Cell signal transduction networks

Summary

Our work aims at the multifactorial study of cellular physiological parameters that help to interpret and understand intracellular signaling pathways from the point of view of systemic biology. To reach this goal, we use different tools that allow the reading of multiple intracellular factors. Our group uses mouse spermatozoa as the main cell model. Being spermatozoa transcriptional and translationally inactive, it is presented as an exceptional model for the study of intracellular signaling mechanisms based on post-translational modification. In addition, the studies derived from the basic research in mouse sperm allow the extrapolation tu human sperm and other mammalian models, with implications in the reproductive clinic, and on the other hand, to commercially interesting species.

In mammals, spermatozoa lack fertilizing capacity at the time of ejaculation. These cells acquire the “capacitated” state within the reproductive female tract. However, sperm capacitation can be accomplished in the laboratory by incubating spermatozoa for as little as one hour (for mouse sperm) in defined culture media. Current knowledge indicates that the functional changes that lead to capacitation respond to a combination of both simultaneous and sequential processes. Some of these processes occur as soon as the sperm are released from the epididymis, while other changes occur more slowly being fired only after a certain period of time. At the biological level, sperm capacitation is associated with both a change in the mode of flagellar beating called hyperactivation and the ability of the sperm to trigger the process called acrosomal exocytosis upon contact with a physiological agonist.

Protein Kinase PKA (PKA) plays a key role in both the rapid and slow events associated with capacitation. These events include: 1) hyperpolarization of the sperm plasma membrane; 2) A cytoplasmic increase of the Ca2+ concentration; 3) Activation of Tyr kinases; 4) Both inactivation and activation of different Thr/Ser phosphatases; 5) Modification of the lipid constitution of the plasma membrane; 6) Actin polymerization. With these events in mind, it is clear that sperm represent an excellent model for the study of signaling cascades, considering the processes that are triggered within one hour, together with the ease of obtaining the samples.

Our laboratory is currently focused on understanding the mechanisms by which PKA activity is regulated during capacitation, specifically related to hyperpolarization of the plasma membrane. This hyperpolarization, as in other cell types, is key to enabling subsequent processes, such as acrosomal exocytosis in the sperm. The mechanisms by which PKA promotes hyperpolarization are not fully understood. Added to this, the question that still persists is how, after immediate activation of PKA in capacitation medium  (coincident with the increase of cAMP derived from sAC), hyperpolarization takes longer times to achieve This question is extensive to a series of events that, being commanded by PKA, take longer to occur, such as actin polymerization and the activation of Tyr kinases as Src. Our group identified that PKA promotes the activation of Src, then allowing its full activation by Src-Tyr416 phosphorylation. In turn, by heterologous expression of Slo3 in Xenopus oocytes, we saw that Src is fundamental for the sensitization of the potassium BK channel Slo3, responsible for the hyperpolarization of Em. Results derived from these studies help to understand the regulation of these events in different cell types, for example in the regulation of Src in different types of cancer.

Our objective is to understand the regulation of various signaling cascades, that while triggered by same mechanisms, are controlled both temporally and spatially in a dissimilar way in cellular systems. Our group, made up of a network of collaborators, integrates different techniques, combining the analysis of physiological parameters derived from single cell studies such as flow cytometry and super-resolution microscopy, with population measurements such as cell fluorimetry and mass spectrometry .

Selected Publications

La Spina FA, Puga Molina LC, Romarowski A, Vitale AM, Falzone TL, Krapf D, Hirohashi N, Buffone MG. “Mouse sperm begin to undergo acrosomal exocytosis in the upper isthmus of the oviduct.” Developmental Biology (2016) Feb 10.

Roy J, Kim B, Hill E, Visconti P, Krapf D, Vinegoni C, Weissleder R, Brown D, Breton S. “Tyrosine kinase-mediated axial motility of basal cells revealed by intravital imaging.” Nature Communications (2016) Feb 12;7:10666. doi: 10.1038/ncomms10666.

Cintia Stival, Florenza La Spina, Carolina Baró Graf, Enid Arcelay, Silvia E Arranz, Juan J Ferreira, Sylvie Le Grand, Viktor A Dzikunu, Celia M Santi, Pablo E Visconti, Mariano G Buffone, Dario Krapf. “Src Kinase Is the Connecting Player between Protein Kinase A (PKA) Activation and Hyperpolarization through SLO3 Potassium Channel Regulation in Mouse Sperm.” Journal of Biological Chemistry (2015)  Jul 24;290(30):18855-64

Romarowski A, Battistone MA, La Spina FA, Puga Molina LD, Luque GM, Vitale AM, Cuasnicu PS, Visconti PE, Krapf D, Buffone MG. “PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis.”Developmental Biology (2015) Jul 10. pii: S0012-1606(15)30046-4.

Felipe A Navarrete, Francisco A García‐Vázquez, Antonio Alvau, Jessica Escoffier, Dario Krapf, Claudia Sánchez‐Cárdenas, Ana M Salicioni, Alberto Darszon, Pablo E Visconti. “Biphasic Role of Calcium in Mouse Sperm Capacitation Signaling Pathways”Journal of cellular Physiology  (2015) 230(8):1758-69

Mariano G Buffone, Eva V Wertheimer, Pablo E Visconti, Dario Krapf. “Central role of soluble adenylyl cyclase and cAMP in sperm physiology”Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease (2014) 1842(12 Pt B):2610-20

Dario Krapf, Emma O’Brien, María P. Maidagán, Enrique O. Morales, Pablo E. Visconti and Silvia E. Arranz. “Calcineurin Regulates Progressive Motility Activation of Rhinella (Bufo) arenarum Sperm Through Dephosphorylation of PKC Substrates.”Journal of Cellular Physiology (2014) 229(10):1378-86.

Wertheimer E, Krapf D, Vega-Beltran JL, Sánchez-Cárdenas C, Navarrete F, Haddad D, Escoffier J, Salicioni AM, Levin LR, Buck J, Mager J, Darszon A, Visconti PE. Compartmentalization of Distinct cAMP Signaling Pathways in Mammalian Sperm. Journal of Biological Chemistry (2013) 288(49); 35307–35320

Tateno H, Krapf D, Hino T, Sánchez-Cárdenas C, Darszon A, Yanagimachi R, Visconti PE. Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways. Proceedings of the National Academy of Sciences of the United States of America (2013) 110 (46), pp. 18543-18548 8

MA Batisttone, VG Da Ros, AM Salicioni, F Navarrette, D Krapf, PE Visconti, PS Cuasnicú. “Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases” Molecular Human Reproduction (2013) 19(9):570-80

de la Vega-Beltran JL, Sanchez-Cardenas C, Krapf D, Hernandez-Gonzalez EO, Wertheimer E, Trevino CL, Visconti PE, Darszon A. “Mouse sperm membrane potential hyperpolarization is necessary and sufficient to prepare sperm for the acrosome reaction.” Journal of Biological Chemistry (2012) 287(53):44384-93

D Krapf, Y C Ruan, E V Wertheimer, M A Battistone, J B Pawlak, A Sanjay, S H Pilder, P Cuasnicu, S Breton, P E Visconti. “cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit”. Developmental Biology (2012) 1;369(1):43-53.

P E Visconti, D Krapf, J L de la Vega-Beltran, J J Acevedo and A Darszon. “Ion channels, phosphorylation and mammalian sperm capacitation.” Asian Journal of Andrology (2011) 13, 395–405

D Krapf, E Arcelay, E V Wertheimer, A Sanjay, S H Pilder, A M Salicioni and P E Visconti. “Inhibition of ser/thr phosphatases induces capacitation-associated signaling in the presence of Src kinase inhibitors” Journal of Biological Chemistry (2010) 12;285(11):7977-85 .

C Stival, L del Puga Molina, B Paudel, MG Buffone, PE Visconti PE, D Krapf. “Sperm Capacitation and Acrosome Reaction in Mammalian Sperm.” Advances in Anatomy, Embryology and Cell Biology (2016) 220:93-106

Collaborators

Dr Mariano G Buffone. Instituto de Biología y Medicina Experimental (CONICET), Buenos Aires, Argentina

Dr Pablo E. Visconti. Department of Veterinaty and Animal Sciences, University of Massachusetts. Amherst, MA, EEUU

Dr Diego Krapf.  School Of Biomedical Engineering, Colorado State University, CO, EEUU

Dra Silvia E Arranz. Plataforma de Biotecnología Acuatica. IBR, Rosario, Argentina

Dr Matias Zurbriggen. Institute of Mathematics and Natural Sciences

University of Düsseldorf, Alemania

Grants

Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT).

Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).

Director

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Dario Krapf
Core CCT
Email: krapf@ibr-conicet.gov.ar
Phone: +54 341 4237070
Office Extension: 648
Laboratory Extension: 614

Postdoctoral fellows

Carla Ritagliati

Doctoral fellows

Cintia Stival

Carolina Baró Graf