Abstract. Myosin II purified from mammalian nonmuscle cells is phosphorylated on the 20-kD light chain subunit (MLC2o) by the Cat+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) . The importance of MLC2o phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Cat+/calmodulin-independent, constitutively active form of MLCK (MK -) into macrophages . The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MKAb compared to macrophages containing control antibodies . Moreover, there is an C ELLULAR locomotion by mammalian cells is essential for embryogenesis, cell-mediated killing (4), and the formation of metastatic colonies by cancer cells. Cell motility is a complex process that requires the coordinated regulation and the interaction of numerous reactions. ATP hydrolysis by actin and myosin II and myosin II polymerization/depolymerization are thought to be among the reactions involved in mediating translational motility (2, 23) . In mammalian nonmuscle cells, both ATP hydrolysis (3,30,31) and filament formation (1Q 27) by myosin II are regulated by phosphorylation of the 20-kD light chain of myosin (MLC2o)' by myosin light chain kinase (MLCK) (2) . Therefore, MLCK and MLC20 phosphorylation are thought to play critical roles in regulating cell motility.However, the role of filamentous myosin (myosin II) in cell motility is unclear. Experiments on the slime mold Dictyostelium discoideum have questioned the importance of filamentous myosin in cell motility (14,22) . Dictyostelium contains two myosins designated myosin I and myosin II (24) . Myosin I is a single-headed myosin with a short heavy chain that does not form filaments (24) . Myosin I associates with lipids (1) and has been localized in the leading edges of lamellipodia of migrating Dictyostelium ameba (17 inverse relationship between the number of cells that migrate and the amount of MKAb introduced into cells . Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK-introduced into cells. Other experiments demonstrated that MKAb decreased intracellular MLC 20 phosphorylation while MK-increased MLC2o phosphorylation . MK-also increased the amount of myosin associated with the cytoskeleton . These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC2o phosphorylation must be maintained within narrow limits during translational motility by mammalian cells.II is similar to mammalian muscle and nonmuscle myosins in that it has two globular heads, a coiled-coiled tail, and an ability to form filaments . In contrast to mammalian nonmuscle and smooth muscle myosin II, ATP hydrolysis and filament formation by Dictyostelium myosin II are regulated by both heavy chain and light chain phosphorylation (24) . Interestingly, Diciyostelium in which myosin II heavy chain expres...

The introduction of impermeant probes such as antibodies and other proteins into living cells without compromising physiological function is an important approach for studying cellular regulatory mechanisms. Many techniques including direct microinjection, liposome-mediated delivery, fusion of red cell ghosts, and osmotic lysis of pinocytic vesicles have been used to introduce proteins into intact cells. We have used a modification of the voltage-discharge technique to introduce antibodies and other proteins into living physiologically responsive pheochromocytoma and other cultured cells. In this technique, called electroinjection, a single discharge of relatively low field strength is used to transiently permeabilize the plasma membrane. Our experiments demonstrate that electroinjection permits the introduction of large amounts (microM) of probe into 2-5 x 10(6) cells simultaneously without compromising cell viability or physiological responsiveness when performed under carefully defined conditions. They also demonstrate that electroinjection results in a single population of loaded cells and that protein incorporation is a function of field strength, capacitance, molecular weight of the protein, and the concentration of the protein in the electroinjection buffer. Interestingly, a significant fraction of the protein electroinjected into cells is trapped in the plasma membrane when cells are shocked at high capacitance. These results demonstrate that electroinjection appears to be an efficient method for loading exogenous proteins into cells while maintaining the integrity of the physiological properties of the cell.

Cellular locomotion results from a series of spatially and temporally integrated reactions. The coordinated regulation of these reactions requires sensitive intracellular signaling mechanisms. Because protein phosphorylation reactions represent important signaling mechanisms in mammalian cells, we investigated the effect of okadaic acid, a phosphoprotein phosphatase inhibitor, on protein phosphorylation and macrophage motility. Okadaic acid was applied to rat alveolar macrophages, and motility was quantitated by a directed chemotaxis assay. Okadaic acid inhibits macrophage motility in a dose-dependent fashion; the concentrations for 50 and 100% inhibition were 3 and 25 microM, respectively. Protein phosphorylation studies demonstrated a 2.5-fold increase in total protein phosphorylation in macrophages treated with 25 microM okadaic acid. These experiments also demonstrated a dose-dependent increase in the phosphorylation of the 20-kDa light chain of myosin. Moreover, 25 microM okadaic acid 1) maximally increased myosin light chain phosphorylation by 6.6-fold, 2) raised the level of myosin associated with the cytoskeleton from a basal level of 47.0 to 96.7% of the total myosin, and 3) induced profound morphological changes as visualized by scanning electron microscopy. These data correlate an increase in protein phosphorylation with a decrease in macrophage motility. Furthermore, they suggest that phosphoprotein phosphatase inhibition may prevent motility by uncoupling coordinated processes, such as cytoskeletal reorganization, that are essential for macrophage motility.