No abstract

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.

In the simple amoeba Dictyostelium discoideum, myosin II filament assembly is regulated primarily by the action of a set of myosin heavy chain (MHC) kinases and by MHC phosphatase activity. Chemoattractant signals acting via G-protein coupled receptors lead to rapid recruitment of myosin II to the cell cortex, but the structural determinants on myosin necessary for translocation and the second messengers upstream of MHC kinases and phosphatases are not well understood. We report here the use of GFP-myosin II fusions to characterize the domains necessary for myosin II filament assembly and cytoskeletal recruitment during responses to global stimulation with the developmental chemoattractant cAMP. Analysis performed with GFP-myosin fusions, and with latrunculin A-treated cells, demonstrated that F-actin binding via the myosin motor domain together with concomitant filament assembly mediates the rapid cortical translocation observed in response to chemoattractant stimulation. A "headless" GFP-myosin construct lacking the motor domain was unable to translocate to the cell cortex in response to chemoattractant stimulation, suggesting that myosin motor-based motility may drive translocation. This lack of localization contrasts with previous work demonstrating accumulation of the same construct in the cleavage furrow of dividing cells, suggesting that recruitment signals and interactions during cytokinesis differ from those during chemoattractant responses. Evaluating upstream signaling, we find that iplA null mutants, devoid of regulated calcium fluxes during chemoattractant stimulation, display full normal chemoattractant-stimulated myosin assembly and translocation. These results indicate that calcium transients are not necessary for chemoattractant-regulated myosin II filament assembly and translocation.