In vivo dynamics of myosin II in Dictyostelium by fluorescent analogue cytochemistry.

We used fluorescent analogue cytochemistry to study in vivo dynamics of myosin II in Dictyostelium discoideum. We labeled myosin with biotin or tetramethyl-rhodamine iodoacetamide (IATR). The labeled myosin shows normal activities as reversible filament assembly and Ca2+ and actin-activatable Mg(2+)-ATPase. We used the biotin-myosin as a probe examining the effects of microinjection on the amoebae and the ability to associate with endogenous actin cytoskeleton. The biotin-myosin incorporates into certain actin populations and localizes to the cortex with the highest accumulation in the posterior end of polarized amoebae. The dynamics in live amoebae were probed by TR-myosin. We monitored the dynamics for a long period to determined the dynamic reorganization corresponding specific cellular behaviors. The TR-myosin converges into a discrete actin- and myosin-rich structure located at the posterior end ("myosin-organizing center"). The rod-shaped TR-myosin exhibits linear orderly arrays emanating from the organizing center which extend about two-thirds of the cell length. The myosin arrays show a dynamic reorganization when the amoebae move. To examine if the observed myosin dynamics are related to filamentous (F-) actin, we disrupted the F-actin by cytochalasin D. The ratioed image of TR-myosin (vs. FITC-dextran) demonstrates that myosin in these cells accumulates in the cortex but does not form the organizing center. Overall, the results suggest that the filamentous myosin organizes into orderly arrays in the live cytoplasm and its translocation occurs by means of F-actin cables, converging into the organizing center.