Sequence of Events Leading to Membrane Fusion during Acrosomal Exocytosis in Human spermatozoa

G.A. DE BLAS, C.M. ROGGERO, S.A. BELMONTE, C.N. TOMES, L.S. MAYORGA

Mérida, México

Congreso; Howard Hughes Medical Institute Meeting of International Research Scholars; 2005

Howard Hughes Medical Institute

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to stimuli, it undergoes a special type of calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. SNARE (SNAP receptor) complexes play a central role in most membrane fusion events. Acrosomal exocytosis has several unique features that allow for a detailed characterization of the dynamics (and its regulation) of assembly and disassembly of SNARE complexes during membrane fusion. The release of the acrosomal content is an all-or-nothing event that occurs as a synchronized wave, with no recycling of components, and requires neurotoxin-sensitive SNAREs. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca2+ chelator, we have shown that SNAREs are associated in cis complexes in resting sperm. Upon Ca2+ entry into the cytoplasm, Rab3A is activated and anchored to the membranes in a process that is modulated by cholesterol. Interestingly, this sterol does not affect late steps of the membrane fusion process. Active Rab3A triggers the disassembly of cis SNARE complexes by means of N-ethlymaleimide–sensitive factor (NSF) and alphaSNAP (soluble NSF attachment protein). SNAREs are then free to reassemble in loose trans complexes that are resistant to NSF/alphaSNAP action. At this point, Ca2+ must be released from inside the acrosome to initiate the final steps of membrane fusion, which require fully assembled trans SNARE complexes and synaptotagmin VI. The function of this Ca2+-sensing protein is regulated by PKC-mediated phosphorylation of a conserved polybasic domain necessary for interaction with multiple effectors. Synaptotagmin desphosphorylation is probably an early event during exocytosis, allowing the interaction of this protein with other components of the fusion machinery. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drives membrane fusion during the acrosomal exocytosis of human spermatozoa.2+ chelator, we have shown that SNAREs are associated in cis complexes in resting sperm. Upon Ca2+ entry into the cytoplasm, Rab3A is activated and anchored to the membranes in a process that is modulated by cholesterol. Interestingly, this sterol does not affect late steps of the membrane fusion process. Active Rab3A triggers the disassembly of cis SNARE complexes by means of N-ethlymaleimide–sensitive factor (NSF) and alphaSNAP (soluble NSF attachment protein). SNAREs are then free to reassemble in loose trans complexes that are resistant to NSF/alphaSNAP action. At this point, Ca2+ must be released from inside the acrosome to initiate the final steps of membrane fusion, which require fully assembled trans SNARE complexes and synaptotagmin VI. The function of this Ca2+-sensing protein is regulated by PKC-mediated phosphorylation of a conserved polybasic domain necessary for interaction with multiple effectors. Synaptotagmin desphosphorylation is probably an early event during exocytosis, allowing the interaction of this protein with other components of the fusion machinery. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drives membrane fusion during the acrosomal exocytosis of human spermatozoa.