Stabilization of Exocytosis by Dynamic F-actin Coating of Zymogen Granules in Pancreatic Acini

Abstract

Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca 2؉ -dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a cagedCa 2؉ compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.

Vesicle-associated Membrane Protein 8 (VAMP8) Is a SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Selectively Required for Sequential Granule-to-granule Fusion

“…First, SNARE movement has been shown to occur and, although our data argue that plasma membrane Q-SNAREs are not directly involved in granule-to-granule fusion, they could play an indirect signaling role. Second, soon after fusion the primary granules become coated with F-actin (37)(38)(39). This F-actin coating is also observed in oocytes (40) and type 2 pneumocytes (41) and has been suggested to be triggered post-fusion by lipid and/or protein interchange between the plasma membrane and the granule membrane (42).…”

Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy

“…Compound exocytosis has been defined as the process by which large SCGs fuse with SCGs that are already connected with the APM, generating strings of three to four interconnected granules (13). These strings, which have been reported to be stable for more than 20 s, should have been detectable under the imaging conditions used in this study (17,34). However, we cannot exclude that, due to the thin optical slices used to image the SCGs in the GFP mice, some of the interconnections between the fusing SCGs might have been missed.…”

“…2D) (27). This approach mimics the bathing of the explanted glands in small fluorescent probes that has been used to investigate exocytosis in ex vivo models (11,13,14,16,17,35). For this set of experiments, we used larger optical sections (1-1.2 μm, Fig.…”

“…In explanted SG slices and in acinar preparations from other exocrine tissues, both complete fusion events and compound exocytosis have been observed under different experimental conditions by using either differential interference contrast microscopy (9-11) or two-photon microscopy (4,11,(13)(14)(15)(16)(17). Finally, the role of actin during exocytosis has been extensively investigated, and various models have been proposed.…”

New insights into the control of secretion

“…22 Further, a growing number of recent reports, from a wide range of cell types, are reaching a consensus that F-actin and myosin 2 are dynamic regulators of complex vesicle behavior. Work shows that actin polymerization is triggered immediately after vesicle fusion forming an F-actin network around the vesicle [23][24][25][26][27][28] that keeps the fusion pore open 27 and stabilizes the vesicle shape. 23,24,29,30 In the last year two reports show that myosin 2 phosphorylation directly regulates fusion pore opening.…”