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Associations between beta-tubulin and mitochondria in adult isolated heart myocytes as shown by immunofluorescence and immunoelectron microscopy

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Summary

We have investigated the associations between β-tubulin and mitochondria in freshly isolated cardiac myocytes from the rat. Beta-tubulin was identified by using monoclonal antibodies for immunofluorescence and high resolution immunogold electron microscopy. In addition, conventional transmission and scanning electron microscopic studies were performed. After chemical stabilization in a formaldehyde solution, the myocytes were shock-frozen at −150°C, cryosectioned at −70°C and subsequently processed for immunohistochemical and immunocytochemical microscopy. A characteristic of the rod shaped myocytes is the presence of a dense network of microtubules in the cytoplasm displaying a pattern of strong anti-β-tubulin reaction. The complexity of this network however varies considerably among the myocytes reflecting microtubule dynamic instability. Further, our findings demonstrate that the β-tubulin label in rod cells is confined to the perinuclear and interfibrillar spaces and, therefore, is largely colocalized with the cytoplasmic organelles. In myocytes undergoing severe contracture the distribution of β-tubulin is entirely restricted to the outer mitochondrial-containing domain. This implies that, in a cell model with marked segregation of the contractile filaments and organelles, mitochondria are codistributed with microtubules in the total absence of desmin intermediate filaments. Moreover, our immunogold preparations demonstrate anti-β-tubulin labelling in the outer mitochondrial membrane as well as of fibres in close apposition to this membrane. These results indicate the presence of a specific β-tubulin binding to the outer mitochondrial membrane that probably also involves microtubule based translocators and/or MAPs.

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References

  • Ball EH, Singer SJ (1982) Mitochondria are associated with microtubules and not with intermediate filaments in cultured fibroblasts. Proc Natl Acad Sci USA 79:123–126

    Google Scholar 

  • Bayley PM (1990) What makes microtubules dynamic? J Cell Sci 95:329–334

    Google Scholar 

  • Bayley PM, Schilstra MJ, Martin SR (1990) Microtubule dynamic instability: numerical simulation of microtubule transition properties using a lateral cap model. J Cell Sci 95:33–48

    Google Scholar 

  • Bernier-Valentin F, Aunis D, Rousset B (1983) Evidence for tubulin-binding sites on cellular membranes: plasma membranes, mitochondrial membranes, and secretory granule membranes. J Cell Biol 97:209–216

    Google Scholar 

  • Bertier B, Samuel JL, Marotte F, Rappaport L (1984) Microtubules and intermediate filaments are not associated in rat heart myocytes. J Submicrosc Cytol 16:99–100

    Google Scholar 

  • Cambray-Deakin MA, Robson SJ, Burgoyne RD (1988) Colocalization of acetylated microtubules, glial filaments, and mitochondria in astrocytes in vitro. Cell Motil Cytoskeleton 10:438–449

    Google Scholar 

  • Cartwright J Jr, Goldstein MA (1982) Microtubules in soleus muscles of the postnatal and adult rat. J Ultrastruct Res 79:74–84

    Google Scholar 

  • Cleveland DW (1987) The multitubulin hypothesis revisited: what have we learned? J Cell Biol 104:381–383

    Google Scholar 

  • Cleveland D (1990) Microtubule mapping. Cell 60:701–702

    Google Scholar 

  • Dabora SL, Sheetz MP (1988) Cultured cell extracts support organelle movement on microtubules in vitro. Cell Motil Cytoskeleton 10:482–495

    Google Scholar 

  • Farrel KW, Jordan MA, Miller HD, Wilson L (1987) Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling. J Cell Biol 104:1035–1046

    Google Scholar 

  • Ferrans VJ, Roberts WC (1973) Intermyofibrillar and nuclearmyofibrillar connections in human and canine myocardium. An ultrastructural study. J Mol Cell Cardiol 5:247–257

    Google Scholar 

  • Forbes MS, Sperelakis N (1980) Structures located at the levels of the Z bands in mouse ventricular myocardial cells. Tissue Cell 12:467–489

    Google Scholar 

  • Forman DS, Lynch KJ, Smith RS (1987) Organelle dynamics in lobster axons: anterograde, retrograde and stationary mitochondria. Brain Res 412:96–106

    Google Scholar 

  • Fry DM, Scales D, Inesi G (1979) The ultrastructure of membrane alterations of enzymatically dissociated cardiac myocytes. J Mol Cell Cardiol 11:1151–1163

    Google Scholar 

  • Fulton C, Simpson PA (1976) Selective synthesis and utilization of flagellar tubilin. The multitubulin hypothesis. In: Goldman R, Pollard T, Rosenbaum J (eds) Cell Motility. Cold Spring Harbor Publications, New York, pp 987–1005

    Google Scholar 

  • Goldstein MA, Entman ML (1979) Microtubules in mammalian heart muscle. J Cell Biol 80:183–195

    Google Scholar 

  • Hargreaves AJ, Avila J (1985) Localization and characterization of tubulin-like proteins associated with brain mitochondria: the presence of a membrane-specific isoform. J Neurochem 45:490–496

    Google Scholar 

  • Hatt PY, Berjal G, Moravec J, Swynghedauw B (1970) Heart failure: an electron microscopic study of the left ventricular papillary muscle in aortic insufficiency in the rabbit. J Mol Cell Cardiol 1:235–247

    Google Scholar 

  • Heggeness MH, Simon M, Singer SJ (1978) Association of mitochondria with microtubules in cultured cells. Proc Natl Acad Sci USA 75:3863–3866

    Google Scholar 

  • Jacobson SL, Piper HM (1986) Cell cultures of adult cardiomyocytes as models of the myocardium. J Mol Cell Cardiol 18:661–678

    Google Scholar 

  • Jancsik V, Filliol D, Felter S, Rendon A (1989) Binding of microtubule-associated proteins (MAPs) to rat brain mitochondria: a comparative study of the binding of MAP2, its microtubule binding and projection domains and tau proteins. Cell Motil Cytoskeleton 14:372–381

    Google Scholar 

  • Linden M, Nelson BD, Letterrier J-F (1989) The specific binding of the microtubule-associated protein 2[MAP2] to the outer membrane of rat brain mitochondria. Biochem J 261:167–173

    Google Scholar 

  • Mac Rea TH, Langdon CM (1989) Tubulin synthesis, structure and function: what are the relationships? Biochem Cell Biol 67:770–790

    Google Scholar 

  • Menzel D, Schliwa M (1986) Motility in the siphonous green algaBryopsis. I. Spatial organization of the cytoskeleton and organelle movements. Eur J Cell Biol 40:275–285

    Google Scholar 

  • Mittal B, Sanger JM, Sanger JW (1989) Visualization of intermediate filaments in living cells using fluorescently labeled desmin. Cell Motil Cytoskeleton 12:127–138

    Google Scholar 

  • Nag AC, Fischman DA, Aumont MC, Zak R (1977) Studies of isolated adult rat heart cells: the surface morphology and the influence of extracellular calcium ion concentration on cellular viability. Tissue Cell 9:419–436

    Google Scholar 

  • Nakamura Y, Ueda K (1982) Connection between microtubules and mitochondria. Cytologia Tokyo 47:713–715

    Google Scholar 

  • Page E (1966) Tubular systems in Purkinje cells of the cat heart. J Ultrastruct Res 17:72–83

    Google Scholar 

  • Paschal BM, Vallee RB (1987) Retrograde transport by the microtubule-associated protein MAP 1C. Nature 330:181–183

    Google Scholar 

  • Pfister KK, Wagner MC, Stenoien DL, Brady ST, Bloom GS (1989) Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. J Cell Biol 108:1453–1463

    Google Scholar 

  • Piper HM, Probst I, Schwartz P, Hütter FJ, Spieckermann PG (1982) Culturing of calcium stable adult cardiac myocytes. J Mol Cell Cardiol 14:397–412

    Google Scholar 

  • Powell T, Terrar DA, Twist VW (1980) Electrical properties of individual cells isolated from adult rat ventricular myocardium. J Physiol (Lond) 302:131–153

    Google Scholar 

  • Price MG, Gomer RH (1989) Mitoskeletin: a mitochondrial protein found in cytoskeletal preparations. Cell Motil Cytoskeleton 13:274–287

    Google Scholar 

  • Raine CS, Røytta M, Dolich M (1987) Microtubule-mitochondrial associations in regenerating axons after taxol intoxication. J Neurocytol 16:461–468

    Google Scholar 

  • Rappaport L, Samuel JL (1988) Microtubules in cardiac myocytes. In: Bourne GH, Friedlander M, Jeon KW (eds) International review of cytology, vol 113. Academic Press, London, pp 101–143

    Google Scholar 

  • Russo MA, Cittadini A, Dani AM, Inesi G, Terranova T (1981) An ultrastructural study of calcium induced degenerative changes in dissociated heart cells. J Mol Cell Cardiol 13:265–279

    Google Scholar 

  • Samuel J-L, Schwartz K, Lompre A-M, Delcayre C, Marotte F, Swynghedauw B, Rappaport L (1983) Immunological quantitation and localization of tubulin in adult rat heart isolated myocytes. Eur J Cell Biol 31:99–106

    Google Scholar 

  • Schliwa M (1986) The cytoskeleton. An introductory survey. In: Alfert M, Beermann W, Goldstein L, Porter KR (eds) Cell Biology Monographs 13. Springer, Wien, New York

    Google Scholar 

  • Schliwa M, Euteneuer U, Koonce MP (1987) Identification of a protein involved in microtubule-dependent organelle movements of the giant amoeba,Reticulomyxa. In: Wohlfarth-Bottermann KE (ed) Nature and function of cytoskeletal proteins in motility and transport. Fortschr Zool 34, G. Fischer, Stuttgart New York, pp 157–169

    Google Scholar 

  • Schnapp BJ, Vale RD, Sheetz MP, Reese TS (1985) Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell 40:455–462

    Google Scholar 

  • Shpetner HS, Vallee RB (1989) Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules. Cell 59:421–432

    Google Scholar 

  • Smith DS, Jailfors U, Cameron BF (1975) Morphological evidence for the participation of microtubules in axonal transport. Ann NY Acad Sci 253:472–506

    Google Scholar 

  • Soltys BJ, Borisy GG (1985) Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes. J Cell Biol 100:1682–1689

    Google Scholar 

  • Stebbings H (1990) How is microtubule-based organelle translocation regulated? J Cell Sci 95:5–7

    Google Scholar 

  • Sætersdal T, Dalen H, Røli J (1989) Immunofluorescence and immunogold electron microscopy of desmin in isolated adult heart myocytes of the rat. Histochemistry 92:467–473

    Google Scholar 

  • Sætersdal TS, Røli J, Myklebust R, Engedal H (1978) Preservation of shock-frozen myocardial tissue as shown by cryo-ultramicrotomy and freeze-fracture studies. In: Echlin P (ed) Low temperature biological microscopy and microanalysis. The Royal Microscopical Society, Oxford, pp 56–70

    Google Scholar 

  • Thornell L-E, Butler-Browne GS, Carlsson E, Eppenberger HM, Fürst DO, Grove BK, Holmbom B, Small JV (1986) Cryoultramicrotomy and immunocytochemistry in the analysis of muscle fine structure. Scanning Electron Microsc IV:1407–1418

    Google Scholar 

  • Thornell L-E, Ericksson A (1981) Filament systems in the Purkinje fibers of the heart. Am J Physiol 241:H291-H305

    Google Scholar 

  • Vale RD, Reese TS, Sheetz MP (1985) Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42:39–50

    Google Scholar 

  • Vale RD, Scholey JM, Sheetz MP (1986) Kinesin: possible biological functions of a new microtubule motor. Trends Biochem Sci 11:464–468

    Google Scholar 

  • Watkins SC, Samuel JL, Marotte F, Bertier-Savalle B, Rappaport L (1987) Microtubules and desmin filaments during onset of heart hypertrophy in rat: a double immunoelectron microscope study. Circ Res 60:327–336

    Google Scholar 

  • Weiss DG (1987) Visualization of microtubule gliding and organelle transport along microtubules from squid giant axons. Fortschr Zool 34:133–144

    Google Scholar 

  • Zernig G, Wiche G (1985) Morphological integrity of single adult cardiac myocytes isolated by collagenase treatment: immunolocalization of tubulin microtubule-associated proteins 1 and 2, plectin, vimentin, and vinculin. Eur J Cell Biol 38:113–122

    Google Scholar 

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Sætersdal, T., Greve, G. & Dalen, H. Associations between beta-tubulin and mitochondria in adult isolated heart myocytes as shown by immunofluorescence and immunoelectron microscopy. Histochemistry 95, 1–10 (1990). https://doi.org/10.1007/BF00737221

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