DIFFERENTIAL LOCALIZATION OF MYOSIN AND MYOSIN PHOSPHATASE SUBUNITS IN SMOOTH-MUSCLE CELLS AND MIGRATING FIBROBLASTS

Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin -activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20 -kDa subunit. The distribution of M130 and PP1C as well as myosin II w as examined in smooth muscle cells and fibroblasts by immunofluorescen ce microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 w as different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M 130 was recovered in a soluble fraction during permeabilization of cel ls, but the conditions used affected the solubility of both M130 and m yosin. The PP1C alpha isoform colocalized with M130 and also was in th e nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentr ated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated my osin, inhibition of myosin LC20 phosphatase, probably by phosphorylati on of the M130 subunit, may be required for cell migration.