LOCALIZATION OF CALDESMON AND ITS DEPHOSPHORYLATION DURING CELL-DIVISION

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle calde smon to dissociate from microfilaments during prometaphase. (Yamashiro , S., Y. Yamakita, R. Ishikawa, and F. Matsumura. 1990. Nature (Lond.) . 344:675-678; Yamashiro, S., Y. Yamakita, H. Hosoya, and F. Matsumura . 1991. Nature (Lond.) 349:169-172). To explore the functions of calde smon phosphorylation during cytokinesis, we have examined the relation ship between the phosphorylation level, actin-binding, and in vivo loc alization of caldesmon in cultured cells after their release of metaph ase arrest. Immunofluorescence studies have revealed that caldesmon is localized diffusely throughout cytoplasm in metaphase. During early s tages of cytokinesis, caldesmon is still diffusely present and not con centrated in contractile rings, in contrast to the accumulation of act in in cleavage furrows during cytokinesis. In later stages of cytokine sis, most caldesmon is observed to be yet diffusely localized although some concentration of caldesmon is observed in cortexes as well as in cleavage furrows. When daughter cells begin to spread, caldesmon show s complete colocalization with F-actin-containing structures. These ob servations are consistent with changes in the levels of microfilament- associated caldesmon during synchronized cell division. Caldesmon is m issing from microfilaments in prometaphase cells arrested by nocodazol e treatment, as shown previously (Yamashiro, S., Y. Yamakita, R. Iskik awa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678). The level o f microfilament-associated caldesmon stays low (12% of that of interph ase cells) when some cells start cytokinesis at 40 min after the relea se of metaphase arrest. When 60% of cells finish cytokinesis at 60 min , the level of microfilament-associated caldesmon is recovered to 50% of that of interphase cells. The level of microfilament-associated cal desmon is then gradually increased to 80% when cells show spreading at 120 min. Dephosphorylation appears to occur during cytokinesis. It st arts when cells begin to show cytokinesis at 40 min and completes when most cells finish cytokinesis at 60 min. These results suggest that c aldesmon is not associated with microfilaments of cleavage furrows at least in initial stages of cytokinesis and that dephosphorylation of c aldesmon appears to couple with its reassociation with microfilaments. Because caldesmon is known to inhibit actomyosin ATPase and/or regula te actin assembly, its continued dissociation from microfilaments may be required for the assembly and/or activation of contractile rings.