Involvement of Rho GTPases in calcium-regulated exocytosis from adrenal chromaffin cells

The Rho GTPase family, including Rho, Rac and Cdc42 proteins, is implicated in various cell functions requiring the reorganization of actin-based stru ctures. In secretory cells, cytoskeletal rearrangements are a prerequisite for exocytosis. We previously described that, in chromaffin cells, the trim eric granule-bound Go protein controls peripheral actin and prevents exocyt osis in resting cells through the regulation of RhoA. To provide further in sight into the function of Rho proteins in exocytosis, we focus here on the ir intracellular distribution in chromaffin cells. By confocal immunofluore scence analysis, we found that Rad and Cdc42 are exclusively localized in t he subplasmalemmal region in both resting and nicotine-stimulated cells. In contrast, RhoA is associated with the membrane of secretory granules. We t hen investigated the effects of clostridial toxins, which differentially im pair the function of Rho GTPases, on the subplasmalemmal actin network and catecholamine secretion. Clostridium difficile toxin B, which inactivates R ho, Rac and Cdc42, markedly altered the distribution of peripheral actin fi laments; Neither Clostridium botulinum C3 toxin, which selectively ADP-ribo sylates Rho, nor Clostridium sordellii lethal toxin, which inactivates Rac, affected cortical actin, suggesting that Cdc42 plays a specific role in th e organization of subplasmalemmal actin. Indeed, toxin B strongly reduced s ecretagogue-evoked catecholamine release. This effect on secretion was not observed in cells having their actin cytoskeleton depolymerized by cytochal asin E or Clostridium botulinum C2 toxin, suggesting that the inhibition of secretion by toxin B is entirely linked to the disorganization of actin. C . sordellii lethal toxin also inhibited catecholamine secretion, but this e ffect was not related to the actin cytoskeleton as seen in cells pretreated with cytochalasin E or C2 toxin. In contrast, C3 exoenzyme did not affect secretion. We propose that Cdc42 plays an active role in exocytosis by coup ling the actin cytoskeleton to the sequential steps underlying membrane tra fficking at the site of exocytosis.