Specific localization of serine 19 phosphorylated myosin II during cell locomotion and mitosis of cultured cells - PubMed (original) (raw)
Specific localization of serine 19 phosphorylated myosin II during cell locomotion and mitosis of cultured cells
F Matsumura et al. J Cell Biol. 1998.
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.
Figures
Figure 3
Bimodal distribution of P-myosin in a motile fibroblast of REF-2A. A motile REF-2A cell was double labeled with pp2b (A), and the monoclonal myosin antibody (B). (C) Phase contrast image. (D) Merged image (green, pp2b; red, anti–heavy chain antibody). (E) Ratio image of A/B. As shown in the merged image of D, both anterior (arrowhead) and posterior (the tail in particular) regions of the cell are greenish whereas the middle area is reddish, indicating that P-myosin is enriched in both anterior and posterior regions. The ratio image of E supports this notion. Asterisk, nucleus. A large white arrow in C shows the direction of cell locomotion. Bar, 10 μm.
Figure 1
Specificity of pp2b antibody against S19-phosphorylated regulatory myosin light chain. (a) The pp2b antibody reacts only with RMLC phosphorylated by MLCK (lane 2), but not with unphosphorylated RMLC (lane 1) nor RMLC phosphorylated by protein kinase C (lane 3). The identical samples of phosphorylated or unphosphorylated RMLC were separated by SDS-PAGE, transferred (lanes 1 and 4, unphosphorylated RMLC; lanes 2 and 5, RMLC phosphorylated by MLCK; lanes 3 and 6, RMLC phosphorylated by PKC), and then immunoblotted with either pp2b (lanes 1–3) or a monoclonal antibody against RMLC (lanes 4–6). Note that pp2b reacted only with RMLC phosphorylated by MLCK (lane 2), whereas the monoclonal antibody reacted with all kinds of RMLC (lanes 4–6). (b) Immunoblot of total cell lysates with pp2b antibody. The antibody reacted with a band whose relative molecular mass corresponds to RMLC (arrowhead). Note that the reactivity was considerably increased when the phosphorylation of RMLC in the total cell lysates is increased in vitro by incubation with MLCK (lane 2), confirming that the band is indeed phosphorylated RMLC. A band with an approximate molecular weight of 50,000 (asterisk) cross-reacted slightly with pp2b. The identity of the 50-kD band is currently unknown. (c) pp2b reacts predominantly with mono-phosphorylated RMLC. Urea/glycerol gel analyses were performed to separate unphosphorylated (0-p), mono-phosphorylated (1-p), di-phosphorylated (2-p), and myosin light chain–1 (a). Lanes 1 and 3, unphosphorylated myosin light chains; lanes 2 and 4, phosphorylated myosin light chains. Lanes 1 and 2 were stained with Coomassie blue, and lanes 3 and 4 were immunoreacted with pp2b. Note that pp2b reacted strongly with mono-phosphorylated (1-p), but not with unphosphorylated (0-p) RMLC. The reactivity to di-phosphorylated (2-p) RMLC was very weak.
Figure 2
Localization of S19-phosphorylated myosin (P-myosin) in motile fibroblasts. Gerbil fibroma cells were double stained with pp2b and a monoclonal antibody against myosin heavy chain. A shows immunofluorescence with pp2b; B shows immunofluorescence with the monoclonal myosin antibody; C shows a phase contrast image; and D shows a merged image (green, pp2b; red, monoclonal anti– heavy chain antibody). Note that P-myosin is enriched in microfilament bundles near membrane ruffles (arrow). The anterior region containing a nucleus (asterisk) is greenish, indicating that P-myosin is also enriched in this region. The upper side of each figure contains a well spread, relatively nonmotile cell (arrowhead). This cell is less stained with pp2b than with the monoclonal myosin antibody, indicating that the level of phosphorylation is low in such cell (see Fig. 4). Bar, 10 μm.
Figure 4
Localization of P-myosin in a fibroblast with a well-spread, nonpolarized morphology. A gerbil fibroma cell with well-developed stress fibers was double stained with pp2b (A) and the monoclonal myosin antibody (B). (C) A merged image (green, pp2b; red, the monoclonal myosin antibody). Note that cortical filaments (arrow) are stained more strongly with pp2b than with the monoclonal myosin antibody, indicating that P-myosin is enriched in cortical filaments. Membrane protrusions (arrowhead), as well as the area containing nucleus (asterisk) are also enriched with P-myosin. In contrast, stress fibers are red, indicating a lower level of myosin phosphorylation. Bar, 10 μm.
Figure 5
P-myosin is also enriched in peripheral microfilament bundles of a leading edge of a motile epithelial cell sheet. A motile epithelial cell sheet of NRK cells was double labeled with pp2b (A) and the monoclonal myosin antibody (B). (C) A merged image (green, pp2b; red, the monoclonal myosin antibody). Note that there is a discontinuous gradient of myosin phosphorylation; pp2b stains the microfilament bundles close to the cell periphery (arrow) more strongly than those present in an inner region of the cell (arrowhead). Also note that circumferential microfilament bundles adjacent to cell–cell contacts (asterisk) was less stained with pp2b than with the monoclonal myosin antibody. The merged image of C clearly shows that the most peripheral microfilament bundles are greenish whereas the circumferential bundles are reddish. Bar, 10 μm.
Figure 6
Localization of P-myosin during wound closing of epithelial cells. NRK cells were stained with pp2b (A) and the monoclonal myosin antibody (B). (C) Phase contrast. (D) Merged image (green, pp2b; red, the monoclonal myosin antibody). pp2b stains the region of closing (arrowhead) more strongly than the microfilament bundles in an inner region (asterisk), again indicating a discontinuous gradient of myosin phosphorylation. Bar, 10 μm.
Figure 7
Specific localization of P-myosin at a small opening of an epithelial sheet. (A) Staining with pp2b. (B) Staining with the biotinylated platelet myosin antibody. (C) A merged image (green, pp2b; red, platelet myosin antibody). Note that the ring or arc surrounding the opening (arrow) was strongly stained with pp2b, suggesting that myosin phosphorylation and contraction are required for the closure of an opening. The pp2b staining of circumferential actin bundles adjacent to cell–cell contacts (asterisk) is much weaker than is the staining with the anti–heavy chain antibody, indicating that phosphorylated myosin is not enriched in these circumferential actin bundles. Bar, 10 μm.
Figure 8
Immunolocalization of phosphorylated myosin in NRK cells at different mitotic stages. Top, phase-contrast; middle, stained with pp2b; bottom, stained with the biotinylated platelet myosin antibody. (A–C) Metaphase. Only spindle poles stained strongly (arrowhead). (D–F) Anaphase cell on the left. The midzone between the separating chromosomes was strongly stained (arrowhead). The neighboring cell was at prophase and only spindle pole staining was observed. (G–I) Telophase. A cleavage furrow (arrowhead) was strongly stained. Bar, 10 μm.
Figure 9
Simultaneous localization of phosphorylated myosin and F-actin in a mitotic cell at the beginning of late anaphase. (A) Stained with pp2b. (B) Stained with rhodamine phalloidin. (C) A phase contrast image. Note that actin accumulation occurs at approximately the same time as phosphorylated myosin appears.
Figure 10
Localization of P-myosin in dividing cells. Dividing REF-2A (A) and NRK epithelial (C) cells were double stained with pp2b and the monoclonal myosin antibody and color images are synthesized (green, pp2b; red, monoclonal myosin antibody). B and D, corresponding phase-contrast images. A, phosphorylated myosin is enriched in a cleavage furrow of an REF-2A cell at telophase (arrow). On the other hand, a cell at metaphase (arrowhead) is red, indicating that the phosphorylation level is low during metaphase. (C) Phosphorylated myosin is enriched in a cleavage furrow (arrow) of a dividing NRK cell. Note that stress fibers as well as circumferential bundles of surrounding cells are reddish, indicating that the phosphorylation level is low with these structures. Bar, 10 μm.
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