Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells.

Abstract

Abstract. Although the actin cytoskeleton has been implicated in vesicle trafficking, docking and fusion, its site of action and relation to the Ca2÷-mediated activation of the docking and fusion machinery have not been elucidated. In this study, we examined the role of actin filaments in regulated exocytosis by introducing highly specific actin monomer-binding proteins, the ~-thymosins or a gelsolin fragment, into streptolysin O-permeabilized pancreatic acinar cells. These proteins had stimulatory and inhibitory effects. Low concentrations elicited rapid and robust exocytosis with a profile comparable to the initial phase of regulated exocytosis, but without raising [Ca2+], and even when [Ca 2÷] was clamped at low levels by EGTA. No additional cofactors were required.Direct visualization and quantitation of actin filaments showed that B-thymosin, like agonists, induced actin depolymerization at the apical membrane where exocytosis occurs. Blocking actin depolymerization by phalloidin or neutralizing fl-thymosin by complexing with exogenous actin prevented exocytosis. These findings show that the cortical actin network acts as a dominant negative clamp which blocks constitutive exocytosis. In addition, actin filaments also have a positive role. High concentrations of the actin depolymerizing proteins inhibited all phases of exocytosis. The inhibition overrides stimulation by agonists and all downstream effectors tested, suggesting that exocytosis cannot occur without a minimal actin cytoskeletal structure.T hE final steps of regulated exocytosis involve vesicle docking, triggering, and membrane fusion. There is now increasing evidence that regulated exocytosis employs a constitutively operating fusion machinery shared by many vesicular trafficking processes and specialized clamps to prevent fusion until the appropriate signals are received (4,40,41). The actin network under the plasma membrane has long been proposed as a physical barrier to granule docking because it transiently depolymerizes during exocytosis (3,30,42,43). The cortical actin can therefore be considered as a part of the clamping apparatus. However, in many cell types, drugs which depolymerize actin do not elicit exocytosis but can potentiate agonist-evoked responses (23,25,38). On the basis of such evidence, it was suggested that dissolution of the actin cytoskeleton is a necessary but not sufficient part of regulated exocytosis. Nevertheless, the exact role of actin in exocytosis remains unclear, since contradictory results were obtained in other cells (1) and between intact and permeabilized cells (19). Furthermore, some cells have cytochalasin-insensitive pools of actin filaments (8). A large part of the uncertainty is due to the nonspecific nature of some of the drugs, which precludes unequivocal conclusions.Address all correspondence to Dr. Helen L. Yin, Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, 648-8685. In the present study, we used a different approach to examine th...

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