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

Natasha Behrendorff,Subhankar Dolai,Wanjin Hong,Herbert Y Gaisano,Peter Thorn

doi:10.1074/jbc.m111.265199

Abstract

Compound exocytosis is found in many cell types and is the major form of regulated secretion in acinar and mast cells. Its key characteristic is the homotypic fusion of secretory granules. These then secrete their combined output through a single fusion pore to the outside. The control of compound exocytosis remains poorly understood. Although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as syntaxin 2, SNAP23 (synaptosome-associated protein of 23 kDa), and SNAP25 have been suggested to play a role, none has been proven. Vesicle-associated membrane protein 8 (VAMP8) is a SNARE first associated with endocytic processes but more recently has been suggested as an R-SNARE in regulated exocytosis. Secretion in acinar cells is reduced when VAMP8 function is inhibited and is less in VAMP8 knock-out mice. Based on electron microscopy experiments, it was suggested that VAMP8 may be involved in compound exocytosis. Here we have tested the hypothesis that VAMP8 controls homotypic granule-to-granule fusion during sequential compound exocytosis. We use a new assay to distinguish primary fusion events (fusion with the cell membrane) from secondary fusion events (granule-granule fusion). Our data show the pancreatic acinar cells from VAMP8 knock-out animals have a specific reduction in secondary granule fusion but that primary granule fusion is unaffected. Furthermore, immunoprecipitation experiments show syntaxin 2 association with VAMP2, whereas syntaxin 3 associates with VAMP8. Taken together our data indicate that granule-to-granule fusion is regulated by VAMP8 containing SNARE complexes distinct from those that regulate primary granule fusion.

Highlights

In the case of acinar cells the apical plasma membrane area is relatively small compared with the total membrane area and is defined by tight junctional boundaries [3]
We confirmed this with singlecell phase contrast imaging and observe that the tight clustering of zymogen granules in the luminal region of the acinar cells in wild type (WT) animals changes to a cell-wide distribution of granules in Vesicle-associated membrane protein 8 (VAMP8) knock-out acinar cells (Fig. 1)
Identification of Compound Exocytosis—To image singlegranule fusion events, we employ two-photon imaging of the entry of extracellular dyes in to fused granules, a method that can identify compound exocytic events [5, 6]. In these studies compound exocytosis was determined on the basis of the spatial appearance of granule fusion events, i.e. fusion of granules apparently far away from the cell membrane is ascribed as a compound exocytic event

Summary

Introduction

In the case of acinar cells the apical plasma membrane area is relatively small compared with the total membrane area and is defined by tight junctional boundaries [3]. Evidence in pancreatic ␤ cells that shows that another SNARE, SNAP25, moves selectively into primary granules as a prelude to secondary fusion [10]. Perhaps the best evidence for SNARE control of compound exocytosis is the observation in mast cells that function blocking antibodies inhibit translocation of SNAP25 into secretory granules and significantly knock down the secretory response [11].

Results

Conclusion