Small GTPases in acrosomal exocytosis

Regulated exocytosis employs a conserved molecular machinery in all secretory cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Rab superfamilies are members of this machinery. Rab proteins are small GTPases that organize membrane microdomains on organelles by recruiting specific effectors that strongly influence the movement, fusion and fission dynamics of intracellular compartments. Rab3 and Rab27 are the prevalent exocytotic isoforms. Many events occur in mammalian spermatozoa before they can fertilize the egg, one of them is the acrosome reaction (AR), a type of regulated exocytosis. The AR relies on the same fusion machinery as all other cell types, which includes members of the exocytotic SNARE and Rab superfamilies. Here, we describe in depth two protocols designed to determine the activation status of small G proteins. One of them also serves to determine the subcellular localization of active Rabs, something not achievable with other methods. By means of these techniques, we have reported that Rab27 and Rab3 act sequentially and are organized in a RabGEF cascade during the AR. Although we developed them to scrutinize the exocytosis of the acrosome in human sperm, the protocols can potentially be extended to study other Ras-related proteins in virtually any cellular model.