Filamentous Actin Regulates Insulin Exocytosis through Direct Interaction with Syntaxin 4

Wei Luo,Jenna L Jewell,Debbie C Thurmond,Eunjin Oh,Zhanxiang Wang

doi:10.1074/jbc.m709876200

Abstract

Glucose-induced insulin exocytosis is coupled to associations between F-actin and SNARE proteins, although the nature and function of these interactions remains unknown. Toward this end we show here that both Syntaxin 1A and Syntaxin 4 associated with F-actin in MIN6 cells and that each interaction was rapidly and transiently diminished by stimulation of cells with d-glucose. Of the two isoforms, only Syntaxin 4 was capable of interacting directly with F-actin in an in vitro sedimentation assay, conferred by the N-terminal 39-112 residues of Syntaxin 4. The 39-112 fragment was capable of selective competitive inhibitory action, disrupting endogenous F-actin-Syntaxin 4 binding in MIN6 cells. Disruption of F-actin-Syntaxin 4 binding correlated with enhanced glucose-stimulated insulin secretion, mediated by increased granule accumulation at the plasma membrane and increased Syntaxin 4 accessibility under basal conditions. However, no increase in basal level Syntaxin 4-VAMP2 association occurred with either latrunculin treatment or expression of the 39-112 fragment. Taken together, these data disclose a new underlying mechanism by which F-actin negatively regulates exocytosis via binding and blocking Syntaxin 4 accessibility, but they also reveal the existence of additional signals and/or steps required to trigger the subsequent docking and fusion steps of exocytosis.

Highlights

F-actin was originally shown to function as a “cell web” in islet beta cells [7,8,9,10]
We have previously demonstrated that, in rodent islet beta cells, glucose-induced insulin exocytosis is coupled to the interaction of F-actin with t-SNARE proteins [21]
Similar to rat islets, isolated mouse islets showed a robust response to 2 h of glucose stimulation, which was further potentiated by latrunculin (Fig. 1A)

Summary

Introduction

F-actin was originally shown to function as a “cell web” in islet beta cells [7,8,9,10]. In three inde- taxin 1A, Syntaxin 4 was found to have some homology to a pendent sets of fractions, latrunculin induced an ϳ2.5-fold motif known as “spectrin-like.” To determine whether Synincrease in VAMP2-bound insulin granules localized to the PM, taxin 4 might bind in a direct manner to F-actin, an in vitro with a coordinate ϳ40% loss from the SG pool (Fig. 2, C and D).

Results

Conclusion