Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction.

Department of Biochemistry Faculty of Science Charles University Vinicna 7 12844 Prague 2 Czech Republic

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources University of Life Sciences Prague Kamycka 129 16500 Prague 6 Czech Republic

Department of Zoology Faculty of Science Charles University Vinicna 7 12844 Prague 2 Czech Republic

Group of Cell Biology and Innovative Therapies Centre for Molecular Biophysics University of Orleans UPR4301 45071 Orleans France

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Prumyslova 595 25250 Vestec Czech Republic

Laboratory of Structural Bioinformatics of Proteins Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Prumyslova 595 25250 Vestec Czech Republic

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Almeida E.A., Huovila A.P., Sutherland A.E., Stephens L.E., Calarco P.G., Shaw L.M., Mercurio A.M., Sonnenberg A., Primakoff P., Myles D.G., et al. Mouse egg integrin alpha 6 beta 1 functions as a sperm receptor. Cell. 1995;81:1095–1104. doi: 10.1016/S0092-8674(05)80014-5. PubMed DOI

Miyado K., Yamada G., Yamada S., Hasuwa H., Nakamura Y., Ryu F., Suzuki K., Kosai K., Inoue K., Ogura A., et al. Requirement of CD9 on the Egg Plasma Membrane for Fertilization. Science. 2000;287:321–324. doi: 10.1126/science.287.5451.321. PubMed DOI

Ziyyat A., Rubinstein E., Monier-Gavelle F., Barraud V., Kulski O., Prenant M., Boucheix C., Bomsel M., Wolf J. CD9 controls the formation of clusters that contain tetraspanins and the integrin α6β1, which are involved in human and mouse gamete fusion. J. Cell Sci. 2006;119:416–424. doi: 10.1242/jcs.02730. PubMed DOI

Lozahic S., Christiansen D., Manié S., Gerlier D., Billard M., Boucheix C., Rubinstein E. CD46 (membrane cofactor protein) associates with multiple β1 integrins and tetraspans. Eur. J. Immunol. 2000;30:900–907. doi: 10.1002/1521-4141(200003)30:3<900::AID-IMMU900>3.0.CO;2-X. PubMed DOI

Rezcallah M.S., Hodges K., Gill D.B., Atkinson J.P., Wang B., Cleary P.P. Engagement of CD46 and α5β1 integrin by group A streptococci is required for efficient invasion of epithelial cells. Cell. Microbiol. 2005;7:645–653. doi: 10.1111/j.1462-5822.2004.00497.x. PubMed DOI

Hynes R.O. Integrins: Bidirectional, allosteric signaling machines. Cell. 2002;110:673–687. doi: 10.1016/S0092-8674(02)00971-6. PubMed DOI

Gupta V., Alonso J.L., Sugimori T., Issafi M., Xiong J.-P., Arnaout M.A. Role of the β-Subunit Arginine/Lysine Finger in Integrin Heterodimer Formation and Function. J. Immunol. 2008;180:1713–1718. doi: 10.4049/jimmunol.180.3.1713. PubMed DOI PMC

Takada Y., Ye X., Simon S. The integrins. Genome Biol. 2007;8:215. doi: 10.1186/gb-2007-8-5-215. PubMed DOI PMC

Kassner P.D., Kawaguchi S., Hemler M.E. Minimum alpha chain cytoplasmic tail sequence needed to support integrin-mediated adhesion. J. Biol. Chem. 1994;269:19859–19867. PubMed

Melker A.A., Sterk L.M., Delwel G.O., Fles D.L., Daams H., Weening J.J., Sonnenberg A. The A and B variants of the alpha 3 integrin subunit: Tissue distribution and functional characterization. Lab. Investig. 1997;76:547–563. PubMed

Lu C., Shimaoka M., Zang Q., Takagi J., Springer T.A. Locking in alternate conformations of the integrin αLβ2 I domain with disulfide bonds reveals functional relationships among integrin domains. Proc. Natl. Acad. Sci. USA. 2001;98:2393–2398. doi: 10.1073/pnas.041618598. PubMed DOI PMC

Sen M., Yuki K., Springer T.A. An internal ligand-bound, metastable state of a leukocyte integrin, alphaxbeta2. J. Cell Biol. 2013;203:629–642. doi: 10.1083/jcb.201308083. PubMed DOI PMC

Zhu J., Luo B.H., Xiao T., Zhang C., Nishida N., Springer T.A. Structure of a complete ntegrin ectodomain in a physiologic resting state and activation and deactivation by applied forces. Mol. Cell. 2008;32:849–861. doi: 10.1016/j.molcel.2008.11.018. PubMed DOI PMC

Takagi J., Springer T.A. Integrin activation and structural rearrangement. Immunol. Rev. 2002;186:141–163. doi: 10.1034/j.1600-065X.2002.18613.x. PubMed DOI

Wiche G., Gromov D., Donovan A., Castañón M.J., Fuchs E. Expression of plectin mutant cDNA in cultured cells indicates a role of COOH-terminal domain in intermediate filament association. J. Cell Biol. 1993;121:607–619. doi: 10.1083/jcb.121.3.607. PubMed DOI PMC

Yang Y., Dowling J., Yu Q.C., Kouklis P., Cleveland D.W., Fuchs E. An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments. Cell. 1996;86:655–665. doi: 10.1016/S0092-8674(00)80138-5. PubMed DOI

Svitkina T.M., Verkhovsky A.B., Borisy G.G. Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton. J. Cell Biol. 1996;135:991–1007. doi: 10.1083/jcb.135.4.991. PubMed DOI PMC

Glander H.J., Schaller J. Beta 1-integrins of spermatozoa: A flow cytophotometric analysis. Int. J. Androl. 1993;16:105–111. doi: 10.1111/j.1365-2605.1993.tb01162.x. PubMed DOI

Glander H.J., Rohwedder A., Henkel R. Adhesion molecules and matrix proteins on human spermatozoa. Andrologia. 1998;30:289–296. doi: 10.1111/j.1439-0272.1998.tb01173.x. PubMed DOI

Klentzeris L.D., Fishel S., McDermott H., Dowell K., Hall J., Green S. A positive correlation between expression of beta 1-integrin cell adhesion molecules and fertilizing ability of human spermatozoa in vitro. Hum. Reprod. 1995;10:728–733. doi: 10.1093/oxfordjournals.humrep.a136023. PubMed DOI

Reddy V.R.K., Rajeev S.K., Gupta V. α6β1 Integrin is a potential clinical marker for evaluating sperm quality in men. Fertil. Steril. 2003;79:1590–1596. doi: 10.1016/S0015-0282(03)00368-6. PubMed DOI

Barraud-Lange V., Naud-Barriant N., Saffar L., Gattegno L., Ducot B., Drillet A.-S., Bomsel M., Wolf J.P., Ziyyat A. Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization. BMC Dev. Biol. 2007;7:102. doi: 10.1186/1471-213X-7-102. PubMed DOI PMC

Boissonnas C.C., Montjean D., Lesaffre C., Auer J., Vaiman D., Wolf J.-P., Ziyyat A. Role of sperm alphavbeta3 integrin in mouse fertilization. Dev. Dyn. 2010;239:773–783. doi: 10.1002/dvdy.22206. PubMed DOI

Frolikova M., Sebkova N., Ded L., Dvorakova-Hortova K. Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction. Sci. Rep. 2016;6:33714. doi: 10.1038/srep33714. PubMed DOI PMC

Johnson P.M., Clift L.E., Andrlikova P., Jursova M., Flanagan B.F., Cummerson J.A., Stopka P., Dvorakova-Hortova K. Rapid sperm acrosome reaction in the absence of acrosomal CD46 expression in promiscuous field mice (Apodemus) Reproduction. 2007;134:739–747. doi: 10.1530/REP-07-0363. PubMed DOI

Yang J., Yan R., Roy A., Xu D., Poisson J., Zhang Y. The i-TASSER suite. Protein structure and function prediction. Nat. Methods. 2015;12:7–8. doi: 10.1038/nmeth.3213. PubMed DOI PMC

Kozakov D., Beglov D., Bohnuud T., Mottarella S.E., Xia B., Hall D.R., Vajda S. How good is automated protein docking? Proteins. 2013;81:2159–2166. doi: 10.1002/prot.24403. PubMed DOI PMC

Kozakov D., Brenke R., Comeau S.R., Vajda S. PIPER: An FFT-based protein docking program with pairwise potentials. Proteins. 2006;65:392–406. doi: 10.1002/prot.21117. PubMed DOI

Pozzi A., Yurchenco P.D., Iozzo R.V. The nature and biology of basement membranes. Matrix Biol. Basement Membr. Health Dis. 2017;57–58:1–11. doi: 10.1016/j.matbio.2016.12.009. PubMed DOI PMC

Yazlovitskaya E.M., Viquez O.M., Tu T., De Arcangelis A., Georges-Labouesse E., Sonnenberg A., Pozzi A., Zent R. The laminin binding α3 and α6 integrins cooperate to promote epithelial cell adhesion and growth. Matrix Biol. 2018 doi: 10.1016/j.matbio.2018.08.010. PubMed DOI PMC

Kierszenbaum A.L., Rivkin E., Tres L.L. Molecular biology of sperm head shaping. Soc. Reprod. Fertil. Suppl. 2007;65:33–43. PubMed

Guyonnet B., Zabet-Moghaddam M., SanFrancisco S., Cornwall G.A. Isolation and Proteomic Characterization of the Mouse Sperm Acrosomal Matrix. Mol. Cell. Proteomics. 2012;11:758–774. doi: 10.1074/mcp.M112.020339. PubMed DOI PMC

Göb E., Schmitt J., Benavente R., Alsheimer M. Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes. PLoS ONE. 2010;5:e12072. doi: 10.1371/journal.pone.0012072. PubMed DOI PMC

Luo B.H., Springer T.A. Integrin structures and conformational signaling. Curr. Opin. Cell Biol. 2006;18:579–586. doi: 10.1016/j.ceb.2006.08.005. PubMed DOI PMC

Askari J.A., Buckley P.A., Mould A.P., Humphries M.J. Linking integrin conformation to function. J. Cell Sci. 2009;122:165–170. doi: 10.1242/jcs.018556. PubMed DOI PMC

Li H., Deng Y., Sun K., Yang H., Liu J., Wang M., Zhang Z., Lin J., Wu C., Wei Z., et al. Structural basis of kindlin-mediated integrin recognition and activation. Proc. Natl. Acad. Sci. USA. 2017;114:9349–9354. doi: 10.1073/pnas.1703064114. PubMed DOI PMC

Su Y., Xia W., Li J., Walz T., Humphries M.J., Vestweber D., Cabañas C., Lu C., Springer T.A., et al. Relating conformation to function in integrin α5β1. Proc. Natl. Acad. Sci. USA. 2016;113:E3872–E3881. doi: 10.1073/pnas.1605074113. PubMed DOI PMC

Tarone G., Russo M.A., Hirsch E., Odorisio T., Altruda F., Silengo L., Siracusa G. Expression of beta 1 integrin complexes on the surface of unfertilized mouse oocyte. Development. 1993;117:1369–1375. PubMed

Spiess M., Hernandez-Varas P., Oddone A., Olofsson H., Blom H., Waithe D., Lock J.G., Lakadamyali M., Strömblad S. Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions. J. Cell Biol. 2018;217:1929–1940. doi: 10.1083/jcb.201707075. PubMed DOI PMC

Chang Y.-F., Lee-Chang J.S., Panneerdoss S., MacLean J.A., Rao M.K. Isolation of Sertoli, Leydig, and spermatogenic cells from the mouse testis. BioTechniques. 2011;51:341–344. doi: 10.2144/000113764. PubMed DOI PMC

Laemmli U.K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. PubMed DOI

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Söderberg O., Leuchowius K.J., Gullberg M., Jarvius M., Weibrecht I., Larsson L.G., Landergen U. Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods. 2008;45:227–232. doi: 10.1016/j.ymeth.2008.06.014. PubMed DOI

Eastman P., Pande V.S. OpenMM: A hardware-independent framework for molecular simulations. Comput. Sci. Eng. 2010;12:34–39. doi: 10.1109/MCSE.2010.27. PubMed DOI PMC

Van der Spoel D., Lindahl E., Hess B., Groenhof G., Mark A.E., Berendsen H.J.C. GROMACS: Fast, flexible, and free. J. Comput. Chem. 2005;26:1701–1718. doi: 10.1002/jcc.20291. PubMed DOI

Schrodinger L.L.C. The pyMOL Molecular Graphics System. 2015. version 1.8.

Bolte S., Cordelières F.P. A guided tour into subcellular colocalization analysis in light microscopy. Pt 3J. Microsc. 2006;224:213–232. doi: 10.1111/j.1365-2818.2006.01706.x. PubMed DOI

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