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.

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.

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.