Abstract
The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle—the acrosome—is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca2+ chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca2+ entry into the cytoplasm, Rab3 is activated and triggers NSF/α-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/α-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP)–specific neurotoxins. Ca2+ must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis.
Highlights
Regulated exocytosis is a sophisticated process that requires the specific attachment of secretory granules to the plasma membrane and the opening of fusion pores connecting the interior of the granule to the extracellular medium [1]
O-nitrophenyl EGTA–acetoxymethyl ester (NP-EGTA-AM ) is a photolabile Ca2þ chelator that prevents inducer-triggered Acrosomal exocytosis (AE) in sperm permeabilized by streptolysin-O (SLO); NPEGTA-AM does this by entering the cytosol, diffusing through the outer acrosomal membrane, and accumulating inside the acrosome [14]
Antibodies against syntaxin1A, synaptosome-associated protein of 25 kD (SNAP25), VAMP2, and synaptotagmin VI were able to abrogate exocytosis even when added after the inducer (Figure 1)
Summary
Regulated exocytosis is a sophisticated process that requires the specific attachment of secretory granules to the plasma membrane and the opening of fusion pores connecting the interior of the granule to the extracellular medium [1]. At the core of this paradigm, Rabs promote the tethering—loose and reversible attachment—of the compartments that will fuse [3]. The assembly of heterotrimeric trans SNARE complexes brings about the docking— tight and irreversible attachment—of the fusing membranes [4]. Docking is followed by the opening and expansion of the fusion pore. In regulated exocytosis, this final stage requires an increase in cytoplasmic Ca2þ and the action of Ca2þ sensor proteins such as synaptotagmin [1]. SNAREs remain engaged in heterotrimeric cis complexes. Of the latter is achieved by the concerted action of a-SNAP and NSF, and is required to prepare SNAREs for subsequent rounds of fusion
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