The distribution of actin and myosin in Dictyostelium amebae at different developmental stages was studied by improved immunofluorescence ("agar-overlay" technique). Both were localized at the cortical region of amebae in all early developmental stages. In amebae with polarized morphology, bright fluorescence with antiactin was seen in the anterior pseudopode. The cortex in the posterior end was also stained with antiactin. On the other hand, very specific crescent-shaped staining with antimyosin was seen at the posterior cortex. In cells in contact with each other, actin was concentrated at the contact region, whereas myosin was localized specifically in the cortex on the other side of the contact region. At the aggregation stage, when monopodial amebae migrate forming streams, actin staining was seen all around the cell periphery, with intense fluorescence in the anterior pseudopode. On the other hand, specific staining of myosin was seen only at the posterior cortex. The cleavage furrow of cells performing cytokinesis displayed distinct myosin staining, and this staining represented the filamentous structure aligned in parallel to the axis of constriction. These findings indicate that myosin staining reflects the portion of the cell cortex where contraction occurs and the motive force of ameboid movement is generated at the posterior cortex of a migrating cell.Actin and myosin are thought to play significant roles in the biological machinery in nonmuscle cell movement. However, the locomotory mechanism of a cell has not been totally elucidated. Dictyostelium is a good material for studying cell motility because it migrates by ameboid movement, one of the most conservative mechanisms of cell motility. Biochemical and electron microscopic studies have revealed the significant concomitance of actin, myosin, and their associated proteins in cellular motile events of Dictyostelium (6, 12).The solation-contraction coupling hypothesis of Hellewell and Taylor (13) suggests a structural requirement of local breakdown of the gel for contraction in the motile extract of Dictyostelium. However, the structural organization of the contractile components in intact cells has not been fully clarified.Initial immunofluorescence using antiactin showed that vegetative amebae were stained uniformly whereas actively migrating cells are stained strongly at their leading edges (8). Recently, Bazari and Clarke (2) demonstrated that calmodulin and myosin are localized in the peripheral region. Condeelis et al. (5) and Brier et al. (3), using conventional immunofluorescence, found that 120-and 95-kdalton actin-binding proteins are also localized at the cell periphery.Partially because of the round shape and small size of Dictyoslelium amebae, no detailed information on the spatial organization of cytoskeletal components has been provided by conventional immunofluorescence. We thus improved the technique and identified the localization of microtubules (17,30). In the present study, we document the localization of actin and myos...

Myosin is thought to act as a major mechanochemical transducer in non-muscle cell motility, but the in situ organization of the molecules has not yet been determined. Here we report the localization of myosin 'rods', analogous to the thick filaments of muscle, by ameliorated immunofluorescence and demonstrate the dynamic translocation of these rods in response to exogenously added cyclic AMP, which is a chemoattractant for Dictyostelium amoebae. On addition of cyclic AMP, we observed instantaneous shedding of the endoplasmic myosin followed by an increase in cortical rods, the original distribution being recovered in a few minutes. We conclude that myosin filaments mediate Dictyostelium cell movement, probably by an assembly/disassembly cycle of the molecules in response to a chemotactic stimulus.

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.

Myosin II is a major component of a contractile ring. To examine if myosin II turns over in contractile rings, fluorescence of GFP–myosin II expressed in Dictyostelium cells was bleached locally by laser illumination, and the recovery was monitored. The fluorescence recovered with a half time of 7.01 ± 2.62 s. This recovery was not caused by lateral movement of myosin II from the nonbleached area, but by an exchange with endoplasmic myosin II. Similar experiments were performed in cells expressing GFP–3ALA myosin II, of which three phosphorylatable threonine residues were replaced with alanine residues. In this case, recovery was not detected within a comparable time range. These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation. Because GFP–3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time. Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings.

No abstract

Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A ؊ /B ؊ /C ؊ triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified. INTRODUCTIONMyosin II plays fundamental roles in a variety of cellular contractile processes, ranging from cytokinesis to cell migration and developmental morphogenesis (Ridley et al., 2003;Baumann, 2004;Van Haastert and Devreotes, 2004). A critical feature of nonmuscle myosin II isoforms is that cellular function requires the assembly of this motor protein into bipolar filament structures that can interact with cortical actin arrays. Filament assembly in nonmuscle cells is dynamic and subject to both spatial and temporal regulation. In mammalian nonmuscle cells, phosphorylation of the myosin II regulatory light chain (RLC) is a widely cited model for assembly regulation, with RLC phosphorylation favoring filament assembly (Scholey et al., 1980). However, there is also strong experimental support for other models of mammalian nonmuscle myosin II assembly control, including evidence for monomer sequestration by the S100 protein metastasin 1 (Li et al., 2003), and biochemical evidence that MHC phosphorylation may modulate filament assembly (Murakami et al., 1998;Murakami et al., 2000). Definitive in vivo studies to distinguish relative contributions of each mechanism have as yet not been performed.The simple amoeba Dictyostelium discoideum contains a single myosin II heavy chain gene, which serves cellular roles that are conserved with those of myosin II in other organisms. Biochemical and cellular studies in this system have established that myosin II filament assembly in vivo involves a dynamic equilibrium between a large pool of disassembled molecules and a pool of assembled filaments that associate with the cortical cytoskeleton. This equilibrium appears regulated by a variety of events ranging from chemoattractant receptor st...

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.

We observed the dynamics of actin foci in live Dictyostelium cells expressing GFP-actin. Actin foci were dynamic structures, but they were fixed on the substratum during cell migration. Interference reflection microscopy revealed that the ventral cell membrane was closer to the substratum at sites of actin foci. Furthermore, some actin foci were incorporated into the retraction fibers, ripped off from the cells and eventually shed on the substratum after the cells moved away. The velocity of the cells was inversely proportional to the number of actin foci. Measurement of traction force using a silicone substratum demonstrated that the traction force was transmitted to the substratum through actin foci. Taken together, several lines of evidence strongly suggest that actin foci function as the active `feet' of Dictyostelium cells. We also found evidence suggesting that changing step is regulated in a coordinated manner during cell migration. Possible mechanisms by which these cells migrate across substrata are discussed in this context.