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 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...

Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 microM blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 microM), which inhibits Rho-kinase, was similar to 30 microM blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 microM blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 microM blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis.

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