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Calpain inhibition prevents flotillin re-ordering and Src family activation during capacitation

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Prior to fertilization, mammalian sperm undergo several molecular, biochemical and physiological changes in a process termed capacitation. However, the mechanisms explaining the involvement of cytoskeletal remodeling and membrane re-ordering in each process prior to fertilization remain poorly understood. We found that the migration of both flotillin microdomains and Src family kinases towards the apical ridge of guinea pig sperm occurs under capacitating conditions. This re-ordering is associated with spectrin cleavage by calpain. Moreover, Src, Fyn, Lyn and Hck interact with flotillin-1; this interaction increases in a capacitation-dependent manner and the increased autophosphorylation of these kinases is linked to flotillin-1 association. The aforementioned results are prevented by the inhibition of calpain by calpeptin. Thus, spectrin cytoskeleton cleavage during capacitation seems to precede the reorganization of flotillin microdomains and Src family kinases towards the apical ridge of the sperm head in order to initiate the signaling cascade required for proper capacitation and further acrosome reaction. The significance of the Src family kinase reorganization for capacitation is demonstrated by the inhibition of calpain during capacitation also preventing the Src-family-kinase-dependent phosphorylation of FAK at Tyr576/577. Our work further highlights the scaffolding properties of flotillin microdomains and reveals the importance of their large-scale segregation during capacitation.

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  1. Aitken RJ, Nixon B (2013) Sperm capacitation: a distant landscape glimpsed but unexplored. Mol Hum Reprod 19:785–793

  2. Angeles-Floriano T, Roa-Espitia AL, Baltiérrez-Hoyos R, Cordero-Martínez J, Elizondo G, Hernández-González EO (2016)Absence of aryl hydrocarbon receptor alters CDC42 expression and prevents actin polymerization during capacitation. Mol Reprod Dev 83:1015–1026

  3. Aoyama T, Ozaki Y, Aoki K, Kunimatsu M, Tada T, Sasaki M, Suzumori K (2001)Involvement of mu-calpain in human sperm capacitation for fertilization. Am J Reprod Immunol 45:12–20

  4. Asano A, Nelson-Harrington JL, Travis AJ (2013) Phospholipase B is activated in response to sterol removal and stimulates acrosome exocytosis in murine sperm. J Biol Chem 288:28104–28115

  5. Austin CR (1952) The capacitation of the mammalian sperm. Nature 170:326

  6. Azamar Y, Uribe S, Mujica A (2007) F-actin involvement in guinea pig sperm motility. Mol Reprod Dev 74:312–320

  7. Babuke T, Tikkanen R (2007)Dissecting the molecular function of reggie/flotillin proteins.Eur J Cell Biol 86:525–532

  8. Bailey JL (2010) Factors regulating sperm capacitation. Syst Biol Reprod Med 56:334–348

  9. Baker MA, Hetherington L, Aitken RJ (2006) Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa. J Cell Sci 119:3182–3192

  10. Baltierrez-Hoyos R, Roa-Espitia AL, Hernandez-Gonzalez EO (2012) The association between CDC42 and caveolin-1 is involved in the regulation of capacitation and acrosome reaction of guinea pig and mouse sperm. Reproduction 144:123–134

  11. Bastian Y, Roa-Espitia AL, Mujica A, Hernandez-Gonzalez EO (2010) Calpain modulates capacitation and acrosome reaction through cleavage of the spectrin cytoskeleton. Reproduction 140:673–684

  12. Ben-Aharon I, Brown PR, Etkovitz N, Eddy EM, Shalgi R (2005) The expression of calpain 1 and calpain 2 in spermatogenic cells and spermatozoa of the mouse. Reproduction 129:435–442

  13. Bennett V, Healy J (2009) Membrane domains based on ankyrin and spectrin associated with cell-cell interactions. Cold Spring Harb Perspect Biol 1:a003012

  14. Bennett V, Lorenzo DN (2016) An adaptable spectrin/ankyrin-based mechanism for long-range organization of plasma membranes in vertebrate tissues. Curr Top Membr 77:143–184

  15. Boerke A, Brouwers JF, Olkkonen VM, van de Lest CH, Sostaric E, Schoevers EJ, Helms JB, Gadella BM (2013)Involvement of bicarbonate-induced radical signaling in oxysterol formation and sterol depletion of capacitating mammalian sperm during in vitro fertilization.Biol Reprod 88:21

  16. Boerke A, Lit J van der, Lolicato F, Stout TA, Helms JB, Gadella BM (2014) Removal of GPI-anchored membrane proteins causes clustering of lipid microdomains in the apical head area of porcine sperm. Theriogenology 81:613–624

  17. Bou Khalil M, Chakrabandhu K, Xu H, Weerachatyanukul W, Buhr M, Berger T, Carmona E, Vuong N, Kumarathasan P, Wong PT, Carrier D, Tanphaichitr N (2006) Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid. Dev Biol 290:220–235

  18. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  19. Bragado MJ, Gil MC, Martin-Hidalgo D, Hurtado de Llera A, Bravo N, Moreno AD, Garcia-Marin LJ (2012) Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa. Reproduction 144:67–75

  20. Brener E, Rubinstein S, Cohen G, Shternall K, Rivlin J, Breitbart H (2003) Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction. Biol Reprod 68:837–845

  21. Briz V, Baudry M (2016) Calpains: master regulators of synaptic plasticity. Neuroscientist 2016:1073858416649178, doi:10.1177/1073858416649178

  22. Brown MT, Cooper JA (1996) Regulation, substrates and functions of src. Biochim Biophys Acta 1287:121–149

  23. Calalb MB, Polte TR, Hanks SK (1995) Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol 15:954–963

  24. Camatini M, Colombo A, Bonfanti P (1991) Identification of spectrin and calmodulin in rabbit spermiogenesis and spermatozoa. Mol Reprod Dev 28:62–69

  25. Chang MC (1951) Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168:697–698

  26. Cross NL (2004) Reorganization of lipid rafts during capacitation of human sperm. Biol Reprod 71:1367–1373

  27. Domingues CC, Ciana A, Buttafava A, Casadei BR, Balduini C, Paula E de, Minetti G (2010) Effect of cholesterol depletion and temperature on the isolation of detergent-resistant membranes from human erythrocytes. J Membr Biol 234:195–205

  28. Etkovitz N, Tirosh Y, Chazan R, Jaldety Y, Daniel L, Rubinstein S, Breitbart H (2009) Bovine sperm acrosome reaction induced by G-protein-coupled receptor agonists is mediated by epidermal growth factor receptor transactivation. Dev Biol 334:447–457

  29. Finkelstein M, Megnagi B, Ickowicz D, Breitbart H (2013) Regulation of sperm motility by PIP2(4,5) and actin polymerization. Dev Biol 381:62–72

  30. Foster LJ, De Hoog CL, Mann M (2003) Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci U S A 100:5813–5818

  31. Gadella BM, Boerke A (2016) An update on post-ejaculatory remodeling of the sperm surface before mammalian fertilization. Theriogenology 85:113–124

  32. Gangwar DK, Atreja SK (2015) Signalling events and associated pathways related to the mammalian sperm capacitation. Reprod Domest Anim 50:705–711

  33. Gestel RA van, Brewis IA, Ashton PR, Helms JB, Brouwers JF, Gadella BM (2005) Capacitation-dependent concentration of lipid rafts in the apical ridge head area of porcine sperm cells. Mol Hum Reprod 11:583–590

  34. Goupil S, La Salle S, Trasler JM, Bordeleau LJ, Leclerc P (2011) Developmental expression of SRC-related tyrosine kinases in the mouse testis. J Androl 32:95–110

  35. Head BP, Patel HH, Insel PA (2014) Interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling. Biochim Biophys Acta 1838:532–545

  36. Hebert-Chatelain E (2013) Src kinases are important regulators of mitochondrial functions.Int J Biochem Cell Biol 45:90–98

  37. Hernandez-Gonzalez EO, Lecona-Valera AN, Escobar-Herrera J, Mujica A (2000) Involvement of an F-actin skeleton on the acrosome reaction in guinea pig spermatozoa. Cell Motil Cytoskeleton 46:43–58

  38. Hinkovska-Galcheva V, Srivastava PN (1993) Phospholipids of rabbit and bull sperm membranes: structural order parameter and steady-state fluorescence anisotropy of membranes and membrane leaflets. Mol Reprod Dev 35:209–217

  39. Hitosugi T, Sato M, Sasaki K, Umezawa Y (2007) Lipid raft specific knockdown of SRC family kinase activity inhibits cell adhesion and cell cycle progression of breast cancer cells. Cancer Res 67:8139–8148

  40. Kato N, Nakanishi M, Hirashima N (2006) Flotillin-1 regulates IgE receptor-mediated signaling in rat basophilic leukemia (RBL-2H3) cells. J Immunol 177:147–154

  41. Kierszenbaum AL, Rivkin E, Talmor-Cohen A, Shalgi R, Tres LL (2009) Expression of full-length and truncated Fyn tyrosine kinase transcripts and encoded proteins during spermatogenesis and localization during acrosome biogenesis and fertilization. Mol Reprod Dev 76:832–843

  42. Krapf D, Arcelay E, Wertheimer EV, Sanjay A, Pilder SH, Salicioni AM, Visconti PE (2010) Inhibition of Ser/Thr phosphatases induces capacitation-associated signaling in the presence of Src kinase inhibitors. J Biol Chem 285:7977–7985

  43. Lang DM, Lommel S, Jung M, Ankerhold R, Petrausch B, Laessing U, Wiechers MF, Plattner H, Stuermer CA (1998) Identification of reggie-1 and reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons. J Neurobiol 37:502–523

  44. Langhorst MF, Reuter A, Luxenhofer G, Boneberg EM, Legler DF, Plattner H, Stuermer CA (2006) Preformed reggie/flotillin caps: stable priming platforms for macrodomain assembly in T cells. FASEB J 20:711–713

  45. Langhorst MF, Solis GP, Hannbeck S, Plattner H, Stuermer CA (2007) Linking membrane microdomains to the cytoskeleton: regulation of the lateral mobility of reggie-1/flotillin-2 by interaction with actin. FEBS Lett 581:4697–4703

  46. Le Rumeur E, Winder SJ, Hubert JF (2010) Dystrophin: more than just the sum of its parts. Biochim Biophys Acta 1804:1713–1722

  47. Liu J, Deyoung SM, Zhang M, Dold LH, Saltiel AR (2005) The stomatin/prohibitin/flotillin/HflK/C domain of flotillin-1 contains distinct sequences that direct plasma membrane localization and protein interactions in 3T3-L1 adipocytes. J Biol Chem 280:16125–16134

  48. Ludwig A, Otto GP, Riento K, Hams E, Fallon PG, Nichols BJ (2010) Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment. J Cell Biol 191:771–781

  49. Machnicka B, Grochowalska R, Boguslawska DM, Sikorski AF, Lecomte MC (2012) Spectrin-based skeleton as an actor in cell signaling. Cell Mol Life Sci 69:191–201

  50. Machnicka B, Czogalla A, Hryniewicz-Jankowska A, Boguslawska DM, Grochowalska R, Heger E, Sikorski AF (2014) Spectrins: a structural platform for stabilization and activation of membrane channels, receptors and transporters. Biochim Biophys Acta 1838:620–634

  51. Miranda PV, Allaire A, Sosnik J, Visconti PE (2009) Localization of low-density detergent-resistant membrane proteins in intact and acrosome-reacted mouse sperm. Biol Reprod 80:897–904

  52. Mitchell LA, Nixon B, Baker MA, Aitken RJ (2008) Investigation of the role of SRC in capacitation-associated tyrosine phosphorylation of human spermatozoa. Mol Hum Reprod 14:235–243

  53. Morrow IC, Rea S, Martin S, Prior IA, Prohaska R, Hancock JF, James DE, Parton RG (2002) Flotillin-1/reggie-2 traffics to surface raft domains via a novel Golgi-independent pathway. Identification of a novel membrane targeting domain and a role for palmitoylation. J Biol Chem 277:48834–48841

  54. Nebl T, Pestonjamasp KN, Leszyk JD, Crowley JL, Oh SW, Luna EJ (2002) Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes. J Biol Chem 277:43399–43409

  55. Neumann-Giesen C, Falkenbach B, Beicht P, Claasen S, Luers G, Stuermer CA, Herzog V, Tikkanen R (2004) Membrane and raft association of reggie-1/flotillin-2: role of myristoylation, palmitoylation and oligomerization and induction of filopodia by overexpression. Biochem J 378:509–518

  56. Neumann-Giesen C, Fernow I, Amaddii M, Tikkanen R (2007) Role of EGF-induced tyrosine phosphorylation of reggie-1/flotillin-2 in cell spreading and signaling to the actin cytoskeleton. J Cell Sci 120:395–406

  57. Nixon B, Aitken RJ (2009) The biological significance of detergent-resistant membranes in spermatozoa. J Reprod Immunol 83:8–13

  58. Okada M (2012) Regulation of the SRC family kinases by Csk. Int J Biol Sci 8:1385–1397

  59. Olson SD, Fauci LJ, Suarez SS (2011) Mathematical modeling of calcium signaling during sperm hyperactivation. Mol Hum Reprod 17:500–510

  60. Ozaki Y, Blomgren K, Ogasawara MS, Aoki K, Furuno T, Nakanishi M, Sasaki M, Suzumori K (2001) Role of calpain in human sperm activated by progesterone for fertilization. Biol Chem 382:831–838

  61. Rivera-Milla E, Stuermer CA, Malaga-Trillo E (2006) Ancient origin of reggie (flotillin), reggie-like, and other lipid-raft proteins: convergent evolution of the SPFH domain. Cell Mol Life Sci 63:343–357

  62. Roa-Espitia AL, Hernandez-Rendon ER, Baltierrez-Hoyos R, Munoz-Gotera RJ, Cote-Velez A, Jimenez I, Gonzalez-Marquez H, Hernandez-Gonzalez EO (2016) Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation. Biol Open 5:1189–1199

  63. Rogers BJ, Yanagimachi R (1975) Retardation of guinea pig sperm acrosome reaction by glucose: the possible importance of pyruvate and lactate metabolism in capacitation and the acrosome reaction. Biol Reprod 13:568–575

  64. Roskoski R Jr (2015) Src protein-tyrosine kinase structure, mechanism, and small molecule inhibitors. Pharmacol Res 94:9–25

  65. Salzer U, Prohaska R (2001) Stomatin, flotillin-1, and flotillin-2 are major integral proteins of erythrocyte lipid rafts. Blood 97:1141–1143

  66. Schlaepfer DD, Hauck CR, Sieg DJ (1999) Signaling through focal adhesion kinase. Prog Biophys Mol Biol 71:435–478

  67. Selvaraj V, Asano A, Buttke DE, McElwee JL, Nelson JL, Wolff CA, Merdiushev T, Fornes MW, Cohen AW, Lisanti MP, Rothblat GH, Kopf GS, Travis AJ (2006) Segregation of micron-scale membrane sub-domains in live murine sperm. J Cell Physiol 206:636–646

  68. Shadan S, James PS, Howes EA, Jones R (2004) Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa. Biol Reprod 71:253–265

  69. Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31–39

  70. Solis GP, Hoegg M, Munderloh C, Schrock Y, Malaga-Trillo E, Rivera-Milla E, Stuermer CA (2007) Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains. Biochem J 403:313–322

  71. Sosnik J, Miranda PV, Spiridonov NA, Yoon SY, Fissore RA, Johnson GR, Visconti PE (2009) Tssk6 is required for Izumo relocalization and gamete fusion in the mouse. J Cell Sci 122:2741–2749

  72. Spector I, Shochet NR, Blasberger D, Kashman Y (1989) Latrunculins—novel marine macrolides that disrupt microfilament organization and affect cell growth. I. Comparison with cytochalasin D. Cell Motil Cytoskeleton 13:127-144

  73. Stival C, La Spina FA, Baro Graf C, Arcelay E, Arranz SE, Ferreira JJ, Le Grand S, Dzikunu VA, Santi CM, Visconti PE, Buffone MG, Krapf D (2015) Src kinase is the connecting player between protein kinase A (PKA) activation and hyperpolarization through SLO3 potassium channel regulation in mouse sperm. J Biol Chem 290:18855–18864

  74. Stival C, Puga Molina Ldel C, Paudel B, Buffone MG, Visconti PE, Krapf D (2016) Sperm capacitation and acrosome reaction in mammalian sperm. Adv Anat Embryol Cell Biol 220:93–106

  75. Stuermer CA (2011) Reggie/flotillin and the targeted delivery of cargo. J Neurochem 116:708–713

  76. Stuermer CA, Plattner H (2005) The “lipid raft” microdomain proteins reggie-1 and reggie-2 (flotillins) are scaffolds for protein interaction and signalling. Biochem Soc Symp 2005:109-118

  77. Stuermer CA, Lang DM, Kirsch F, Wiechers M, Deininger SO, Plattner H (2001) Glycosylphosphatidyl inositol-anchored proteins and Fyn kinase assemble in noncaveolar plasma membrane microdomains defined by reggie-1 and -2. Mol Biol Cell 12:3031–3045

  78. Suarez SS (2008) Control of hyperactivation in sperm. Hum Reprod Update 14:647–657

  79. Susuki K, Otani Y, Rasband MN (2016)Submembranous cytoskeletons stabilize nodes of Ranvier.Exp Neurol 283:446–451

  80. Tapia S, Rojas M, Morales P, Ramirez MA, Diaz ES (2011) The laminin-induced acrosome reaction in human sperm is mediated by Src kinases and the proteasome. Biol Reprod 85:357–366

  81. Thomas SM, Brugge JS (1997) Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13:513–609

  82. Travis AJ, Merdiushev T, Vargas LA, Jones BH, Purdon MA, Nipper RW, Galatioto J, Moss SB, Hunnicutt GR, Kopf GS (2001) Expression and localization of caveolin-1, and the presence of membrane rafts, in mouse and guinea pig spermatozoa. Dev Biol 240:599–610

  83. Varano G, Lombardi A, Cantini G, Forti G, Baldi E, Luconi M (2008) Src activation triggers capacitation and acrosome reaction but not motility in human spermatozoa. Hum Reprod 23:2652–2662

  84. Ward CR, Storey BT (1984) Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev Biol 104:287–296

  85. Watanabe H, Kondoh G (2011) Mouse sperm undergo GPI-anchored protein release associated with lipid raft reorganization and acrosome reaction to acquire fertility. J Cell Sci 124:2573–2581

  86. Xiao X, Yang WX (2007) Actin-based dynamics during spermatogenesis and its significance. J Zhejiang Univ Sci B 8:498–506

  87. Yasuda K, Nagafuku M, Shima T, Okada M, Yagi T, Yamada T, Minaki Y, Kato A, Tani-Ichi S, Hamaoka T, Kosugi A (2002) Cutting edge: Fyn is essential for tyrosine phosphorylation of Csk-binding protein/phosphoprotein associated with glycolipid-enriched microdomains in lipid rafts in resting T cells. J Immunol 169:2813–2817

  88. Young RM, Holowka D, Baird B (2003) A lipid raft environment enhances Lyn kinase activity by protecting the active site tyrosine from dephosphorylation. J Biol Chem 278:20746–20752

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We thank the staff of Unidad de Microscopía Confocal (Dpto. Biología Celular, CINVESTAV-IPN) for providing confocal facilities. We also thank Dr Liora Shoshani Z. and Dr. Dominique Mornet for their generous antibody donations. This work was supported by a Consejo Nacional de Ciencia y Tecnología (CONACYT) grant (79921) to Enrique O. Hernández-González, and with doctoral scholarships to Deneb Maldonado-García (322154), Tania Reyes-Miguel (262875) and Monica L. Salgado-Lucio (263011).

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Correspondence to Enrique O. Hernández-González.

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Supplementary Figure 1

Chlortetracycline (CTC) staining patterns after capacitation of guinea pig sperm. Sperm were incubated under capacitation conditions and, after 90 min, were stained with CTC. Fluorescence images corresponding to three different CTC staining patterns of the guinea pig sperm. The patterns scored were: F corresponds to non-capacitated sperm, B corresponds to capacitated sperm, AR corresponds to acrosome-reacted sperm. Means ± SEM, n = 3 independent experiments. (JPEG 33.4 kb)

Supplementary Figure 2

Immunoprecipitation of Rac-1 as a negative control. Co-immunoprecipitation assays with Rac-1 were performed with total sperm extracts (500 μg) from non-capacitated and 90-min capacitated sperm. The precipitated proteins were subjected to SDS–PAGE and Western blot analysis probed for Rac-1 as a positive control and probed with anti-flotillin-1 (Flot-1), anti-flotillin-2 (Flot-2), anti-spectrin and anti-SFKs antibodies to determine its associations. Western blot images are representative of three independent experiments. (JPEG 34.1 kb)

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Maldonado-García, D., Salgado-Lucio, M.L., Roa-Espitia, A.L. et al. Calpain inhibition prevents flotillin re-ordering and Src family activation during capacitation. Cell Tissue Res 369, 395–412 (2017). https://doi.org/10.1007/s00441-017-2591-2

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  • Flotillin microdomain aggregation
  • Spectrin and sperm membrane domains
  • Src family kinases
  • Acrosome reaction
  • Role of spectrin in capacitation