CYTOPLASMIC DYNAMICS OF MYOSIN-IIA AND MYOSIN-IIB - SPATIAL SORTING OF ISOFORMS IN LOCOMOTING CELLS

Different isoforms of non-muscle myosin II have different distribution s in vivo, even within individual cells. In order to understand how th ese different distributions arise, the distribution and dynamics of no n-muscle myosins IIA and myosin IIB were examined in cultured cells us ing immunofluorescence staining and time-lapse imaging of fluorescent analogs. Cultured bovine aortic endothelia contained both myosins IIA and IIB, Both isoforms distributed along stress fibers, in linear or p unctate aggregates within lamellipodia, and diffusely around the nucle us. However, the A isoform was preferentially located toward the leadi ng edge of migrating cells when compared with myosin IIB by double imm unofluorescence staining. Conversely, the B isoform was enriched in st ructures at the cells' trailing edges. When fluorescent analogs of the two isoforms were co-injected into living cells, the injected myosins distributed with the same disparate localizations as endogenous myosi ns IIA and IIB, This indicated that the ability of the myosins to 'sor t' within the cytoplasm is intrinsic to the proteins themselves, and n ot a result of localized synthesis or degradation. Furthermore, time-l apse imaging of injected analogs in living cells revealed differences in the rates at which the two isoforms rearranged during cell movement , The A isoform appeared in newly formed structures more rapidly than the B isoform, and was also lost more rapidly when structures disassem bled, These observations suggest that the different localizations of m yosins IIA and IIB reflect different rates at which the isoforms trans it through assembly, movement and disassembly within the cell. The rel ative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based struc tures in that cell.