Dynamics of SNARE Assembly and Disassembly during Sperm Acrosomal Exocytosis

<div><p>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 <em>cis</em> complexes. Upon Ca²⁺ entry into the cytoplasm, Rab3 is activated and triggers NSF/α-SNAP-dependent disassembly of <em>cis</em> SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose <em>trans</em> 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 <em>trans</em> SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis.</p>