IHEM   20887
INSTITUTO DE HISTOLOGIA Y EMBRIOLOGIA DE MENDOZA DR. MARIO H. BURGOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
ARF6 Promotes Calcium-Regulated Exocytosis by Modulating Lipid Turnover and Rab3A Activation
Autor/es:
PELLETÁN, LEONARDO E; SUHAIMAN, LAILA; VAQUER, CINTIA C; VITALE, NICOLAS; MAYORGA, LUIS S; BELMONTE, SILVIA A
Lugar:
Estrasburgo
Reunión:
Simposio; 39th Symposium on Hormones and Cell Regulation. European Society of Endocrinology (ESE). INOSITOL LIPID SIGNALING : from molecular mechanisms to human pathologies; 2014
Institución organizadora:
European Society of Endocrinology
Resumen:
Regulated secretion is a sophisticated and highly regulated biological process that constitutes a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. The acrosome is a large and electron dense membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacologic stimuli, sperm undergo exocytosis of this granule in a synchronized wave, with no recycling of components. This is a calcium-dependent exocytosis termed the acrosome reaction and a prerequisite for fertilization . The role of ARF6 as a GTPase that plays a fundamental role in vesicle secretion was identified even its downstream effectors remain elusive. In this work, we focus on the role of ARF6 in the acrosomal exocytosis. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol (DAG)-induced exocytosis. Results from pull-down assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Unexpectedly, we found that myristoylated and GTPgS-loaded recombinant ARF6 induces sperm exocytosis even in the absence of extracellular calcium. ARF6 stimulates a sperm phospholipase D (PLD) activity, to produce phosphatidic acid (PA), and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). Although both lipids are required for exocytosis they do not induce secretion by themselves . In this report, we unveiled the ARF6 signaling that promotes regulated secretion. We present direct evidence that active ARF6 increases PLC activity, causing PIP2 hydrolysis, and identified a link between ARF6 and PLC signaling. Consistent with this result, ARF6 promotes IP3-dependent intraacrosomal calcium release. Moreover, active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. Both the increase in PLC activity and Rab3A activation depend in part upon the availability of DAG. Our results link ARF6 and DAG in a loop that drives exocytosis. We propose that exocytic stimuli activate ARF6, which induces acrosomal calcium efflux and assembles the fusion machinery leading to membrane fusion. Further, we demonstrate that ARF6 triggers a mechanism involving novel targets, different from those previously described. Our findings prove the physiological importance of ARF6 as a key molecule for human sperm exocytosis and fertilization.