Scaffold protein Lin7 family in membrane skeletal protein complex in mouse seminiferous tubules

  • Akio Kamijo
  • Yurika Saitoh
  • Takeharu Sakamoto
  • Hiroshi Kubota
  • Junji Yamauchi
  • Nobuo TeradaEmail author
Original Paper


The membrane skeletal complex, protein 4.1G–membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell–cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.


Lin7 Membrane skeletal protein Membrane palmitoylated proteins Spermatogonia Seminiferous tubule 



This work was partially supported by a grant from the Japan Society for the Promotion of Science (KAKENHI 16K08463) to N. Terada.

Compliance with ethical standard

Conflict of interest

The authors have no conflicting interests.


  1. Baines AJ, Lu HC, Bennett PM (2013) The protein 4.1 family: hub proteins in animals for organizing membrane proteins. Biochim Biophys Acta 1838:605–619CrossRefGoogle Scholar
  2. Bohl J, Brimer N, Lyons C, Vande Pol SB (2007) The stardust family protein MPP7 forms a tripartite complex with LIN7 and DLG1 that regulates the stability and localization of DLG1 to cell junctions. J Biol Chem 282:9392–9400CrossRefGoogle Scholar
  3. Chapman KM, Medrano GA, Chaudhary J, Hamra FK (2015) NRG1 and KITL signal downstream of retinoic acid in the germline to support soma-free syncytial growth of differentiating spermatogonia. Cell Death Discov 1:15018CrossRefGoogle Scholar
  4. Cohen AR, Wood DF, Marfatia SM, Walther Z, Chishti AH, Anderson M (1998) Human CASK/Lin-2 binds syndecan-2 and protein 4.1 and localizes to the basolateral membrane of epithelial cells. J Cell Biol 142:129–138CrossRefGoogle Scholar
  5. Costa LD, Galimand J, Fenneteau O, Mohandas N (2013) Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders. Blood Rev 27:167–178CrossRefGoogle Scholar
  6. Discher DE, Winardi R, Schischmanoff PO, Parra M, Conboy JG, Mohandas N (1995) Mechanochemistry of protein 4.1 spectrin–actin binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening. J Cell Biol 130:897–907CrossRefGoogle Scholar
  7. Funke L, Dakoji S, Bredt DS (2005) Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions. Annu Rev Biochem 74:219–245CrossRefGoogle Scholar
  8. Hanada T, Takeuchi A, Sondarva G, Chishti AH (2003) Protein 4.1-mediated membrane targeting of human discs large in epithelial cells. J Biol Chem 278:34445–34450CrossRefGoogle Scholar
  9. Hori K, Konno D, Maruoka H, Sobue K (2003) MALS is a binding partner of IRSp53 at cell–cell contacts. FEBS Lett 554:30–34CrossRefGoogle Scholar
  10. Jo K, Derin R, Li M, Bredt DS (1999) Characterization of MALS/Velis-1, -2, and -3: a family of mammalian LIN-7 homologs enriched at brain synapses in association with the postsynaptic density-95/NMDA receptor postsynaptic complex. J Neurosci 19:4189–4199CrossRefGoogle Scholar
  11. Kakiuchi K, Taniguchi K, Kubota H (2018) Conserved and non-conserved characteristics of porcine glial cell line-derived neurotrophic factor expressed in the testis. Sci Rep 8:7656. CrossRefGoogle Scholar
  12. Kamberov E, Makarova O, Roh M, Liu A, Karnak D, Straight S, Margolis B (2000) Molecular cloning and characterization of Pals, proteins associated with mLin-7. J Biol Chem 275:11425–11431CrossRefGoogle Scholar
  13. Kamijo A, Saitoh Y, Ohno N, Ohno S, Terada N (2014) Immunohistochemical study of mouse sciatic nerves under various stretching conditions with “in vivo cryotechnique”. J Neurosci Methods 227:181–188CrossRefGoogle Scholar
  14. Kamijo A, Saitoh Y, Ohno S, Ohno N, Terada N (2016) Immunohistochemical study of the membrane skeletal protein, membrane protein palmitoylated 6 (MPP6), in the mouse small intestine. Histochem Cell Biol 145:81–92CrossRefGoogle Scholar
  15. Kanatsu-Shinohara M, Morimoto H, Shinohara T (2012) Enrichment of mouse spermatogonial stem cells by melanoma cell adhesion molecule expression. Biol Reprod 87:1–10CrossRefGoogle Scholar
  16. Kubota H, Avarbock MR, Brinster RL (2004) Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA 101:16489–16494CrossRefGoogle Scholar
  17. Lee S, Fan S, Makarova O, Straight S, Margolis B (2002) A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia. Mol Cell Biol 22:1778–1791CrossRefGoogle Scholar
  18. Lozovatsky L, Abayasekara N, Piawah S, Walther Z (2009) CASK deletion in intestinal epithelia causes mislocalization of LIN7C and the DLG1/Scrib polarity complex without affecting cell polarity. Mol Biol Cell 20:4489–4499CrossRefGoogle Scholar
  19. Maekawa M, Ito C, Toyama Y, Suzuki-Toyota F, Fujita E, Momoi T, Toshimori K (2011) Localization of RA175 (Cadm1), a cell adhesion molecule of the immunoglobulin superfamily, in the mouse testis, and analysis of male infertility in the RA175-deficient mouse. Andrologia 43:180–188CrossRefGoogle Scholar
  20. Misawa H, Kawasaki Y, Mellor J, Sweeney N, Jo K, Nicoll RA, Bredt DS (2001) Contrasting localizations of MALS/LIN-7 PDZ proteins in brain and molecular compensation in knockout mice. J Biol Chem 276:9264–9272CrossRefGoogle Scholar
  21. Murakami S, Sakurai-Yageta M, Maruyama T, Murakami Y (2014) Trans-homophilic interaction of CADM1 activates PI3K by forming a complex with MAGuK-family proteins MPP3 and Dlg. PLoS One 9:e110062. CrossRefGoogle Scholar
  22. Ohno N, Terada N, Yamakawa H, Komada M, Ohara O, Trapp BD, Ohno S (2007) Expression of protein 4.1G in Schwann cells of the peripheral nervous system. J Neurosci Res 84:568–577CrossRefGoogle Scholar
  23. Oliva C, Escobedo P, Astorga C, Molina C, Sierralta J (2012) Role of the MAGUK protein family in synapse formation and function. Dev Neurobiol 72:57–72CrossRefGoogle Scholar
  24. Olsen O, Moore KA, Fukata M, Kazuta T, Trinidad JC, Kauer FW, Streuli M, Misaswa H, Burlingame AL, Nicoll RA, Bred DS (2005) Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex. J Cell Biol 170:1127–1134CrossRefGoogle Scholar
  25. Olsen O, Funke L, Long JF, Fukata M, Kazuta T, Trinidad JC, Moore KA, Misawa H, Welling PA, Burlingame AL et al (2007) Renal defects associated with improper polarization of the CRB and DLG polarity complexes in MALS-3 knockout mice. J Cell Biol 179:151–164CrossRefGoogle Scholar
  26. Onda T, Uzawa K, Nakashima D, Saito K, Iwadate Y, Seki N, Shibahara T, Tanzawa H (2007) Lin-7C/VELI3/MALS-3: an essential component in metastasis of human squamous cell carcinoma. Cancer Res 67:9643–9648CrossRefGoogle Scholar
  27. Reissner C, Missler M (2014) MAGUKs end a tale of promiscuity. Proc Natl Acad Sci USA 111:17350–17351CrossRefGoogle Scholar
  28. Saitoh Y, Ohno N, Yamauchi J, Sakamoto T, Terada N (2017) Deficiency of a membrane skeletal protein, 4.1G, results in myelin abnormalities in the peripheral nervous system. Histochem Cell Biol 148:597–606CrossRefGoogle Scholar
  29. Saitoh Y, Kamijo A, Yamauchi J, Sakamoto T, Terada N (2019) The membrane palmitoylated protein, MPP6, is involved in myelin formation in the mouse peripheral nervous system. Histochem Cell Biol 151:385–394CrossRefGoogle Scholar
  30. Shelly M, Mosesson Y, Citri A, Lavi S, Zwang Y, Melamed-Book N, Aroeti B, Yarden Y (2003) Polar expression of ErbB-2/HER2 in epithelia. Bimodal regulation by Lin-7. Dev Cell 5:475–486CrossRefGoogle Scholar
  31. Shingai T, Ikeda W, Kakunaga S, Morimoto K, Takekuni K, Itoh S, Satoh K, Takeuchi M, Imai T, Monden M et al (2003) Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells. J Biol Chem 278:35421–35427CrossRefGoogle Scholar
  32. Shinoda G, Yvanka de Soysa T, Seligson MT, Yabuuchi A, Fujiwara Y, Huang PY, Hagan JP, Gregory RI, Moss EG, Daley GQ (2013) Lin28a regulates germ cell pool size and fertility. Stem Cells 31:1001–1009CrossRefGoogle Scholar
  33. Stöhr H, Molday LL, Molday RS, Weber BH, Biedermann B, Reichenbach A, Krämer F (2005) Membrane-associated guanylate kinase proteins MPP4 and MPP5 associate with Veli3 at distinct intercellular junctions of the neurosensory retina. J Comp Neurol 481:31–41CrossRefGoogle Scholar
  34. Straight SW, Karnak D, Borg JP, Kamberov E, Dare H, Margolis B, Wade JB (2000) mLin-7 is localized to the basolateral surface of renal epithelia via its NH2 terminus. Am J Physiol Renal Physiol 287:F464–F475CrossRefGoogle Scholar
  35. Straight SW, Pieczynski JN, Whiteman EL, Liu CJ, Margolis B (2006) Mammalian Lin-7 stabilizes polarity protein complexes. J Biol Chem 281:37738–37747CrossRefGoogle Scholar
  36. Takase MH, Nussea R (2016) Paracrine Wnt/β-catenin signaling mediates proliferation of undifferentiated spermatogonia in the adult mouse testis. Proc Natl Acad Sci USA 113:1489–1497CrossRefGoogle Scholar
  37. Terada N, Ohno N, Yamakawa H, Baba T, Fujii Y, Zea Z, Ohara O, Ohno S (2004) Immunohistochemical study of protein 4.1B in the normal and W/Wv mouse seminiferous epithelium. J Histochem Cytochem 52:769–777CrossRefGoogle Scholar
  38. Terada N, Ohno N, Saitoh S, Saitoh Y, Komada M, Kubota H, Ohno S (2010) Involvement of a membrane skeleton protein, 4.1G, for Sertoli/germ cell interaction. Reproduction 139:883–892CrossRefGoogle Scholar
  39. Terada N, Saitoh Y, Ohno N, Komada M, Saitoh S, Peles E, Ohno S (2012) Essential function of protein 4.1G targeting of membrane protein palmitoylated 6 into Schmidt-Lanterman incisures in myelinated nerves. Mol Cell Biol 32:199–205CrossRefGoogle Scholar
  40. Terada N, Saitoh Y, Kamijo A, Ohno S, Ohno N (2016) Involvement of membrane skeletal molecules in the Schmidt–Lanterman incisures in Schwann cells. Med Mol Morphol 49:5–10CrossRefGoogle Scholar
  41. Tseng TC, Marfatia SM, Bryant PJ, Pack S, Zhuang Z, O’Brien JE, Lin L, Hanada T, Chishti AH (2001) VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain. Biochim Biophys Acta 1518:249–259CrossRefGoogle Scholar
  42. Uzawa K, Kasamatsu A, Shimizu T, Saito Y, Baba T, Sakuma K, Fushimi K, Sakamoto Y, Ogawara K, Shiiba M et al (2014) Suppression of metastasis by mirtazapine via restoration of the Lin-7C/β-catenin pathway in human cancer cells. Sci Rep 4(5433):1–8Google Scholar
  43. van der Weyden Arends MJ, Chausiaux OE, Ellis PJ, Lange UC, Surani MA, Affara N, Murakami Y, Adams DJ, Bradley A (2006) Loss of TSLC1 causes male infertility due to a defect at the spermatid stage of spermatogenesis. Mol Cell Biol 26:3595–3609CrossRefGoogle Scholar
  44. Wakayama T, Iseki S (2009) Role of the spermatogenic–Sertoli cell interaction through cell adhesion molecule-1 (CADM1) in spermatogenesis. Anat Sci Int 84:112–121CrossRefGoogle Scholar
  45. Wakayama T, Koami H, Ariga H, Kobayashi D, Sai Y, Tsuji A, Yamamoto M, Iseki S (2003) Expression and functional characterization of the adhesion molecule spermatogenic immunoglobulin superfamily in the mouse testis. Biol Reprod 68:1755–1763CrossRefGoogle Scholar
  46. Yageta M, Kuramochi M, Masuda M, Fukami T, Fukuhara H, Murayama T, Shibuya M, Murakami Y (2002) Direct association of TSLC1 and DAL-1, two distinct tumor suppressor proteins in lung cancer. Cancer Res 62:5129–5133Google Scholar
  47. Ye F, Zeng M, Zhang M (2018) Mechanisms of MAGUK-mediated cellular junctional complex organization. Curr Opin Struct Biol 48:6–15CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Health Science Division, Department of Medical SciencesShinshu University Graduate School of Medicine, Science and TechnologyNaganoJapan
  2. 2.Center for Medical EducationTeikyo University of ScienceTokyoJapan
  3. 3.Division of Cellular and Molecular BiologyThe Institute of Medical Science, The University of TokyoTokyoJapan
  4. 4.Laboratory of Cell and Molecular Biology, Department of Animal Science, School of Veterinary MedicineKitasato UniversityAomoriJapan
  5. 5.Laboratory of Molecular Neuroscience and Neurology, School of Life SciencesTokyo University of Pharmacy and Life SciencesTokyoJapan

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