Specific Localization of Serine 19 Phosphorylated Myosin II during Cell Locomotion and Mitosis of Cultured Cells

Abstract

Phosphorylation of the regulatory light chain of myosin II (RMLC) at Serine 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 time within cells during coordinated cell movements, including cell locomotion and cell division, we generated a phosphorylation-specific antibody.Motile fibroblasts with a polarized cell shape exhibit a bimodal distribution of phosphorylated myosin along the direction of cell movement. The level of myosin phosphorylation is high in an anterior region near membrane ruffles, as well as in a posterior region containing the nucleus, suggesting that the contractility of both ends is involved in cell locomotion. Phosphorylated myosin is also concentrated in cortical microfilament bundles, indicating that cortical filaments are under tension. The enrichment of phosphorylated myosin in the moving edge is shared with an epithelial cell sheet; peripheral microfilament bundles at the leading edge contain a higher level of phosphorylated myosin. On the other hand, the phosphorylation level of circumferential microfilament bundles in cell–cell contacts is low. These observations suggest that peripheral microfilaments at the edge are involved in force production to drive the cell margin forward while microfilaments in cell–cell contacts play a structural role. During cell division, both fibroblastic and epithelial cells exhibit an increased level of myosin phosphorylation upon cytokinesis, which is consistent with our previous biochemical study (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129–137). In the case of the NRK epithelial cells, phosphorylated myosin first appears in the midzones of the separating chromosomes during late anaphase, but apparently before the formation of cleavage furrows, suggesting that phosphorylation of RMLC is an initial signal for cytokinesis.

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

“…It should be noted that the staining of the nucleus, in addition to the filamentous structures, was also observed in the serum-starved cells, suggesting that the phosphorylated MLC 20 at Ser1/Ser2 seems to be present in the nucleus. This observation is similar to the previous reports that the phosphorylated MLC 20 was found in the nucleus (Matsumura et al, 1998;Nagano et al, 2006). After PDGF stimulation, the disassembly of the stress fibers was observed ( Figure 2A, j-l).…”

Dissecting the Role of Rho-mediated Signaling in Contractile Ring Formation

“…It has been reported that MRLC is phosphorylated at the cleavage furrow (Matsumura et al, 1998;Murata-Hori et al, 1998;Ueda et al, 2002). We found that phosphorylated MRLC colocalized with Rho at the equatorial region before furrow ingression ( Figure 3F, Mock).…”

“…It has been reported that MRLC is phosphorylated at the cleavage furrow (Matsumura et al, 1998;Murata-Hori et al, 1998) and that this phosphorylation occurs before furrow ingression (Matsumura et al, 1998). In this study, we have shown that the Rho inhibitor C3 and siRNA to ECT2, MgcRacGAP, or MKLP1 inhibit accumulation of phosphorylated MRLC at the equatorial region.…”

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

“…The broad area of RhoA activation that we detected after 3 min of EGF stimulation is similar to the localization of phosphorylated myosin light chain, which is regulated by myosin light chain kinase and ROCK [40]. To test whether the effect of Rho/ROCK inhibition on protrusion was due to a disruption of the contractile apparatus, we treated the cells with the myosin light chain kinase inhibitor ML-7.…”