Rab GTPases pp 141-160 | Cite as

Small GTPases in Acrosomal Exocytosis

  • Matias A. Bustos
  • Ornella Lucchesi
  • Maria C. Ruete
  • Luis S. MayorgaEmail author
  • Claudia N. Tomes
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)


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.

Key words

Acrosome reaction Exocytosis Rab27 Rab3 Sperm 



This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (grant numbers PICT 2006-1036 and PICT 2010-0342) and Secretaría de Ciencia, Técnica y Posgrado, Universidad Nacional de Cuyo to C.N.T.


  1. 1.
    Sudhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474–477CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Rizo J, Sudhof TC (2012) The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices-guilty as charged? Annu Rev Cell Dev Biol 28:279–308CrossRefPubMedGoogle Scholar
  3. 3.
    Cherfils J, Zeghouf M (2013) Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol Rev 93:269–309CrossRefPubMedGoogle Scholar
  4. 4.
    Wickner W (2010) Membrane fusion: five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles. Annu Rev Cell Dev Biol 26:115–136CrossRefPubMedGoogle Scholar
  5. 5.
    Barr FA (2013) Review series: Rab GTPases and membrane identity: causal or inconsequential? J Cell Biol 202:191–199CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10:513–525CrossRefPubMedGoogle Scholar
  7. 7.
    Nottingham RM, Pfeffer SR (2009) Defining the boundaries: Rab GEFs and GAPs. Proc Natl Acad Sci U S A 106:14185–14186CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Fukuda M (2008) Regulation of secretory vesicle traffic by Rab small GTPases. Cell Mol Life Sci 65:2801–2813CrossRefPubMedGoogle Scholar
  9. 9.
    Yanagimachi R (2011) Mammalian sperm acrosome reaction: where does it begin before fertilization? Biol Reprod 85:4–5CrossRefPubMedGoogle Scholar
  10. 10.
    Tomes CN (2007) Molecular mechanisms of exocytosis. In: Regazzi R (ed) Molecular mechanisms of membrane fusion during acrosomal exocytosis, 65th edn. Landes Biosciences and Springer Science + Bussiness Media LLC, New York, pp 275–291Google Scholar
  11. 11.
    Mayorga LS, Tomes CN, Belmonte SA (2007) Acrosomal exocytosis, a special type of regulated secretion. IUBMB Life 59:286–292CrossRefPubMedGoogle Scholar
  12. 12.
    Florman HM, Jungnickel MK, Sutton KA (2008) Regulating the acrosome reaction. Int J Dev Biol 52:503–510CrossRefPubMedGoogle Scholar
  13. 13.
    Visconti PE, Krapf D, de la Vega-Beltran JL et al (2011) Ion channels, phosphorylation and mammalian sperm capacitation. Asian J Androl 13:395–405CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Costello S, Michelangeli F, Nash K et al (2009) Ca2+-stores in sperm: their identities and functions. Reproduction 138:425–437CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Gadella BM, Luna C (2014) Cell biology and functional dynamics of the mammalian sperm surface. Theriogenology 81:74–84CrossRefPubMedGoogle Scholar
  16. 16.
    Buffone MG, Ijiri TW, Cao W et al (2012) Heads or tails? Structural events and molecular mechanisms that promote mammalian sperm acrosomal exocytosis and motility. Mol Reprod Dev 79:4–18CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Aitken RJ, Nixon B (2013) Sperm capacitation: a distant landscape glimpsed but unexplored. Mol Hum Reprod 19:785–793CrossRefPubMedGoogle Scholar
  18. 18.
    Buffone MG, Hirohashi N, Gerton GL (2014) Unresolved questions concerning mammalian sperm acrosomal exocytosis. Biol Reprod 90:112CrossRefPubMedGoogle Scholar
  19. 19.
    Pocognoni CA, De Blas GA, Heuck AP et al (2013) Perfringolysin O as a useful tool to study human sperm physiology. Fertil Steril 99:99–106CrossRefPubMedGoogle Scholar
  20. 20.
    Lopez CI, Belmonte SA, De Blas GA et al (2007) Membrane-permeant Rab3A triggers acrosomal exocytosis in living human sperm. FASEB J 21:4121–4130CrossRefPubMedGoogle Scholar
  21. 21.
    Yunes R, Tomes C, Michaut M et al (2002) Rab3A and calmodulin regulate acrosomal exocytosis by mechanisms that do not require a direct interaction. FEBS Lett 525:126–130CrossRefPubMedGoogle Scholar
  22. 22.
    Branham MT, Bustos MA, De Blas GA et al (2009) Epac activates the small G proteins Rap1 and Rab3A to achieve exocytosis. J Biol Chem 284:24825–24839CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Bustos MA, Lucchesi O, Ruete MC et al (2012) Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis. Proc Natl Acad Sci U S A 109:E2057–E2066CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Bustos MA, Roggero CM, De la Iglesia PX et al (2014) GTP-bound Rab3A exhibits consecutive positive and negative roles during human sperm dense-core granule exocytosis. J Mol Cell Biol 6:286–298CrossRefPubMedGoogle Scholar
  25. 25.
    Ruete MC, Lucchesi O, Bustos MA et al (2014) Epac, Rap and Rab3 act in concert to mobilize calcium from sperm's acrosome during exocytosis. Cell Commun Signal 12:43PubMedCentralPubMedGoogle Scholar
  26. 26.
    Lopez CI, Pelletan LE, Suhaiman L et al (2012) Diacylglycerol stimulates acrosomal exocytosis by feeding into a PKC- and PLD1-dependent positive loop that continuously supplies phosphatidylinositol 4,5-bisphosphate. Biochim Biophys Acta 1821:1186–1199CrossRefPubMedGoogle Scholar
  27. 27.
    Coppola T, Perret-Menoud V, Luthi S et al (1999) Disruption of Rab3-calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis. EMBO J 18:5885–5891CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Kondo H, Shirakawa R, Higashi T et al (2006) Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets. J Biol Chem 281:28657–28665CrossRefPubMedGoogle Scholar
  29. 29.
    Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143CrossRefPubMedGoogle Scholar
  30. 30.
    Sekiya K, Satoh R, Danbara H et al (1993) A ring-shaped structure with a crown formed by streptolysin O on the erythrocyte membrane. J Bacteriol 175:5953–5961PubMedCentralPubMedGoogle Scholar
  31. 31.
    Sekiya K, Danbara H, Futaesaku Y (1993) Mechanism of pore formation on erythrocyte membrane by streptolysin-O. Kansenshogaku Zasshi 67:736–740CrossRefPubMedGoogle Scholar
  32. 32.
    Sekiya K (1995) Electron-microscopic observation of pore formation in the erythrocyte membrane by streptolysin O. Nihon Saikingaku Zasshi 50:509–517CrossRefPubMedGoogle Scholar
  33. 33.
    Bhakdi S, Tranum-Jensen J, Sziegoleit A (1985) Mechanism of membrane damage by streptolysin-O. Infect Immun 47:52–60PubMedCentralPubMedGoogle Scholar
  34. 34.
    World Health Organization Department of Reproductive Health and Research (2010) WHO laboratory manual for the examination and processing of human semen. WHO Press, GenevaGoogle Scholar
  35. 35.
    Makler A (1980) The improved ten-micrometer chamber for rapid sperm count and motility evaluation. Fertil Steril 33:337–338PubMedGoogle Scholar
  36. 36.
    Mendoza C, Carreras A, Moos J et al (1992) Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil 95:755–763CrossRefPubMedGoogle Scholar
  37. 37.
    Burstein ES, Macara IG (1992) Interactions of the ras-like protein p25rab3A with Mg2+ and guanine nucleotides. Biochem J 282:387–392PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Matias A. Bustos
    • 1
  • Ornella Lucchesi
    • 1
  • Maria C. Ruete
    • 1
  • Luis S. Mayorga
    • 1
    Email author
  • Claudia N. Tomes
    • 1
  1. 1.Instituto de Histología y Embriología (IHEM, CONICET/UNCuyo), Facultad de Ciencias Médicas, CC56Universidad Nacional de CuyoMendozaArgentina

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