Dynamics of SNARE assembly and disassembly during sperm acrosomal exocytosis.
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/alpha-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/alpha-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.
De Blas
Gerardo A
GA
Instituto de Histología y Embriología, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina.
Roggero
Carlos M
CM
Tomes
Claudia N
CN
Mayorga
Luis S
LS
D000177:Acrosome / Q000502:physiology*; D020101:Acrosome Reaction / Q000502:physiology; D001905:Botulinum Toxins / Q000494:pharmacology; D019274:Botulinum Toxins, Type A; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D010539:Permeability / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D050600:SNARE Proteins / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D050857:Synaptotagmins / Q000502:physiology; D013744:Tetanus Toxin / Q000494:pharmacology; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism; D020826:rab3A GTP-Binding Protein / Q000502:physiology*
D002614:Chelating Agents; D005029:Ethylenediamines; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D050988:Vesicle-Associated Membrane Protein 2; C508966:o-nitrophenylegtazic acid acetoxymethyl ester; C096323:rimabotulinumtoxinB; D050857:Synaptotagmins; D000001:Calcimycin; D004533:Egtazic Acid; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D020826:rab3A GTP-Binding Protein; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium; C096326:botulinum toxin type E