INVESTIGADORES
MAYORGA Luis Segundo
artículos
Título:
Small GTPases in acrosomal exocytosis.
Autor/es:
BUSTOS MA; LUCCHESI, O; RUETE, MC; MAYORGA LS; TOMES C.N.
Revista:
METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.)
Editorial:
Springer Human Press
Referencias:
Año: 2015 vol. 1298 p. 141 - 160
ISSN:
1064-3745
Resumen:
Regulated secretion is a process that employs a conserved molecular machinery in all secretory cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) superfamilies and Rabs are members of this machinery. Rab proteins constitute a subfamily of Ras monomeric small GTPases that organize membrane microdomains on subcellular organelles by recruiting specific effector factors that strongly influence the movement, fusion and fission dynamics of intracellular compartments. During regulated exocytosis, a set of secretory Rabs present in secretory vesicles play an essential role recruiting tethering and docking factors required for membrane recognition and fusion. Many essential events occur in mammalian spermatozoa before they can fertilize the egg, one of them is the acrosome reaction (AR), a type of regulated exocytosis. To study the role of Rab3 and Rab27 during the AR, we have employed a variety of techniques such as Western blot, far-immunofluorescence, functional and pull-down assays. Here, we describe in depth the far-immunofluorescence and pull-down protocols because both have been designed to determine the activation status of small G proteins. In addition to the activation status, the far-immunofluorescence protocol that we have recently described serves to determine simultaneously the subcellular localization of active Rabs, something not achievable with other methods. By means of these techniques, we have determined that Rab27 and Rab3 act sequentially and are organized in a RabGEF cascade. In addition, we have established that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study. This chapter provides protocols to analyze how Rab3 and Rab27 cycle. We applied them for scrutinize the exocytosis of the acrosome in human sperm. Nevertheless, the protocols can be extended to study other Ras-related proteins in virtually any cellular model.