Laboratory Name: Fusion and signaling molecules involved in sperm exocytosis

PI: Claudia Nora Tomes, Ph.D.

Position: Staff Researcher, Argentine Research Council (Investigador Independiente, CONICET)
at the Instituto de Histología y Embriología de Mendoza (IHEM-CONICET)
School of Medicine Cuyo National University, PO Box 56 5500, Mendoza Argentina.

Instructor at the Department of Morpho-Physiology, School of Medicine, Cuyo National University

e-mail: ctomes@fcm.uncu.edu.ar

Phone: 54-261-449-4143, ext 2744

Fax: 54-261-449-4117

Group members: Dr María Celeste Ruete (Investigador Asistente CONICET), Molecular Biologist Matías Alberto Bustos (graduate student, recipient of CONICET type II fellowship), Molecular Biologist Ornella Lucchesi (graduate student, recipient of CONICET type I fellowship) and Biochemist María Florencia Quevedo (recipient of CONICET type I fellowship)

Research interests

            Sexual reproduction to perpetuate a given species occurs through fertilization, during which a diploid zygote is formed to produce a genetically distinct individual.  To this end, the haploid sperm and haploid egg must collide to allow entry of the sperm head delivering the male chromatin into the egg cytoplasm.  Both the male and female gametes undergo regulated exocytosis —termed the acrosome reaction (AR) and the cortical reaction respectively—at different times during their encounter.  The success of fertilization depends on these exocytoses.

            In all exocytotic cell lines, membrane fusion is governed by an exquisite and highly conserved protein machinery responsible for avoiding premature secretion and guarantee instantaneous release at the same time.  Some components of this machinery are integral proteins localized to the vesicle (Rabs, R-SNAREs, synaptotagmins) and plasma membrane (Q-SNAREs), whereas others do not have transmembrane domains or lipid modifications (α-SNAP, PTP1B, NSF, Munc18, complexins).  In sperm the AR requires calcium, cyclic AMP and all the fusion proteins mentioned above, orchestrated in a highly regulated protein-protein interaction network.  The relevant cAMP target is Epac, a guanine nucleotide exchange factor for the small GTPase Rap.  A soluble adenylyl cyclase, regulated by calcium and bicarbonate, synthesizes the cAMP required for the AR.  Epac sits at a critical point during the exocytotic cascade after which the pathway splits into two limbs, one (Rab3A-PTP1B-α-SNAP/NSF-SNAREs) that assembles the fusion machinery into place, and another (Rap1-phospholipase C) that mobilizes intracellular calcium through IP3-sensitive channels.  My lab is currently concerned with understanding more deeply the nature and regulation of these signalling pathways, determining at what level/s do these pathways cross talk, and where do they converge to achieve exocytosis.

            The discovery that very similar versions of the proteins involved play the same roles in virtually all membrane fusion models has greatly simplified our thinking and means that these mechanisms need not be studied in detail in all cell types; instead, future work can concentrate on the analysis of a few favorable secretory cell models.  The AR is a relatively simple model and because of the universality of mechanisms underlying exocytosis, the knowledge arising from studying sperm exocytosis needs not to be restricted to the Reproductive Biology field but could be extended to more complex membrane fusion scenarios.

Mentoring/Student supervision (current)

- Thesis advisor, Matías Alberto Bustos (MS, Molecular Biology).  Ph.D. student in School of Pharmacy and Biochemistry, University of Buenos Aires.  Recipient of an ANPCyT (2009-2011) and a National Research Council (CONICET, 2011-2013) doctoral fellowships.  Project title: Biochemical definition of the sequence of steps leading to regulated exocytosis using mammalian sperm as model system

- Thesis advisor, Ornella Lucchesi (MS, Molecular Biology).   Ph.D. student in the School of Sciences, University of Córdoba. Recipient of a National Research Council (CONICET) doctoral fellowship, 2010-2013.  Project title: Molecular characterization of calcium mobilization-related fusion stages during the acrosome reaction.

- Supervisor, María Celeste Ruete, Ph.D., Biochemistry. Junior Staff Researcher (Investigadora Asistente) at National Research Council (CONICET), 2010-on.

- Supervisor, María Florencia Quevedo (MS, Biochemistry).  Recipient of a National Research Council (CONICET) doctoral fellowship, 2012-2015.  Project title: Characterization of the role of exocytic Rabs during the acrosome reaction.

Supervised theses (past)

- María Teresita Branham, Ph.D. Project title: Membrane fusion: role of EPAC—a novel cAMP target—in acrosomal exocytosis.  Dr Branham obtained her Ph.D. in PROBIOL (Postgraduate Program in Biology), Cuyo National University on March 29, 2010, she graduated with the highest grades. Previously she had been the Recipient of a Dr Osvaldo Fustinoni (CEDIQUIFA) graduate student fellowship in 2003 and of a National Research Council (CONICET) doctoral fellowship, 2005-2010.

- Valeria Eugenia Paola Zarelli, Ph.D. Project title: Protein tyrosine phosphorylation/dephosphorylation and their role in membrane fusion.  Dr Zarelli obtained her Ph.D. in the School of Pharmacy and Biochemistry, University of Buenos Aires on April 28, 2009, she graduated with the highest grades. Previously she had been the Recipient of a National Research Council (CONICET) doctoral fellowship, 2004-2008.

- Matías Alberto Bustos.  Thesis to opt for a degree in Molecular Biology (equivalent to MS).  Title: Proteins involved in the human sperm acrosome reaction.  Graduated in June 2008 with the highest grades, School of Sciences, National University of San Luis, San Luis, Argentina.

- Eugenia Farré.  Thesis to opt for a degree in Molecular Biology (equivalent to MS).  Title: Membrane fusion during the acrosome reaction.  Graduated in 2002 with the highest grades, School of Sciences, National University of San Luis, San Luis, Argentina.

- Juan Facundo Rodríguez Ayala, Ph.D.  Project title: SNAREs, α-SNAP and Munc18-1 during human sperm acrosomal exocytosis. Dr Rodríguez obtained his Ph.D. in PROBIOL (Postgraduate Program in Biology), Cuyo National University on March 14, 2012, he graduated with the highest grades. Previously he had been the Recipient of National Research Council (CONICET) doctoral fellowships, 2007-2012.

Funding (current)

a) As Principal Investigator

SeCTyP (Secretaría de Ciencia y Técnica, Universidad Nacional de Cuyo) 06/J416. Title:Molecular characterization of membrane fusion late stages during the acrosome reaction: signaling pathways and cross talk. 2011-2013
- PICT-2010-0342, Agencia Nacional de Promoción Científica y Tecnológica (FONCYT). Title: A detailed molecular and morphological analysis of exocytosis stages using human sperm as model system. 2012-2015

b) As team member

CONICET (National Research Council of Argentina).  Title: Mapping of early and late stages and lipid- and protein-linked signal transduction pathways during regulated exocytosis.  PI Dr SA Belmonte.  2010-2012.

Invited Speaker (selected)

1) Speaker at the Symposium Secretory events during fertilization. Gordon Conference in Fertilization and Activation of Development.  New Hampshire, USA. 2001.

2) Departmental Seminar series, Department of Biochemistry & Molecular Biology, Dalhousie University. Halifax, NS, Canada. 1999 and 2001.

3) Speaker at the Symposium Biology of Reproduction.  First Joint Meeting of Seven Biomedical Societies.  Mar del Plata, Argentina, 2004.

4) Seminar series, Departament of Neurobiology, Max Planck Institute for Biophysical Chemistry.  Goettingen, Germany, July 14 2006.

5) Speaker at the Symposium on signalling and sperm function in the 10th International Symposium on Spermatology. Madrid, Spain,17- 22 September 2006.

6) Speaker at the Sperm Signaling Symposium, Gordon Research Conference (GRC) on Fertilization and the Activation of Development. Organizer: GRC. Plymouth, NH, USA, July 2007.

7) Plenary (International) Lecture at the American Society of Andrology (ASA) Meeting. 33rd ASA Annual Meeting. Organized by the ASA.  Albuquerque, New Mexico, USA, April 2008.

8) Speaker at the SymposiumSignal Transduction. XLVII Annual Meeting Argentine Society for Biochemistry and Molecular Biology (SAIB). Potrero de los Funes, San Luis. 30 Oct-2 Nov de 2011

Publications (selected)

1) Tomes CN, Roggero CM, De Blas GA, Saling PM and Mayorga LS (2004).  Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis.  Developmental Biology, 265, 399-415.

2) Tomes C, De Blas GA, Michaut MA, Farré E, Chertihin O, Visconti PE and Mayorga LS (2005).  Alpha-SNAP and NSF are required in a priming step during human sperm exocytosis.  Molecular Human Reproduction, 11, 43-51.

3) Belmonte S, López C, Roggero C, De Blas G, Tomes C and Mayorga L (2005).  Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association.  Developmental Biology, 285, 393-408.

4) Roggero CM, Tomes CN, De Blas GA, Castillo J, Michaut MA, Fukuda M, and Mayorga LS (2005).  Protein Kinase C-mediated Phosphorylation of Synaptotagmin VI C2A and C2B Domains Regulates Their Function in Acrosomal Exocytosis.  Developmental Biology, 285, 422-435.

5) De Blas GA, Roggero CM, Tomes CN*, and Mayorga LS* (*both authors contributed equally to this work and share the senior author position) (2005).  Dinamycs of SNARE assembly and disassembly during sperm acrosomal exocytosis. PLoS Biology, 3, e323, pp1801-1812.

6) Branham MT, Mayorga LS and Tomes C (2006).  Calcium-induced acrosomal exocytosis requires cAMP acting through a protein kinase A-independent, Epac-mediated pathway.  The Journal of Biological Chemistry 281, 8656-8666.

7) Mayorga LS, Tomes C and Belmonte SA (2007). Acrosomal exocytosis, a special type of regulated secretion. IUBMB Life 59, 286-292.

8) Roggero CM, De Blas GA, Dai H, Tomes CN, Rizo J and Mayorga LS (2007). Complexin/synaptotagmin interplay controls acrosomal exocytosis. The Journal of Biological Chemistry 282, 26335-26343.

9) Tomes C (2007)  Acrosomal exocytosis.  In Molecular mechanisms of exocytosis. Ed. R. Regazzi.  Landes Bioscience/Eurekah.com, Austin, Texas, and Springer Science+Business Media, New York, New York, USA. 117-147

10) Tomes C (2007).  Molecular mechanisms of membrane fusion during acrosomal exocytosis. Proceedings of the 10th Spermatology Symposium  Society for Reproduction and Fertility Supplement 65:275-91. Ed: E. R.S. Roldán and M. Gomendio. Nottingham University Press.  Nottingham,  NG, UK

11) Zarelli VEP, Ruete MC, Roggero CM, Mayorga LS, and Tomes CN (2009).  PTP1B dephosphorylates NSF and elicits SNARE complex disassembly during human sperm exocytosis. The Journal of Biological Chemistry 284, 10491-10503.

12) Bátiz LF,  De BlasGA, Michaut MA, Ramírez AR, Rodríguez F, Ratto MH, Oliver C, Tomes CN, Rodríguez EM, and Mayorga  LS  (2009). A point mutation of α-SNAP causes impaired acrosome reaction and reduced male fertility.  PLoS ONE. 4(3):e4963.

13) Branham MT, Bustos MA, De Blas GA, Rehmann H, Zarelli VEP, Treviño CL, Darszon A, Mayorga LS, and Tomes CN (2009).  Epac activates the small G proteins Rap1 and Rab3A to achieve exocytosis.  The Journal of Biological Chemistry 284, 24825-24839.

14) Rodríguez F, Bustos MA, Zanetti MN, Mayorga LS, and Tomes CN. α-SNAP prevents docking of the acrosome during sperm exocytosis because it sequesters monomeric syntaxin.  PLoS ONE 6 (7): e21925. doi:10.1371/journal.pone.0021925.

Actualizado Abril 2012