Myosin II dynamics in Dictyostelium: determinants for filament assembly and translocation to the cell cortex during chemoattractant responses.
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.
Levi
Stephanie
S
Department of Physiology and Biophysics, Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA.
Polyakov
Mark V
MV
Egelhoff
Thomas T
TT
D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002630:Chemotactic Factors / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004023:Dictyostelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D024462:Myosin Type II / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D000199:Actins; D002630:Chemotactic Factors; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D010749:Phosphoprotein Phosphatases; D024462:Myosin Type II; D002118:Calcium