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Vesicular transport system in myotubes: ultrastructural study and signposting with vesicle-associated membrane proteins

Abstract

Myofibers have characteristic membrane compartments in their cytoplasm and sarcolemma, such as the sarcoplasmic reticulum, T-tubules, neuromuscular junction, and myotendinous junction. Little is known about the vesicular transport that is believed to mediate the development of these membrane compartments. We determined the locations of organelles in differentiating myotubes. Electron microscopic observation of a whole myotube revealed the arrangement of Golgi apparatus, rough endoplasmic reticulum, autolysosomes, mitochondria, and smooth endoplasmic reticulum from the perinuclear region toward the end of myotubes and the existence of a large number of vesicles near the ends of myotubes. Vesicles in myotubes were further characterized using immunofluorescence microscopy to analyze expression and localization of vesicle-associated membrane proteins (VAMPs). VAMPs are a family of seven proteins that regulate post-Golgi vesicular transport via the fusion of vesicles to the target membranes. Myotubes express five VAMPs in total. Vesicles with VAMP2, VAMP3, or VAMP5 were found near the ends of the myotubes. Some of these vesicles are also positive for caveolin-3, suggesting their participation in the development of T-tubules. Our morphological analyses revealed the characteristic arrangement of organelles in myotubes and the existence of transport vesicles near the ends of the myotubes.

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References

  1. Advani RJ, Yang B, Prekeris R et al (1999) VAMP-7 mediates vesicular transport from endosomes to lysosomes. J Cell Biol 146:765–776

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  2. Al-Qusairi L, Laporte J (2011) T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases. Skelet Muscle 1:26. doi:10.1186/2044-5040-1-26

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  3. Bao ZZ, Lakonishok M, Kaufman S, Horwitz AF (1993) Alpha 7 beta 1 integrin is a component of the myotendinous junction on skeletal muscle. J Cell Sci 106:579–589. doi:10.1093/hmg/ddp362

    CAS  PubMed  Google Scholar 

  4. Baumert M, Maycox PR, Navone F et al (1989) Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain. EMBO J 8:379–384

    CAS  PubMed Central  PubMed  Google Scholar 

  5. Braiman L, Alt A, Kuroki T et al (2001) Activation of protein kinase czeta induces serine phosphorylation of VAMP2 in the GLUT4 Compartment and increases glucose transport in skeletal muscle. Mol Cell Biol 21:7852–7861. doi:10.1128/MCB.21.22.7852- 7861.2001

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  6. Burattini S, Ferri P, Battistelli M et al (2004) C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. Eur J Histochem 48:223–233

    CAS  PubMed  Google Scholar 

  7. Chaineau M, Danglot L, Galli T (2009) Multiple roles of the vesicular-SNARE TI-VAMP in post-Golgi and endosomal trafficking. FEBS Lett 583:3817–3826. doi:10.1016/j.febslet.2009.10.026

    CAS  PubMed  Article  Google Scholar 

  8. Curci R, Battistelli M, Burattini S et al (2008) Surface and inner cell behaviour along skeletal muscle cell in vitro differentiation. Micron 39:843–851. doi:10.1016/j.micron.2007.12.007

    CAS  PubMed  Article  Google Scholar 

  9. Flucher BE, Terasaki M, Chin HM et al (1991) Biogenesis of transverse tubules in skeletal muscle in vitro. Dev Biol 145:77–90

    CAS  PubMed  Article  Google Scholar 

  10. Furuta N, Fujita N, Noda T et al (2010) Combinational soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins VAMP8 and Vti1b mediate fusion of antimicrobial and canonical autophagosomes with lysosomes. Mol Biol Cell 21:1001–1010. doi:10.1091/mbc.E09-08-0693

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  11. Golini L, Chouabe C, Berthier C et al (2011) Junctophilin 1 and 2 proteins interact with the L-type Ca2+ channel dihydropyridine receptors (DHPRs) in skeletal muscle. J Biol Chem 286:43717–43725. doi:10.1074/jbc.M111.292755

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  12. Hirokawa N, Niwa S, Tanaka Y (2010) Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease. Neuron 68:610–638. doi:10.1016/j.neuron.2010.09.039

    CAS  PubMed  Article  Google Scholar 

  13. Hong W (2005) SNAREs and traffic. Biochim Biophys Acta 1744:120–144. doi:10.1016/j.bbamcr.2005.03.014

    CAS  PubMed  Article  Google Scholar 

  14. Ishikawa H (1968) Formation of elaborate networks of T-system tubules in cultured skeletal muscle with special reference to the T-system formation. J Cell Biol 38:51–66

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  15. Lee E (2002) Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle. Science 297:1193–1196. doi:10.1126/science.1071362

    CAS  PubMed  Article  Google Scholar 

  16. Lu Z, Joseph D, Bugnard E et al (2001) Golgi complex reorganization during muscle differentiation: visualization in living cells and mechanism. Mol Biol Cell 12:795–808

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  17. McMahon HT, Ushkaryov YA, Edelmann L et al (1993) Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. Nature 364:346–349. doi:10.1038/364346a0

    CAS  PubMed  Article  Google Scholar 

  18. McMahon HT, Kozlov MM, Martens S (2010) Membrane curvature in synaptic vesicle fusion and beyond. Cell 140:601–605. doi:10.1016/j.cell.2010.02.017

    CAS  PubMed  Article  Google Scholar 

  19. Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326. doi:10.1016/j.cell.2010.01.028

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  20. Neville C, Rosenthal N, McGrew M et al (1997) Methods Cell Biol 52:85–116

    CAS  PubMed  Article  Google Scholar 

  21. Parton R, Way M, Zorzi N, Stang E (1997) Caveolin-3 associates with developing T-tubules during muscle differentiation. J Cell Biol 136:137–154

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  22. Proux-Gillardeaux V, Gavard J, Irinopoulou T et al (2005) Tetanus neurotoxin-mediated cleavage of cellubrevin impairs epithelial cell migration and integrin-dependent cell adhesion. Proc Natl Acad Sci USA 102:6362–6367. doi:10.1073/pnas.0409613102

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  23. Randhawa VK, Bilan PJ, Khayat ZA et al (2000) VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts. Mol Biol Cell 11:2403–2417

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  24. Rappoport JZ (2008) Focusing on clathrin-mediated endocytosis. Biochem J 412:415. doi:10.1042/BJ20080474

    CAS  PubMed  Article  Google Scholar 

  25. Rossetto O (1996) VAMP/synaptobrevin isoforms 1 and 2 are widely and differentially expressed in nonneuronal tissues. J Cell Biol 132:167–179

    CAS  PubMed  Article  Google Scholar 

  26. Rossi D, Barone V, Giacomello E et al (2008) The sarcoplasmic reticulum: an organized patchwork of specialized domains. Traffic 9:1044–1049. doi:10.1111/j.1600-0854.2008.00717.x

    CAS  PubMed  Article  Google Scholar 

  27. Roth TF, Porter KR (1964) Yolk protein uptake in the oocyte of the mosquito Aedes aegypti L. J Cell Biol 20:313–332

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  28. Sanes JR, Lichtman JW (2001) Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Nat Rev Neurosci 2:791–805. doi:10.1038/35097557

    CAS  PubMed  Article  Google Scholar 

  29. Segev N (2011) Coordination of intracellular transport steps by GTPases. Semin Cell Dev Biol 22:33–38. doi:10.1016/j.semcdb.2010.11.005

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  30. Singhal N, Martin PT (2011) Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction. Dev Neurobiol 71:982–1005. doi:10.1002/dneu.20953

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  31. Skalski M, Yi Q, Kean MJ et al (2010) Lamellipodium extension and membrane ruffling require different SNARE-mediated trafficking pathways. BMC Cell Biol 11:62. doi:10.1186/1471-2121-11-62

    PubMed Central  PubMed  Article  Google Scholar 

  32. Sorrentino V (2011) Sarcoplasmic reticulum: structural determinants and protein dynamics. Int J Biochem Cell Biol 43:1075–1078. doi:10.1016/j.biocel.2011.04.004

    CAS  PubMed  Article  Google Scholar 

  33. Steegmaier M, Klumperman J (1999) Vesicle-associated membrane protein 4 is implicated in trans-Golgi network vesicle trafficking. Mol Biol Cell 10:1957–1972

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  34. Stenoien DL, Knyushko TV, Londono MP et al (2007) Cellular trafficking of phospholamban and formation of functional sarcoplasmic reticulum during myocyte differentiation. Am J Physiol Cell Physiol 292:C2084–C2094. doi:10.1152/ajpcell.00523.2006

    CAS  PubMed  Article  Google Scholar 

  35. Südhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474–477. doi:10.1126/science.1161748

    PubMed Central  PubMed  Article  Google Scholar 

  36. Tajika Y, Sato M, Murakami T et al (2007) VAMP2 is expressed in muscle satellite cells and up-regulated during muscle regeneration. Cell Tissue Res 328:573–581. doi:10.1007/s00441-006-0376-0

    CAS  PubMed  Article  Google Scholar 

  37. Tajika Y, Murakami T, Sato M et al (2008) VAMP2 is expressed in myogenic cells during rat development. Dev Dyn 237:1886–1892. doi:10.1002/dvdy.21596

    PubMed  Article  Google Scholar 

  38. Tajika Y, Takahashi M, Hino M et al (2010) VAMP2 marks quiescent satellite cells and myotubes, but not activated myoblasts. Acta Histochem Cytochem 43:107–114. doi:10.1267/ahc.10010

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  39. Takahashi M, Tajika Y, Khairani AF et al (2013) The localization of VAMP5 in skeletal and cardiac muscle. Histochem Cell Biol 139:573–582. doi:10.1007/s00418-012-1050-0

    CAS  PubMed  Article  Google Scholar 

  40. Takekura H, Flucher BE, Franzini-Armstrong C (2001) Sequential docking, molecular differentiation, and positioning of T-tubule/SR junctions in developing mouse skeletal muscle. Developmental Biology 239:204–214. doi:10.1006/dbio2001.0437

    CAS  PubMed  Article  Google Scholar 

  41. Tayeb MA, Skalski M, Cha MC et al (2005) Inhibition of SNARE-mediated membrane traffic impairs cell migration. Exp Cell Res 305:63–73. doi:10.1016/j.yexcr.2004.12.004

    CAS  PubMed  Article  Google Scholar 

  42. Tortorella LL, Pilch PF (2002) C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression. Am J Physiol Endocrinol Metab 283:E514–E524. doi:10.1152/ajpendo.00092.2002

    CAS  PubMed  Google Scholar 

  43. Towler MC, Kaufman SJ, Brodsky FM (2004) Membrane traffic in skeletal muscle. Traffic 5:129–139. doi:10.1111/j.1600-0854.2003.00164.x

    CAS  PubMed  Article  Google Scholar 

  44. Trimble WS, Cowan DM, Scheller RH (1988) VAMP-1: a synaptic vesicle-associated integral membrane protein. Proc Natl Acad Sci USA 85:4538–4542

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  45. van Kerkhof P, Lee J, McCormick L et al (2005) Sorting nexin 17 facilitates LRP recycling in the early endosome. EMBO J 24:2851–2861. doi:10.1038/sj.emboj.7600756

    PubMed Central  PubMed  Article  Google Scholar 

  46. Veale KJ, Offenhäuser C, Lei N et al (2011) VAMP3 regulates podosome organisation in macrophages and together with Stx4/SNAP23 mediates adhesion, cell spreading and persistent migration. Exp Cell Res 317:1817–1829. doi:10.1016/j.yexcr.2011.04.016

    CAS  PubMed  Article  Google Scholar 

  47. Wang C-C, Ng CP, Shi H et al (2010) A role for VAMP8/endobrevin in surface deployment of the water channel aquaporin 2. Mol Cell Biol 30:333–343. doi:10.1128/MCB.00814-09

    PubMed Central  PubMed  Article  Google Scholar 

  48. Zeng Q, Subramaniam VN, Wong SH et al (1998) A novel synaptobrevin/VAMP homologous protein (VAMP5) is increased during in vitro myogenesis and present in the plasma membrane. Mol Biol Cell 9:2423–2437

    CAS  PubMed Central  PubMed  Article  Google Scholar 

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Acknowledgments

We thank Ms. Harumi Matsuda and Mr. Yoshihiro Morimura (Department of Anatomy, Gunma University Graduate School of Medicine) for both technical and secretarial assistance and Mr. Hiroyuki Seo and Dr. Touko Hirano (Laboratory for Analytical Instruments, Education and Research Support Center, Gunma University Graduate School of Medicine) for their support for the EM preparation and observation. We also thank Mr. Hisao Yajima and Mr. Ryota Koyama (Gunma University School of Medicine) for cooperating in technical matters as a part of Practical Course on Basic Medical Science. This work was supported by MEXT KAKENHI Grant Numbers 21790175, 23590230, and 25860138.

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Correspondence to Hiroshi Yorifuji.

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Tajika, Y., Takahashi, M., Khairani, A.F. et al. Vesicular transport system in myotubes: ultrastructural study and signposting with vesicle-associated membrane proteins. Histochem Cell Biol 141, 441–454 (2014). https://doi.org/10.1007/s00418-013-1164-z

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Keywords

  • Skeletal muscle
  • Myotube
  • SNARE
  • VAMP
  • T-tubule