Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C (original) (raw)

1992, The Journal of Cell Biology

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified . A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC . It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils . Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBDphallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin . [31P]ATP was used to detect protein phosphorylation in electroporated cells . Exposure of neutrophils to 4/3-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the xPOSURE of neutrophils to a variety of agents including chemotactic factors and phorbol esters initiates a coordinated series ofbiological responses . Several of these responses, including shape change, projection of membrane ruffles and pseudopodia, cell motility, and phagocytosis, are dependent on the mechanical displacement of part or all of the cell. This in turn requires the assembly and reorganization ofcytoskeletal microfilaments, which are composed primarily of actin (reviewed by . Actin also plays a major structural role in resting and activated neutrophils . Despite the importance of actin-containing microfilaments, the molecular basis of the processes that signal their assembly and reorganization