (Wessels et al., 1992). (C,D) Centroid tracks of the representative JH10 cell and representative S13A cell through the three successive natural waves (1,2,3) in which the deduced peak plus back portions (phases C plus D) are boxed. Arrows point in the direction of the interpreted aggregation centers. (E,F) Amplified centroid tracks through one wave and associated cell morphologies. Similar results were obtained for nine additional S13A-1 cells and ten S13A-2 cells analyzed in a similar fashion.

View larger version (19K): [in a new window]   Fig. 11. S13A cells respond abnormally to the deduced peak and back of natural waves generated by JH10 cells in mixed cultures. Tracks are presented of a representative labeled S13A-1 cell and a representative unlabeled neighboring JH10 cell in an aggregation territory that includes S13A-1 and JH10 cells in a ratio of 1:9. JH10 cells exhibited tracks similar to those in homogeneous JH10 cell populations, suggesting that the waves relayed in the mixed population conformed to that of the majority JH10 cell type. Note that the labeled S13A-1 cell continued to translocate in a persistent fashion, albeit at reduced velocity, at the peak and in the back of deduced waves. Note also how the S13A cell veers off track. The decrease in tracking efficiency was observed in a majority of labeled S13A-1 cells in JH10 aggregation territories. Reverse labeling experiments were performed that demonstrated that labeling did not contribute to the observed effects.

View larger version (73K): [in a new window]   Fig. 12. Streams of S13A cells late in aggregation fragment. Homogeneous populations of JH10 and S13A-1 cells were video-recorded at low magnification late in aggregation during stream formation. Stream formation and fragmentation are obvious in the S13A-1 cultures. In repeat experiments, S13A-1 and S13A-2 streams formed and fragmented, as in the representative panels in B. Zero minutes represents the time at which video-recording was initiated.

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