SPECIFIC LOCALIZATION OF SERINE-19 PHOSPHORYLATED MYOSIN-II DURING CELL LOCOMOTION AND MITOSIS OF CULTURED-CELLS

Phosphorylation of the regulatory light chain of myosin II (RMLC) at S erine 19 by a specific enzyme, MLC kinase, is believed to control the contractility of actomyosin in smooth muscle and vertebrate nonmuscle cells, To examine how such phosphorylation is regulated in space and t ime within cells during coordinated cell movements, including cell loc omotion and cell division, we generated a phosphorylation-specific ant ibody. Motile fibroblasts with a polarized cell shape exhibit a bimoda l distribution of phosphorylated myosin along the direction of cell mo vement. The level of myosin phosphorylation is high in an anterior reg ion near membrane ruffles, as well as in a posterior region containing the nucleus, suggesting that the contractility of both ends is involv ed in cell locomotion, Phosphorylated myosin is also concentrated in c ortical microfilament bundles, indicating that cortical filaments are under tension, The enrichment of phosphorylated myosin in the moving e dge is shared with an epithelial cell sheet; peripheral microfilament bundles at the leading edge contain a higher level of phosphorylated m yosin, On the other hand, the phosphorylation level of circumferential microfilament bundles in cell-cell contacts is low, These observation s suggest that peripheral microfilaments at the edge are involved in f orce production to drive the cell margin forward while microfilaments in cell-cell contacts play a structural role, During cell division, bo th fibroblastic and epithelial cells exhibit an increased level of myo sin phosphorylation upon cytokinesis, which is consistent with our pre vious biochemical study (Yamakita, Y., S. Yamashiro, and F. Matsumura, 1994. J. Cell Biol. 124:129-137). In the case of the NRK epithelial c ells, phosphorylated myosin first appears in the midzones of the separ ating chromosomes during late anaphase, but apparently before the form ation of cleavage furrows, suggesting that phosphorylation of RMLC is an initial signal for cytokinesis.