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

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1 Research Article 1733 Phosphorylation of the myosin regulatory light chain plays a role in motility and polarity during Dictyostelium chemotaxis Hui Zhang 1, Deorah Wessels 1, Petra Fey 2, Karla Daniels 1, Rex L. Chisholm 2 and David R. Soll 1, * 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 *Author for correspondence ( david-soll@uiowa.edu) Accepted 29 January 2002 Journal of Cell Science 115, (2002) The Company of Biologists Ltd Summary The myosin regulatory light chain (RLC) of Dictyostelium discoideum is phosphorylated at a single serine site in response to chemoattractant. To investigate the role of the phosphorylation of RLC in oth motility and chemotaxis, mutants were generated in which the single phosphorylatale serine was replaced with a nonphosphorylatale alanine. Several independent clones expressing the mutant RLC in the RLC null mutant, mlcr, were otained. These S13A mutants were sujected to high resolution computer-assisted motion analysis to assess the asic motile ehavior of cells in the asence of a chemotatic signal, and the chemotactic responsiveness of cells to the spatial, temporal and concentration components of natural camp waves. In the asence of a camp signal, mutant cells formed lateral pseudopods less frequently and crawled faster than wild-type cells. In a spatial gradient of camp, mutant cells chemotaxed more efficiently than wild-type cells. In the front of simulated temporal and natural waves of camp, mutant cells responded normally y suppressing lateral pseudopod formation. However, unlike wild-type cells, mutant cells did not lose cellular polarity at the peak and in the ack of either wave. Since depolarization at the peak and in the descending phase of the natural wave is necessary for efficient chemotaxis, this deficiency resulted in a decrease in the capacity of S13A mutant cells to track natural camp waves relayed y wild-type cells, and in the fragmentation of streams late in mutant cell aggregation. These results reveal a regulatory pathway induced y the peak and ack of the chemotactic wave that alters RLC phosphorylation and leads to cellular depolarization. We suggest that depolarization requires myosin II rearrangement in the cortex facilitated y RLC phosphorylation, which increases myosin motor function. Key words: Myosin light chain, Myosin phosphorylation, Cell motility, Chemotaxis, Dictyostelium discoideum Introduction In a translocating Dictyostelium amoea, myosin II forms thick filaments that localize to the cortex of the cell ody (Yumura and Fukui, 1985; Fukui, 1990). Myosin II does not localize to an expanding pseudopod, although it does appear transiently in a retracting pseudopod (Moores et al., 1996). Through the motion analysis of myosin heavy-chain-deficient mutants (DeLozanne and Spudich, 1987; Knecht and Loomis, 1987), it has een demonstrated that mysoin II is necessary for normal cell polarity, cell motility and chemotaxis, and plays a role in the spatial distriution and dynamics of pseudopod extension and retraction (Wessels et al., 1988; Wessels and Soll, 1990; Spudich, 1989; Sheldon and Knecht, 1996). It has een proposed that myosin II plays a role in the suppression of lateral pseudopod formation (Wessels et al., 1988, Wessels et al., 2000; Stites et al., 1998; Chung and Firtel, 1999), presumaly through the generation of cortical tension (Clarke and Spudich, 1974; Fukui and Yumura, 1986; Pasternak et al., 1989; Egelhoff et al., 1996). During chemotaxis in natural aggregation territories of Dictyostelium, the regulation of lateral pseudopod formation and polarity play key roles in the ehavioral responses of cells to the different phases of each natural camp wave (Wessels et al., 1992; Wessels et al., 2000a; Wessels et al., 2000). Since myosin II is involved in oth pseudopod formation and cell polarity, it must play an underlying role in chemotaxis. The rapid addition of the chemoattractant camp to cells in uffer results in phosphorylation of the myosin heavy chain (Berlot et al., 1985; Berlot et al., 1987), which in turn results in the depolymerization of myosin II thick filaments (Kuczmarski and Spudich, 1980; Cote and McCrea, 1987; Ravid and Spudich, 1989). Conversion of the three mapped threonine phosphorylation sites in the MHC tail to nonphosphorylatale alanines in the mutant 3XALA results in an increase in myosin II localization to the cell cortex and increased cortical tension (Egelhoff et al., 1996). It also results in ehavioral defects in uffer and in spatial gradients of camp consistent with an increase in cortical tension, and a significant decrease in chemotactic efficiency (Stites et al., 1998). Together, these results suggest that the phosphorylation/dephosphorylation of MHC plays a critical role in the maintenance of cell shape and motility in uffer, and in chemotaxis in a spatial gradient of camp. Cyclic AMP also stimulates myosin regulatory light chain (RLC) phosphorylation (Kuczmarski and Spudich, 1980; Berlot et al., 1985; Berlot et al., 1987), increasing myosin s actin-activated Mg 2+ ATPase activity (Griffith et al., 1987;

2 1734 Journal of Cell Science 115 (8) Tryus, 1989). To investigate the role of RLC phosphorylation in motility and chemotaxis, we expressed either wild-type RLC or RLC in which serine 13 was sustituted with alanine (S13A) in an RLC null mutant (Ostrow et al., 1994). Myosin II from mutant S13A cells exhiited only 30% of the actin-activated Mg 2+ ATPase activity of wild-type myosin II (Ostrow et al., 1994). Nevertheless, S13A cells underwent cell division, localized myosin II to the cortex of locomoting cells and formed fruiting odies (Ostrow et al., 1994), suggesting that RLC phosphporylation was not essential for growth or cytoskeletal organization during locomotion and development. Under the assumption that a modification of myosin II mediated through occupancy of the camp receptor must play a role in chemotaxis, we sujected S13A cells to high resolution computer-assisted motion analysis, employing a set of experimental protocols that tested, first, the asic motile ehavior of mutant cells in the asence of an extracellular camp signal and, second, the responses of mutant cells to the different spatial, temporal and concentration components of the natural camp wave (Fig. 1). The results of these experiments demonstrate that RLC phosphorylation plays a role in the asic motile ehavior of cells in the asence of an extracellular chemotactic signal, and in the normal response of cells to the peak and ack of a natural chemotactic wave. The incapacity of mutant S13A cells to phosphorylate RLC at the peak and in the ack of the wave results in less efficient chemotaxis in natural waves early in mutant cell aggregation, and to the fragmentation of streams late in aggregation. These results support a model of chemotactic regulation in which independent regulatory pathways emanating from the distinct phases of the natural chemotactic wave elicit a sequence of specific cellular ehaviors that together represent the natural chemotactic response. Materials and Methods Origin of strains The RLC null mutant mlcr was generated from parental strain JH10 y targeting the RLC gene (mlcr) as previously descried (Chen et al., 1994). The S13A mutants, which contained unphosphorylatale RLC, were generated y transforming mlcr with the Dictyostelium integrating vector pbvn5115, which contained a mutated version of RLC [in which Ser13 (TCA) was changed to Ala (GCC), under the regulation of the actin 15 promoter] and a neomycin resistance gene for G418 selection of transformed cells (Ostrow et al., 1994). Three S13A mutants were generated in independent transformations, S13A- 1, S13A-2 and S13A-3. A control strain, WT-res, representing the RLC deletion strain rescued with wild-type RLC was generated y transforming mlcr with the Dictyostelium integrating vector pbvn5133, which contained a wild-type version of RLC under the regulation of the actin 15 promoter, and a neomycin resistance gene for the selection of transformed cells (Ostrow et al., 1994). It was demonstrated that the WT-res RLC, ut not the S13A RLC, could e phosphorylated with the myosin regulatory light chain kinase MLCK in vitro (Ostrow et al., 1994). In the computer-assisted analysis of mutant ehavior, the first characterizations were performed in detail on mutant strain S13A-1, and aerrant ehaviors then verified less rigorously in mutant strains S13A-2 and S13A-3. Maintenance and development of control, mutant and rescued strains Spores of JH10, S13A and WT-res strains were frozen in 10% glycerol and stored at 80 C. For experimental purposes, cultures were generated from spores every three weeks (Sussman, 1987). Cells were initially grown in HL-5 medium alone for two days, then in HL-5 medium containing 10 µg per ml of G418, to a final cell concentrations of per ml. To initiate development, cells were washed in uffered salt solution (BSS) containing 20 mm KCl, 2.5 mm MgCl 2 and 20 mm KH 2PO 4 (ph 6.4) and dispersed on a lack filter pad saturated with BSS at a density of cells per cm 2 (Soll, 1987). For all analyses of single cell ehavior, except those in which the developmental regulation of motility was monitored, cells were harvested at the ripple stage, which in dense cultures represents the onset of aggregation (Soll, 1979), the time at which Dictyostelium amoeae attain their highest average velocity (Varnum et al., 1986). Analysis of the asic motile ehavior of mutant cells (protocol 1, Fig. 1B) The ehavior of cells in the asence of an extracellular camp signal, which we will refer to henceforth as the asic motile ehavior of a cell, was analyzed according to methods previously descried (Varnum et al., 1985; Varnum-Finney et al., 1987a; Wessels et al., 2000a; Wessels et al., 2000). In rief, 1.1 ml of dilute cell suspension were inoculated into a Sykes-Moore chamer (Bellco Glass, Vineland, NJ). The chamer was then inverted and positioned on the stage of an upright microscope fitted with long-range ojectives and condenser. For motion analysis, cell ehavior was either video-recorded or digitized directly through a 10 ojective or 25 ojective. The chamer was perfused with BSS at a rate that replaced the liquid volume every 15 seconds to ensure that cells did not condition the medium. This flow rate was demonstrated not to interfere with normal cellular translocation. Analysis of mutant cell chemotaxis in a spatial gradient of camp (protocol 2, Fig. 1B) The motile ehavior of cells in a spatial gradient of camp generated in a single cell spatial gradient chamer (Zigmond, 1977) was analyzed according to methods previously descried (Varnum and Soll, 1984; Varnum-Finney et al., 1987; Wessels et al., 2000a; Wessels et al., 2000). In rief, cells were dispersed on the ridge of a Plexiglas gradient chamer, in which one of the two troughs ordering the ridge contained BSS (sink) and the other trough contained BSS plus 10 6 M camp (source). Cells were videorecorded through a 25 ojective with right field optics for a 10 minute period following an initial 5 minute incuation period necessary for estalishing a steep gradient (Shutt et al., 1998). Analysis of mutant cell ehavior in temporal waves of camp (protocol 3, Fig. 1B) The motile ehavior of cells in a series of temporal waves of camp, which simulate the temporal dynamics of natural waves in the asence of spatial gradients, was analyzed according to methods previously descried (Varnum et al., 1985; Varnum-Finney et al., 1987a; Wessels et al., 2000). In rief, cells were inoculated into a Sykes-Moore chamer as descried for the analysis of cell ehavior in uffer. To generate temporal waves of camp, cells were perfused with increasing, then decreasing, temporal gradients of camp, and the process repeated three times. Cells were first perfused with 5 ml of BSS, then with 2 ml of BSS containing M camp over a 30 second period. At 30 second intervals thereafter, cells were perfused with 2 ml of a new solution containing twice the camp concentration of the preceding solution, terminating at 10 6 M camp, the last step in the increasing phase. Cells were then treated with 2 ml increments of BSS containing half the previous concentration of camp at 30 second intervals, terminating at 10 8 M camp. The second, third and fourth waves were generated in a similar fashion. The periodicity of

3 Myosin regulatory light chain and motility 1735 A. Behavioral responses to the different phases of the natural wave. Direction of relayed wave Rapid, directed translocation towards source of wave; suppression of lateral pseudopods (response to increasing temporal gradient of camp.) Direction towards aggregation center estalished, cellular polarization (response to positive spatial gradient of camp). PHASE A Tested Behavior PHASE B Front of Wave: Increasing temporal and increasing spatial gradients of camp PHASE C Peak Cell depolarization, cessation of translocation (response to peak concentration of camp). PHASE D Back of Wave: Decreasing temporal and decreasing spatial gradients of camp simulated temporal waves was, therefore, 7 minutes. Fields of cells were video-recorded or directly digitized through a 10 or 25 ojective. The concentration of camp in the chamer through the four simulated waves was assessed y fluorescent dye experiments as previously descried (Wessels et al., 2000). Analysis of mutant cell ehavior after the rapid addition of 10 6 M camp (protocol 4, Fig. 1B) The motile ehavior of cells efore and after the rapid addition of 10 6 Re-extension of pseudopods in random directions; maintenance of depolarized state; no net movement in any direction (response to decreasing temporal gradient of camp) B. Experimental protocols for determining the ehavioral defects of S13A mutants in asic motile ehavior and chemotaxis. 1. Basic motile ehavior in the asence of an extracellular camp signal. 2. Capacity to assess the direction of a spatial gradient of camp (Phase A). 3. Behavior in the middle of the front of the wave in response to increasing temporal gradient of camp (Phase B), the response to the concentration of camp at the peak of the wave (Phase C) and the ehavior in the ack of the wave (Phase D). x 4. Response to the concentration of camp at the peak of the wave (Phase C). 5. Behavior in all phases (A, B, C and D) of self-generated natural waves of camp. 6. Behavior in response to all phases (A, B, C and D) of natural waves generated y wild type cells. Direction of aggregation center Experimental Protocol 1. Perfusion of amoeae with uffer in perfusion chamer. 2. Spatial gradient of camp generated on the ridge of a single cell spatial gradient chamer. 3. Exposure to a sequence of interspersed increasing and decreasing temporal gradients of camp that mimic the temporal dynamics of natural waves in the asence of estalished spatial gradients. 4. Rapid addition of 10-6 M camp to cells crawling in uffer. 5. Aggregation in a dilute monolayer on a plastic surface. 6. Aggregation in which fluorescently stained mutant cells are mixed with unstained wild type cells in a 1 to 9 ratio. Fig. 1. (A) The ehavioral responses of wild-type cells to the spatial, temporal and concentration components of the different phases of the natural camp wave, derived from results otained in prior studies (Varnum-Finney et al., 1987a; Varnum-Finney et al., 1987; Wessels et al., 1992; Wessels et al., 2000). (B) Protocols used in this study to determine the ehavioral defects of S13A mutant cells. M camp was analyzed according to methods descried aove for analysis of the asic motile ehavior in uffer with one modification. That is, following perfusion for 10 minutes with BSS, the perfusion solution was rapidly switched to BSS containing 10 6 M camp. The concentration of camp in the Sykes-Moore chamer was assessed y fluorescent dye experiments as previously descried (Wessels et al., 2000). Cell ehavior prior to and after addition of camp was continuously video recorded or digitized directly through a 10 or 25 ojective. Analysis of mutant cell ehavior in self-generated waves of camp (protocol 5, Fig. 1B) The motile ehavior of cells in selfgenerated waves of camp was analyzed according to methods previously descried (Escalante et al., 1997), with the exception that the plastic surface of the tissue culture dish was not coated with agar (Wessels et al., 2000). In rief, 2 ml of a cell suspension ( per ml BSS) were dispersed on a 35 mm tissue culture dish. After 30 minutes of incuation, 1.0 ml of fluid was withdrawn and the dish placed on the stage of an inverted microscope. Cell ehavior was continuously video-recorded or directly digitized through a 10 ojective. Individual cells positioned in the same area of the field that exhiited no cell-cell contacts were selected for analysis. For streaming experiments late in aggregation, a 2.5 ojective was employed. Analysis of mutant cell ehavior in wild-type waves of camp (protocol 6, Fig. 1B). The motile ehavior of mutant cells in natural waves of camp generated y wild-type cells was analyzed according to methods previously descried (Wessels et al., 2000a; Wessels et al., 2000). In rief, S13A cells were stained with the vital dye DiI (Molecular Proes, Eugene, OR), mixed with a majority of unstained JH10 cells, at a ratio of 1:9, and 2 ml of the cell mixture ( per ml BSS) dispersed on a 35 mm tissue culture dish. After 30 minutes, 1 ml of fluid was withdrawn and the dish positioned on the stage of an Axiovert 100STV Zeiss microscope equipped for epifluorescent analysis. Cell ehavior was analyzed with rightfield and fluorescence microscopy according to methods previously descried (Wessels et al., 2000). In a control experiment, unstained mutant cells were mixed with stained wild-type cells.

4 1736 Journal of Cell Science 115 (8) Inst. Vel. (µm/min) JH10 S13A WT-res Onset of Aggregation Time (hrs) 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 chamer and analyzed for cell velocity over a 10 minute period while perfused with uffer. The mean instantaneous velocity (Inst. Vel.) was computed at each time point from the average instantaneous velocity of 20 to 30 amoeae selected at random without a velocity threshold. Results similar to those for S13A-1 cells were otained for cells of the independent mutant strains S13A-2 and S13A-3. Computer-assisted analysis of cell motility Video images were digitized at a rate of 15 frames per minute (i.e. at 4 second intervals) onto the hard disc of a Macintosh G4 computer (Apple Computers, Cupertino, CA) equipped with a Data Translation framegraer oard (Data Translation Inc., Marloro, MA) and 2D- DIAS software (Soll, 1995; Soll and Voss, 1998). Perimeters were automatically outlined and converted to eta-spline replacement images (Soll, 1995; Soll and Voss, 1998; Soll et al., 2000). Motility parameters were computed from centroid positions and morphology parameters from perimeter contours (Soll, 1995). Instantaneous velocity of a cell in frame n was computed y drawing a line from the centroid in frame n-1 to the centroid in frame n+1 and dividing the length of the line y twice the interval time (15 seconds) etween frames. For simplicity, instantaneous velocity will e referred to simply as velocity in the text. Directional change was computed as the direction in the interval (n-1, n) minus the direction in the interval (n, n+1). Directional change values >180 were sutracted from 360, providing a positive value etween 0 and 180. Difference pictures were generated y superimposing the image in frame n on the image in frame n-1. The regions of the cell image in frame n not overlapping the cell image in frame n-1 were considered the expansion zones. The summed area in the expansion zones of a difference picture divided y the total cell area in frame n and multiplied y 100 represents positive flow. The period etween overlapping images in difference pictures was 1 minute. This parameter provides a measure of cellular translocation that is independent of cell centroid movement (Soll, 1995; Soll and Voss, 1998). Maximum length was the longest chord etween any two points along the perimeter of a cell. Roundness was computed y the formula 10 4π area/perimeter 2. Chemotactic index (CI) in a spatial gradient of chemoattractant was the net distance moved towards the source of chemoattractant divided y the total distance moved in the same time period. Percent positive chemotaxis was the proportion of the cell population exhiiting a positive CI over the period of analysis. In measuring the frequency of lateral pseudopod formation, a lateral pseudopod was considered to e a projection formed from the main axis of translocation at an angle 30 that attained a minimum of 5% total cell area and initially contained nonparticulate cytoplasm. The main axis of translocation was determined y drawing a line etween the centroid of the cell in the frame 15 seconds earlier and the centroid of the cell in the present frame (Wessels et al., 1996; Wessels et al., 2000a; Wessels et al., 2000). For the analysis of instantaneous velocity as a function of developmental time, all cells in the population were motion analyzed. For all other experiments, motion analysis parameters were computed at 4 second intervals only for those cells crawling at instantaneous velocities aove 3 µm per minute. For all strains in all tested situations, this represented over 70% of each population. Myosin II localization Cells were stained for myosin II according to methods previously descried (Wessels et al., 2000). In rief, cells were sujected to three simulated temporal waves of camp. Midway through the increasing phase, at the peak and midway through the decreasing phase of the last of these waves in independent cultures, the chamers were perfused with 4% paraformaldehyde in phosphate uffer solution supplemented with 0.01% saponin. After an antigen retrieval 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 oth cell types. Time is indicated in seconds in upper left hand corners of panels. Similar results were otained for cells of strains WT-res, S13A-2 and S13A-3.

5 Myosin regulatory light chain and motility 1737 Tale 1. Lateral pseudopod formation y cells crawling in uffer or in a spatial gradient of camp* Average frequency of Numer 0 Lateral pseudopods 1 Lateral pseudopod 2 Lateral pseudopods >2 lateral pseudopods lateral pseudopod Condition Cell type of cells per 10 min (%) per 10 min (%) per 10 min (%) per 10 min (%) per cell per 10 min Buffer JH S13A Spatial gradient JH S13A *Cells were imaged at 25 magnification. For the definition of a lateral pseudopod, see Materials and Methods. Cells were analyzed in all cases for 10 minutes. A Chi square test was performed etween JH10 and S13A cells on the comined data of the four categories of lateral pseudopod formation. The difference etween JH10 and S13A cells oth in uffer and spatial gradients of camp was found to e highly significant (10 12 and , respectively). procedure (Wessels et al., 2000), cells were incuated with rait anti-myosin II antiody, a generous gift of Arturo DeLozanne (University of Texas, Austin, TX), and stained with FITC-laeled antirait antiody (Jackson ImmunoResearch, West Grove, PA). DIC and confocal images were captured at 1 µm intervals eginning at the sustratum with a Zeiss 510 laser-scanning confocal microscope in the Central Microscopy Facility at the University of Iowa. To measure the distriution of myosin II across a cell, intensity plots were derived along a line that did not cross the cell nucleus, using Zeiss 510 software. Results Strains analyzed To otain mutant cells containing a myosin regulatory light chain (RLC) that cannot e phosphorylated, the RLC deletion mutant mlcr, derived from the parental wild-type strain JH10, was rescued with a mutated form of RLC, S13A, in which serine 13 (TCA) was sustituted with alanine (GCC), under the control of the constitutive actin 15 promoter (Ostrow et al., 1994). Three independent S13A mutants were generated, S13A-1, S13A-2 and S13A-3. In addition, the mlcr mutant was rescued with wild-type RLC to generate the control strain WTres. The parent strain JH10, the mutant strains S13A-1, S13A- 2 and S13A-3, and the rescued strain WT-res were then analyzed for asic motile ehavior and chemotaxis. In every condition in which aerrant ehavior was demonstrated in strain S13A-1, it was also demonstrated in the additional mutants S13A-2 and S13A-3. For simplicity, mutant S13A-1 will e referred to as strain S13A in the Results, and as strain S13A-1 in the figure legends and tales, where corroorative results with strain S13A-2 and S13A-3 are reported. The asic motile ehavior of mutant cells is aerrant During Dictyostelium development, the velocity of individual wild-type amoeae increases to a maximum at the onset of aggregation (Varnum et al., 1986; Wessels et al., 2000; Escalante et al., 1997). To test whether mutant cells ehaved similarly, JH10, S13A and WT-res cells were removed from developing cultures at various times and analyzed for mean instantaneous velocity in uffer. All three strains attained maximum instantaneous velocity at the onset of aggregation, etween 8 and 9 hours of development (Fig. 2), demonstrating that the developmental regulation of velocity was intact in the asence of RLC phosphorylation. However, the maximum instantaneous velocity achieved y S13A cells at the onset of aggregation was at least 30% higher than that of either JH10 or WT-res cells (Fig. 2). The difference in oth cases was significant (P<0.01, Student t-test). To address the possiility that the increase simply reflected the proportion of motile cells, the mean instantaneous velocity of only those cells moving faster than 3 µm per minute was computed for all three cell lines. This velocity threshold has een used previously to eliminate cells not persistently translocating (Wessels et al., 1996; Wessels et al., 2000a; Wessels et al., 2000). When applied, the peak velocity (±s.d.) of JH10, S13A and WT-res cells was 8.3±5.6 (n=46), 10.3±5.3 (n=38) and 7.4±4.6 (n=52) µm per minute, respectively. Again, the peak of S13A cells was 24% higher than that of JH10 cells and 39% higher than that of WT-res cells. These differences were significant (P<0.02, Student t-test). Cell velocity can e affected y the rate of pseudopod expansion (Cox et al., 1992; Cox et al., 1996) and the frequency of lateral pseudopod formation, the latter correlating with the frequency of turning (Varnum-Finney et al., 1987). In uffer, the directional change parameter, an indicator of turning frequency (Soll, 1995; Soll and Voss, 1998), was Tale 2. Motility, dynamic morphology and chemotaxis parameters in a spatial gradient of camp Instantaneous Directional Percent velocity Positive flow change Area Maximum length positive Chemotactic Cell type Cell numer (µm/min) (%/min) (deg./min) (µm 2 ) (µm) Roundness (%) chemotaxis index JH ± ±4.6 25±13 100±18 17±3 68± ±0.32 S13A ± ± ±9 88±17 18±3 57± ±0.24 WT-res ± ±6.8 22±15 95±27 18±5 63± ±0.47 P values* JH10 vs S13A S13 vs WT-res NS(0.01) NS(0.03) NS NS 0.04 NS(0.02) *Significance was determined y the Student t-test for all measured parameters except percent positive chemotaxis. A P value greater than 0.05 was considered non-significant (NS), ut values close to 0.05 are shown in parenthesis. A Chi-square test found the difference etween JH10 and S13A close to significant and the difference etween WT-res and S13A significant.

6 1738 Journal of Cell Science 115 (8) 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 (> ) more frequently than JH10 cells or WT-res cells. The numer of JH10, S13A-1 and WT-res cells analyzed was 20, 28 and 29, respectively. Results similar to those for S13A-1 cells were otained for S13A-2 and S13A-3 cells. consistently 10-20% lower in S13A cells than JH10 cells translocating in uffer. Increased turning can depress instantaneous velocity, while decreased turning can elevate it. We tested whether the increase in velocity of S13A cells in uffer was accompanied y a decrease in lateral pseudopod formation y counting the numer of lateral pseudopods formed over a 10 minute period. JH10 and S13A cells retracted old anterior pseudopods and extended new lateral pseudopods in a qualitatively similar manner (Fig. 3A and B, respectively). However, S13A cells formed lateral pseudopods at only onethird the rate of JH10 cells (Tale 1). These results suggest that the increase in velocity of S13A cells in uffer may e due, at least in part, to the decreased rate of lateral pseudopod formation. Mutant cells chemotax efficiently in a spatial gradient of camp To test whether S13A cells chemotax efficiently in a spatial gradient of camp, the mechanism presumed to e asic to the directional decision in phase A of the natural wave (Fig. 1A), the ehavior of JH10, S13A, and WT-res cells were compared in spatial gradients of camp generated in a chamer consisting of a ridge that supports the cells, and two ordering troughs, one filled with attractant (the source) and the other with uffer (the sink) (Zigmond, 1977; Varnum and Soll, 1984; Shutt et al., 1998). Cell ehavior was analyzed in a 10 minute time window (the 5-15 minute period following filling of the chamer troughs), when the evolving gradient of camp across the ridge elicits the maximum chemotactic response (Shutt et al., 1998). S13A cells translocated in spatial gradients of camp at a velocity significantly higher than that of JH10 or WT-res cells (Tale 2). S13A cells also exhiited a mean positive flow value, approximately twice that of either JH10 or WT-res cells (Tale 2). Positive flow is a measure of area displacement in a 4 second period that provides a measure of translocation that is independent of the cell centroid (Soll, 1995). Furthermore, S13A cells exhiited a directional change 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 otained for strains S13A-2 and S13A-3 cells. parameter 60% that of JH10 cells and 70% that of WT-res cells (Tale 2), indicating that S13A cells turned less frequently than the other two cell types during chemotaxis. Finally, oth the mean chemotactic index and the proportion of the population exhiiting a positive chemotactic index (percent positive chemotaxis) were higher in S13A cells (Tale 2). The higher mean chemotactic index (Tale 2) was due to the very high proportion of S13A cells with chemotactic indices >0.8, as demonstrated in the histogram in Fig. 4. The differences in oth velocity and chemotactic efficiency were reflected in perimeter tracks. The perimeter tracks of the three S13A cells with the highest chemotactic indices in a spatial gradient of camp were more persistent in the direction of the source of chemoattractant and included fewer sharp turns (Fig. 5B) than the perimeter tracks of the three JH10 cells (Fig. 5A) and the three WT-res cells (Fig. 5C) with the highest

7 Myosin regulatory light chain and motility 1739 A. JH Inst. Vel. (µm/min) Inst. Vel. (µm/min) B. S13A Time (mins) camp conc. (Mol/L) camp conc. (Mol/L) Fig. 6. Mutant S13A cells respond to a sequence of simulated temporal waves of camp generated in the asence of spatial gradients y 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., 2000), 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 otained for nine additional cells of each respective cell line. Results similar to those for S13A-1 cells were otained with S13A-2 cells. chemotactic indices. In addition, the perimeters of S13A cells (Fig. 5B) were less tightly stacked than those of JH10 cells (Fig. 5A) or WT-res cells (Fig. 5C), indicating higher average velocities. The reduction in sharp turns in the S13A perimeter tracks suggested that, as in uffer, S13A cells formed fewer lateral pseudopods per unit time in a spatial gradient than either JH10 or Wt-res cells. To test this prediction, direct counts were made of the numer of lateral pseudopods formed in a 10 minute period. The results demonstrated that the frequency of lateral pseudopod formation y S13A cells was half that of JH10 cells during chemotaxis in a spatial gradient of camp (Tale 1). Mutant cells respond anormally to the peak and ack of temporal waves The results otained in spatial gradient chamers suggest that mutant cells, which cannot phosphorylate the myosin regulatory light chain in response to a camp signal, can still orient and chemotax efficiently up a spatial gradient of camp, the presumed mechanism for orientation and polarization in phase A of the natural wave (Fig. 1A). The ehavior of cells in phases B, C and D of the natural wave, however, are in response to the temporal and concentration characteristics of the wave (Wessels et al., 1992) (Fig. 1A). In response to the increasing temporal gradient in the front of each wave (phase B), cells suppress lateral pseudopods (Varnum-Finney et al., 1987a; Wessels et al., 1992; Wessels et al., 2000) and move in a highly persistent and directional manner towards the aggregation center. When cells encounter the high concentration of camp at the peak of the wave (phase C), they round up, lose polarity and stop translocating. Finally, in response to the decreasing temporal gradient in the ack of the wave (phase D), cells again extend pseudopods, ut remain relatively apolar, resulting in little net movement in any direction (Varnum-Finney et al., 1987a; Wessels et al., 1992, 2000). These responses restrict the movement of cells towards the aggregation center during natural aggregation to phase B of the natural wave. The responses to the temporal and concentration components of phases B, C and D of the natural wave are readily assessed y sujecting cells to sequential increasing and decreasing gradients of camp generated in a purfusion chamer (Fig. 1B, protocol 3) (Varnum et al., 1985; Varnum-Finney et al., 1987a; Wessels et al., 1992; Wessels et al., 2000). Because of the round shape of the chamer and perfusion rate, the temporal waves are generated in the asence of spatial gradients. In Fig. 6A and B, the instantaneous velocity of a representative JH10 and S13A cell, respectively, and the estimated concentration of camp, are co-plotted as functions of time through four successive simulated temporal waves. The average velocity of the representative JH10 cell (Fig. 6A) and the S13A cell (Fig. 6B) remained depressed through the first simulated temporal wave, increased at the onset of the second wave, peaked at the midpoint of the increasing phase of the second wave, decreased at the peak of the second wave and remained depressed through the remaining decreasing phase of the second wave. Behaviors in the different phases of the third

8 1740 Journal of Cell Science 115 (8) Fig. 7. Cell morphology and myosin II localization during the different phases of a simulated temporal wave of camp generated in the asence 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 ack, 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 ack, respectively, of a simulated temporal wave of camp. J and K are representative JH10 cells in the front and the ack, respectively, of a simulated temporal wave of camp stained with anti-myosin II antiody (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 ack, 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 ack of simulated waves, and found to exhiit the morphologies of the representative cells in this figure. Nine additional JH10 and S13A-1 cells in the front and ack of simulated temporal waves were found to exhiit the distriution of myosin demonstrated for the representative cells in the figure. and fourth waves were similar to those in the second wave. Similar results were otained with WT-res cells (data not shown). The velocity data suggest, therefore, that S13A cells respond normally to the temporal dynamics of the chemotactic wave. However, scrutiny of cell shape during the different phases of the temporal wave revealed that the instantaneous velocity plots did not provide the full story. In simulated temporal wave two to four, JH10 and WT-res cells exhiited the sequence of shape changes previously reported (Wessels et al., 2000). In the increasing temporal gradient in the front of

9 Myosin regulatory light chain and motility 1741 Fig. 8. S13A cells anormally fail to lose polarity and anormally continue to translocate, aleit at diminished velocity, at the peak and in the ack 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 ack of the second and third waves in a series of four simulated temporal waves generated in the asence of a spatial gradient. (C,D) Perimeter tracks of representative JH10 and S13A cells, respectively, after the rapid addition of 10 6 M camp. waves two to four (phase B), JH10 cells were highly elongate with a dominant anterior pseudopod (Fig. 7A-C). At the peak of the wave (phase C), JH10 rounded up, exhiiting a loss of polarity (Fig. 7D-F). In the decreasing temporal gradient in the ack of the wave (phase D), JH10 cells again extended pseudopods, ut in random directions, reflecting a lack of polarity (Fig. 7G-I). WT-res cells progressed through shape changes identical to those of JH10 cells in phases B, C and D (data not shown). In the increasing temporal gradients in the front of waves two to four (phase B), S13A cells were elongate on average (Fig. 7A -C ), similar to JH10 (Fig. 7A-C) and WTres cells (data not shown). However, the majority of S13A cells were still elongate and polar at the peak of the wave (phase C), each with a dominant anterior pseudopod (Fig. 7D -F ), and remained elongate and polar during the decreasing phase of the wave (phase D) (Fig. 7G -I ). Therefore, although S13A cells exhiited a decrease in instantaneous velocity at the peak and in the decreasing phase of the second to fourth simulated temporal wave in a series, they did not undergo the normally associated loss of cellular polarity. The anormal maintenance of polarity resulted in a defect in the motile ehavior of S13A cells at the peak and in the decreasing phase of temporal waves. The perimeters of JH10 cells (Fig. 8A) and WT-res cells (data not shown) at the peak and in the ack of simulated temporal waves ecame on average relatively round and polar, and tended to stack one on top of the other in a time series, indicating little net translocation in any one direction. However, the perimeters of S13A cells (Fig. 8B) remained on average elongate and polar, and generated tracks with a directional component, indicating that S13A cells continued to crawl anormally at the peak and in the ack of simulated temporal waves, aleit at reduced velocity. Myosin distriution in the peak and ack of temporal waves Myosin II localizes to the cortex of normal elongate cells translocating in the front of a wave and is more generally distriuted in less polar cells not actively suppressing lateral pseudopod formation (Wessels et al., 2000). In the front of simulated temporal waves of camp, myosin II localized to the cortex of the posterior two-thirds of rapidly translocating, elongate JH10 cells (Fig. 7J) and S13A cells (Fig. 7J ). In the ack of simulated temporal waves of camp, although myosin II was more evenly distriuted throughout the cytoplasm of apolar, nontranslocating JH10 cells (Fig. 7K), it was still localized in the cortex of the posterior two-thirds of the anormally elongate S13A cells (Fig. 7K ). Similar results were attained when the same analysis was performed on nine additional JH10 cells and nine additional S13A cells in each phase of the wave. Mutant cells respond anormally to the rapid addition of 10 6 M camp In the increasing phase of a natural wave, a cell experiences an increase in the concentration of camp from less than 10 8 M at the trough to 10 6 M at the peak over a period of several minutes (Tomchik and Deverotes, 1981). At the peak of a wave, a normal cell loses polarity and stops translocating (Wessels et al., 1992). One approach that has een commonly used to assess the cellular response to the peak of the wave is to add camp (10 6 M) rapidly to cells in uffer (e.g. Ross and Newell, 1981; Hall et al., 1988; Wessels et al., 1989). When camp is added rapidly to cells crawling in a perfusion chamer, so that the concentration increases from 0 to 10 6 M in less then 8 seconds, the cells stop translocating, round-up and lose cellular polarity within 20 seconds from the time camp first enters the chamer (Wessels et al., 1989). These ehavioral changes are similar to those of cells responding to the peaks of simulated temporal waves of camp and to the peaks of natural waves (Wessels et al., 1992). JH10 cells responded to the rapid increase in camp in a manner similar to that descried for other wild-type strains of Dictyostelium (Wessels et al., 1989; Wessels and Soll, 1990; Cox et al., 1992; Escalante et al.,

10 1742 Journal of Cell Science 115 (8) A. JH10 B. S13A 10-6 M camp 10-6 M camp 0 M camp 0 M camp 50 µm 50 µm 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 etween centroids is 10 seconds. Similar results were otained for 17 additional JH10 and S13A cells analyzed in the same fashion. Results similar to those of JH10 were otained for WT-res cells analyzed in the same fashion and results similar to those for S13A-1 cells were otained for S13A-2 and S13A-3 cells analyzed in the same fashion. 1997). Prior to the addition of 10 6 M camp, the centroid tracks of JH10 cells reflected relatively persistent and rapid translocation (Fig. 9A). Within 10 seconds after the addition of camp to the chamer, centroids clustered, reflecting the cessation of cellular translocation (Fig. 9A). After the addition of camp, perimeters stacked one on top of the other, again reflecting the cessation of cellular translocation (Fig. 8C). Perimeters also ecame rounder, reflecting the loss of cellular polarity (Fig. 8C). Prior to the addition of 10 6 M camp, the centroid tracks of the S13A cells also reflected persistent translocation (Fig. 9B). However, after the addition of 10 6 M camp the centroids did not cluster tightly like those of JH10 cells. Rather, they reflected continued translocation, aleit at reduced velocity. This interpretation was reinforced in perimeter tracks. After the rapid addition of camp, S13A cells retained their elongate morphologies and translocated in a persistent manner (Fig. 8D), similar to S13A cells responding to the peak and ack of simulated temporal waves (Fig. 8B). Therefore, S13A cells anormally retained an elongate, polar morphology and continued to translocate (aleit at reduced velocity), after the rapid addition of 10 6 M camp, the same anormalities exhiited at the peak of simulated temporal waves of camp. Mutant cells exhiit defects at the peak and in the ack of self-generated natural waves of camp Based on the ehavioral phenotypes of S13A cells in uffer, in a spatial gradient of camp and in simulated temporal waves of camp, one would expect S13A cells to orient correctly at the onset of each natural wave (phase A, Fig. 1A) and translocate in a persistent fashion towards the aggregation center in the front of the wave (phase B, Fig. 1A), ut anormally remain elongate (i.e. not undergo cellular depolarization) and anormally continue to translocate at the peak and in the ack of the wave (phases C,D, Fig. 1A). To assess the ehavior of mutant cells in natural waves, we employed a sumerged culture protocol (Escalante et al., 1997) that allowed comparison of the ehavior of individual S13A, JH10 and WT-res cells in self-generated natural waves of camp with similar average periodicity (5 minutes for S13A cells, 6 minutes for JH10 cells and 5 minutes for WT-res cells). Time plots of velocity for JH10 cells (Fig. 10A), WT-res cells (data not shown) and S13A cells (Fig. 10B) contained peaks and troughs at relatively constant intervals reflecting increased velocity in the front of the waves (phase B) and decreased velocity at the peak (phase C) and in the ack (phase D) of waves. Centroid tracks of oth JH10 and S13A cells pointed in the general direction of their respective aggregation centers during each rapid translocation segment (phase B) (Fig. 10C and 10D, respectively), demonstrating that S13A cells assessed the correct direction of the spatial gradient of camp at the onset of each self-generated natural wave (phase A). However, neighoring S13A centroid tracks did not appear to exhiit on average the overall accuracy of JH10 cells (i.e. maintain the same level of directionality towards the deduced aggregation center; data not shown). In addition, S13A cells anormally retained polarity and continued to translocate, aleit at reduced velocity, in the deduced peak and ack of each self-generated wave, just as they did in the ack of simulated temporal waves. The tracks of JH10 cells (Fig. 10C) included segments in which centroids were separated and aligned in the direction of the aggregation center (arrow), representing ehavior in the front of each wave (phase B), interspersed with segments in which the centroids were highly clustered, reflecting little net translocation in any one direction at the peak (phase C) and in the ack (phase D) of each natural wave (Fig. 10B). Outlined images of a representative JH10 cell through a wave revealed an elongate morphology during the translocation segment in the deduced front of the wave (phase B), and the loss of polarity during centroid clustering at the deduced peak and in the deduced ack of the wave (phase C and D; Fig. 10E). The centroid tracks of S13A cells (Fig. 10E) also included segments in which the centroids were separated and aligned in the general direction of the aggregation center, representing ehavior in front of each wave (phase B), interspersed with contracted segments in which the distances etween centroids were reduced. The contracted segments still exhiited alignment, reflecting slower ut still persistent translocation at the peak and in the ack of the wave, the same anormal ehavior oserved at the peak and in the ack of simulated temporal waves of camp. Outlined images of a representative

11 Myosin regulatory light chain and motility 1743 Fig. 10. S13A cells remain anormally elongate and continue to translocate, aleit at reduced velocity, at the deduced peak and in the deduced ack 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 descried 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 ack portions (phases C plus D) are oxed. 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 otained for nine additional S13A-1 cells and ten S13A-2 cells analyzed in a similar fashion. S13A cell through a wave revealed the anormal maintenance of an elongate, polar morphology at the peak and in the ack of the wave (Fig. 10F), the same anormality exhiited at the peak and in the ack of a simulated temporal wave (Fig. 8B). S13A cells respond anormally to wild-type waves If S13A cells respond anormally to the peaks and acks of self generated camp waves in aggregation territories, they should also respond anormally to the peaks and acks of camp waves generated y wild-type cells. To test this prediction, S13A cells were stained with the vital dye DiI, mixed at a 1:9 ratio with unlaeled JH10 cells, and analyzed y transmitted light and fluorescence microscopy. The results (Fig. 11) were similar to those collected for the two cell types in self generated waves. In the centroid tracks of the dominant cell type JH10, expanded, persistent segments (phase B) were interspersed with highly clustered segments (phase C and D). The net direction of the representative JH10 track in Fig. 11, was towards the aggregation center. The tracks of nine additional JH10 cells analyzed in the same manner exhiited the same general characteristics. In the centroid track of a neighoring S13A cell, expanded persistent segments (phase B) were interspersed with less extensive, ut still persistent segments (phases C and D). As in simulated temporal waves and self-generated natural waves, S13A cells continued to translocate at the peak and in the ack of natural waves generated y JH10 cells. In addition, although the track of the representative S13A cell was in the general direction of the aggregation center, its accuracy was not as great as that of the neighoring JH10 cells (Fig. 11). The tracks of nine additional S13A cells analyzed in the same manner exhiited the same general characteristics as the representative S13A cell in Fig. 11, and were, on average, also less on track in phase B than neighoring JH10 cells (data not shown). Streaming is defective during S13A aggregation In the previous sections, we demonstrated ehavioral defects associated with single cell chemotaxis, which occurs early in the aggregation process. However, late in the aggregation process, cells coalesce into multicellular streams, in which they move, still in a pulsatile fashion, into the final aggregate (Reitdorf et al., 1997). To test whether streaming was normal in late aggregating S13A cell populations, fields of cells were video-recorded at low magnification. Whereas JH10 cells formed normal contiguous streams late in aggregation that grew thicker as aggregation progressed, S13A cells formed streams that fragmented along their lengths (Fig. 12). Discussion Disruption of the gene mlcr, which encodes the myosin regulatory light chain (RLC) in Dictyostelium discoideum, resulted in defects in cytokinesis, morphogenesis and motility (Chen et al., 1994). These defects were similar to those otained with the original disruption of the myosin heavy chain (DeLozanne and Spudich, 1987; Knecht and Loomis, 1987; Wessels et al., 1988). Although it is not clear whether the defects exhiited y the RLC deletion mutant were due to the

12 1744 Journal of Cell Science 115 (8) 1 JH10 c+d c+d c+d c+d 2 Direction of Aggregation Center mislocalization of myosin II or to an alteration in motor function, a recent analysis of mutants haroring RLCs with point mutations suggested that RLC played a direct role in the motor properties of myosin (Chaudoir et al., 1999). The Dictyostelium RLC is phosphorylated in response to the rapid addition of chemoattractant (Kuczmarski and Spudich, 1980; Berlot et al., 1985; Berlot et al., 1987; Griffith et al., 1987), suggesting that phosphorylation plays a role in chemotaxis. However, the gross defects of the mlcr mutant were reversed y the reintroduction of an RLC lacking the single phosphorylation site at serine 13 (Ostrow et al., 1994), suggesting that RLC phosphorylation was not required for myosin II function. In verterates, ending of the smooth muscle and nonmuscle myosin tail is regulated y RLC phosphorylation through a Ca 2+ -calmodulin-dependent myosin light chain kinase, which stimulates assemly and actinactivated ATPase (Suzuki et al., 1978; Somlyo and Somlyo, 1981; Kamm and Stull, 1985; Ikee et al., 1987; Tryus and Lowery, 1987). In Dictyostelium, phosphorylation of RLC regulates only enzymatic activity (Griffith et al., 1987). The oservations that RLC phosphorylation is tightly coupled to camp receptor occupancy, and that it plays a role in enzymatic activity led us to hypothesize that it must play a role in the complex set of responses of Dictyostelium amoeae to the natural chemotactic wave (Wessels et al., 1992; Wessels et al., 2000a; Wessels et al., 2000). Dissecting the complex ehavior of Dictyostelium amoeae in natural chemotactic waves The chemotactic responsiveness of Dictyostelium amoeae has c+d S13A Fig. 11. S13A cells respond anormally to the deduced peak and ack of natural waves generated y JH10 cells in mixed cultures. Tracks are presented of a representative laeled S13A-1 cell and a representative unlaeled neighoring JH10 cell in an aggregation territory that includes S13A-1 and JH10 cells in a ratio of 1:9. JH10 cells exhiited 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 laeled S13A-1 cell continued to translocate in a persistent fashion, aleit at reduced velocity, at the peak and in the ack of deduced waves. Note also how the S13A cell veers off track. The decrease in tracking efficiency was oserved in a majority of laeled S13A-1 cells in JH10 aggregation territories. Reverse laeling experiments were performed that demonstrated that laeling did not contriute to the oserved effects. 3 c+d een assessed y a variety of in vitro protocols, including the rapid addition of 10 6 M camp, slow release of camp from a micropipette and the genesis of a spatial gradient of camp in a gradient chamer. However, the actual chemotactic signal a cell experiences in nature is quite different. In a natural aggregation territory, individual cells respond to nondissipating, symmetrical waves of camp relayed from the aggregation center outwardly through the cell population (Tomchik and Devreotes, 1981). A cell responds to each phase of a natural wave in a relatively different fashion (Fig. 1A) (Varnum et al., 1985; Varnum-Finney et al., 1987a; Wessels et al., 1992; Wessels et al., 2000). In the front of each natural wave, cells experience an increasing spatial gradient of camp (increasing in the direction of the aggregation center) and an increasing temporal gradient of camp (concentration increasing with time). It has een proposed (Wessels et al., 1992) that cells use the direction of the spatial gradient at the onset of the front of the wave to polarize in the direction of the aggregation center. Once that direction is set, cells respond to the associated increasing temporal gradient of camp in the front of the wave y suppressing lateral pseudopod formation, which facilitates rapid and directional movement in a lind fashion in the direction of the aggregation center (Wessels et al., 1992). At the peak of each natural wave, cells experience a camp concentration that has een demonstrated to cause a loss of cellular polarity and a dramatic decrease in instantaneous velocity (Varnum and Soll, 1984; Wessels et al., 1989; Wessels et al., 1992). In the ack of the wave, cells experience a decreasing spatial gradient of camp (decreasing in the direction of the aggregation center) and a decreasing temporal gradient of camp (concentration decreasing with time). The decreasing temporal gradient suppresses cellular repolarization, resulting in the formation of pseudopods in random directions and no net translocation in any direction. This complex sequence of ehavioral responses to the different spatial, temporal and concentration components of the four phases of the natural wave (Fig. 1A) confines directed cellular translocation towards the aggregation center to the front of the wave. The variety of experimental protocols employed in the present study (Fig. 1B) have allowed us to test which, if any, of the phase specific responses involve RLC phosphorylation. S13A cells are faster, even in the asence of a chemotactic signal We have found that, despite their inaility to phosphorylate RLC, S13A cells translocate faster than wild-type cells and form fewer lateral pseudopods in the asence of a chemotactic signal. Although the decrease in pseudopod formation must contriute to the oserved increase in velocity, it does not represent the entire explanation. The increased separation of centroids and perimeters in plotted tracks of S13A cells in uffer, in spatial gradients of camp and in the front of simulated temporal and natural waves of camp suggests that the asic speed of individual mutant cells is greater than that of wild-type cells, independent of lateral pseudopod formation and turning. These results demonstrate that the serine phosphorylation site is necessary for oth the normal frequency of turning and the normal velocity of a translocating cell in the asence of a camp signal, and that phosphorylation/

13 Myosin regulatory light chain and motility 1745 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 ovious 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. dephosphorylation of RLC plays a role in the asic motile ehavior of a cell in the asence of extracellular camp. S13A cells migrate faster and with higher chemotactic efficiency in spatial gradients of camp S13A cells also exhiited a higher average chemotactic index than either of the two control cell types. This oservation was at first counter intuitive, since one would have expected most mutations in cytoskeletal events downstream of camp receptor occupancy to decrease the efficiency of chemotaxis. However, it may not have een completely surprising given the inverse relationship demonstrated etween the efficiency of chemotaxis and the frequency of lateral pseudopod formation. Varnum-Finney et al. demonstrated that as the chemotactic index increases, the rate of lateral pseudopod formation decreases (Varnum-Finney et al., 1987). S13A cells already exhiit depressed rates of lateral pseudopod formation in their asic motile ehavior, which appear to enhance chemotactic efficiency in a spatial gradient. Therefore, if S13A cells can still assess the direction of a spatial gradient and adjust direction y relying more heavily on iased anterior pseudopod expansion than new lateral pseudopod formation, they may chemotax more efficiently. Why, then, does Dictyostelium go to the troule of phosphorylating the RLC? One possile answer is that cells in vivo must assess not only the spatial characteristics, ut also the temporal dynamics of a natural camp wave in order to chemotax properly, and that the phosphorylation/dephosphorylation of RLC is intricately involved in this complex process. S13A cells respond anormally to the peak and ack of simulated temporal and natural waves of camp To test whether S13A cells were defective in responding to the temporal characteristics of natural waves, they were sujected to a series of increasing and decreasing temporal gradients that simulated the temporal dynamics of sequential waves in the asence of spatial gradients (Varnum-Finney et al., 1987a; Wessels et al., 1992). The velocity responses of mutant cells were generally normal. Cells moved at peak velocities in the front of waves, and at trough velocities at the peak and in the ack of waves. However, mutant cells failed to round up at the peak of the wave and remained anormally polarized (elongate) in the ack of waves. Mutant cells continued to move in a directed fashion at the peak and in the ack of simulated temporal waves, although at greatly reduced average velocity. These results demonstrate that phosphorylation of RLC is necessary for the morphological response to the high concentration of camp at the peak of the natural wave, ut is not essential for the general reduction in velocity. Mutant cells also anormally retained polarity in the ack of the wave even though velocity remained generally suppressed. To confirm that the asence of depolarization in response to the peak of the wave represented a failure of mutant cells to respond to the peak concentration of camp (Varnum and Soll, 1984), we tested the response of mutant cells to the rapid addition of 10 6 M camp (Wessels et al., 1989). Although the rapid increase in camp caused a dramatic reduction in velocity, it did not elicit a loss of cellular polarity, as it did in wild-type cells. This defect had an impact on the motile ehavior of mutant cells at the peak and in the ack of oth simulated temporal waves and natural waves of camp. While there is very little net translocation y wild-type cells at the peak and in the ack of simulated temporal camp waves (Varnum et al., 1985; Varnum-Finney et al., 1987a) and deduced natural waves (Wessels et al., 1992), anormally elongate mutant cells continued to translocate in a directed fashion, aleit at reduced velocity. S13A cells respond less efficiently to natural camp waves If normal depolarization at the peak of a natural wave and the normal maintenance of the depolarized state in the ack of the natural wave are necessary components of chemotaxis, then S13A cells must e less efficient in natural chemotaxis. Our results demonstrate that this is indeed the case. S13A cells exhiited the same defects at the peak and in the ack of selfgenerated natural waves as those exhiited at the peak and in the ack of simulated temporal waves. In addition, the tracks of S13A cells, although generally directed at a common aggregation center, were on average less on course than tracks of JH10 cells responding to self-generated natural waves. When a minority of laeled S13A cells were mixed with unlaeled JH10 cells, their tracks were also generally directed towards the aggregation center, ut again were on average less on course than the tracks of neighoring JH10 cells. The