Role of Rabphilin3a in human sperm acrosomal exocitosis?

QUEVEDO MF; BUSTOS MA; TOMES CN

Buenos Aires

Congreso; Argentine Society for Biochemistry and Molecular Biology (SAIB) scientific meeting "Molecular mechanisms in cell signaling and gene expression; 2013

Argentine Society for Biochemistry and Molecular Biology (SAIB)

Mammalian sperm contain a single secretory granule: the acrosome. The acrosome reaction (RA) is a type of regulated exocytosis that releases the acrosomal granule content in the vicinity of the egg during fertilization. Rabphilin is a secretory vesicle protein that interacts with the GTP-bound form of the small GTPases Rab3 and Rab27. Rabphilin consists of an N-terminal Rab binding domain, a linker region of unknown function, and a C-terminal tandem of C2 domains (C2A and C2B) that interact with the SNARE protein SNAP25 and membrane phospholipids. Both Rab3 and Rab27 exhibit crucial roles during the AR but their effectors have not yet been determined. Here, we report that Rabphilin3a is present in human sperm and localizes to the acrosomal region. By means of a functional assay using antibodies in combination with a photosensitive intravesicular calcium chelator, we found that Rabphilin3a participates in an early as well as a late stages during membrane fusion. When introduced into streptolysin O-permeabilized sperm, both wild type and a V61A point mutant (with impaired binding to Rab3) of Rabphilin3a abolished the AR triggered by calcium. We plan to use several other mutants to gather mechanistic information about the specific interactions of Rabphilin during the human sperm AR and demonstrate that Rabphilin3a is a relevant secretory Rabs effector