IMAGING OF PROTEIN-KINASE-C DISTRIBUTION AND TRANSLOCATION IN LIVING VASCULAR SMOOTH-MUSCLE CELLS

The subcellular distribution of protein kinase C was directly imaged in single living smooth muscle cells using a new fluorescent protein kinase C probe. The probe localized prominently to perinuclear organelles and, to a lesser extent, to the cytosol and surface membrane. The perinuclear signal did not detectably change over the time observed. The ratio between the surface membrane intensity and that in the cytosol (R) was measured in parallel with cell shortening. In 1 mM extracellular Ca2+, the time to peak R and time to peak shortening were not significantly different. In Ca2+-free solution, no significant increase in the surface membrane/cytosol fluorescence ratio was observed with time, and shortening was inhibited. These results provide a new method for monitoring protein kinase C localization in living cells and directly demonstrate that protein kinase C moves in a Ca2+-dependent fashion from the cytosol to the surface membrane simultaneously with contraction.