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 structures. In secretory cells, cytoskeletal rearrangements are a prerequisite for exocytosis. We previously described that, in chromaffin cells, the trimeric granule-bound Go protein controls peripheral actin and prevents exocytosis in resting cells through the regulation of RhoA. To provide further insight into the function of Rho proteins in exocytosis, we focus here on their intracellular distribution in chromaffin cells. By confocal immunofluorescence analysis, we found that Rac1 and Cdc42 are exclusively localized in the subplasmalemmal region in both resting and nicotine-stimulated cells. In contrast, RhoA is associated with the membrane of secretory granules. We then investigated the effects of clostridial toxins, which differentially impair the function of Rho GTPases, on the subplasmalemmal actin network and catecholamine secretion. Clostridium difficile toxin B, which inactivates Rho, Rac and Cdc42, markedly altered the distribution of peripheral actin filaments. Neither Clostridium botulinum C3 toxin, which selectively ADP-ribosylates Rho, nor Clostridium sordellii lethal toxin, which inactivates Rac, affected cortical actin, suggesting that Cdc42 plays a specific role in the organization of subplasmalemmal actin. Indeed, toxin B strongly reduced secretagogue-evoked catecholamine release. This effect on secretion was not observed in cells having their actin cytoskeleton depolymerized by cytochalasin 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 effect 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 coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis.