IHEM   20887
INSTITUTO DE HISTOLOGIA Y EMBRIOLOGIA DE MENDOZA DR. MARIO H. BURGOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Biochemical characterization of Rab3 and Rab27 during human sperm exocytosis
Autor/es:
BUSTOS, MATÍAS; LUCCHESI, ORNELLA; RUETE, MARÍA C; TOMES, CLAUDIA
Lugar:
San Luis
Reunión:
Congreso; XLVII Reunión Anual Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2011
Institución organizadora:
SAIB
Resumen:
The two secretory Rabs, Rab3 and Rab27, regulate distinct steps of vesicle exocytosis in neuroendocrine cells. Sperm contain a single, large dense-core granule that is released by regulated exocytosis (the acrosome reaction, AR) during fertilization. The AR utilizes the same fusion machinery as neurons and neuroendocrine cells. Here, we characterize the behaviour of Rab3 in human sperm and show for the first time the requirement of Rab27 during the AR. We detected Rab27 in human sperm by Western blot and indirect immunofluorescence. Calcium was unable to elicit the AR when permeabilized human sperm was loaded with anti-Rab27 antibodies or with Slac2-b, a Rab27 effector, indicating that this small GTPase is essential for exocytosis. Anti-Rab27 and anti-Rab3 antibodies had different effects when added to sperm after cis SNARE complex disassembly and when exocytosis was triggered by high calcium concentrations. Rab27 was predominantly membrane-bound at all times, whereas the population of membrane-bound Rab3 augmented in response to exocytosis inducers. By means of pull down assays with specific protein cassettes we found that a substantial proportion of these Rabs was active (GTP-bound) in resting cells; AR inducers increased the levels even further but to different degrees for each Rab. These results indicate that there are distinct points of action for these two small GTPases