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Dynamic post-translational modification of tubulin in rat cerebral cortical neurons extending neurites in culture: Effects of taxol

Journal of Neurocytology

Summary

Dissociated embryonic (E18–E20) rat cortical neurons were grown in culture and double-labelled by immunofluorescence with antibodies directed against tyrosinated (YL 1/2), detyrosinated (SUP GLU), and acetylated (6-11B-1) α-tubulin. Within 90 min of plating, neurons extended growth cones that were YL 1/2+ but SUP GLU and 6-11B-1. The neurite that forms behind the advancing growth cone is also, initially, YL 1/2+ and SUP GLU/6-11B-1. However, when it has attained a length of about half the cell body diameter, it becomes SUP GLU+ and 6-11B-1+.

The effects of the microtubule polymerizing agent taxol (15 μm) on growth cone and neurite α-tubulins was investigated. Taxol, as reported previously, caused the formation of microtubule loops in the central domain of the growth cone, a loss of filopodia, and the collapse of the growth cone onto the loops. The taxol effects peaked at 60 min, when over 85% of neurites showed microtubule loops, and declined thereafter, so that at 420 min in taxol, only about 23% of neurites had microtubule loops. Over this period there was an inverse correlation between the presence of microtubule loops and growth cones. Taxol had striking effects on the intensity of SUP GLU and 6-11B-1 staining in neurons. In 48 h cultures, a 30 min exposure to taxol enhanced the SUP GLU and 6-11B-1 staining of dendrites and axons and produced a loss of YL 1/2 staining in axons. Immunoblotting experiments confirmed that there was an overall reduction in YL 1/2 immunoreactivity and an increase in SUPGLU immunoreactivity. These observations support previous suggestions that the neurite microtubules are assembled in the growth cone and post-translationally modified in the neurite and, in addition, imply that growth cones can overcome the effects of taxol in the continued presence of the compound.

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References

  • Argarana, C. E., Barra, H. S. &Caputto, R. (1978) Release of [14C]-tyrosine from tubulinyl-[14C]-tyrosine by brain extract. Separation of a carboxypeptidase from tubulin tyrosine ligase.Molecular Cell Biochemistry 19, 17–22.

    Google Scholar 

  • Bamburg, J. R., Bray, D. &Chapman, K. (1986) Assembly of microtubules at the tip of growing axonsNature 321, 788–90.

    Google Scholar 

  • Barra, H. S., Rodriguez, J. A., Arce, C. A. &Caputto, R. A. (1973) A soluble preparation from rat brain that incorporates into its own proteins [14C]-arginine by a ribonuclease-sensitive system and [14C]-tyrosine by a ribonuclease-insensitive system.Journal of Neurochemishy 20, 97–108.

    Google Scholar 

  • Bartlett, W. P. &Banker, G. A. (1984) An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture I. Cells which develop without intercellular contacts.Journal of Neuroscience 4, 1944–53.

    Google Scholar 

  • Bass, P. W. &Black, M. M. (1990) Individual microtubules in the axon consist of domains that differ in both composition and stability.Journal of Cell Biology 111, 495–509.

    Google Scholar 

  • Bershadsky, A. D. &Vasiliev, J. M. (1988)Cytoskeleton. New York: Plenum Press.

    Google Scholar 

  • Black, M. M. (1987) Taxol interferes with the interaction of microtubule-associated proteins with microtubules in cultured neurons.Journal of Neuroscience 7, 3695–702.

    Google Scholar 

  • Black, M. M., Bass, P. W. &Humphries, S. (1989) Dynamics of α-tubulin deacetylation in intact neurons.Journal of Neuroscience 9, 358–68.

    Google Scholar 

  • Cambray-Deakin, M. A. &Burgoyne, R. D. (1987) Post-translational modifications of α-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum.Journal of Cell Biology 104, 1569–74.

    Google Scholar 

  • Daniels, M. (1972) Colchicine inhibition of nerve fiber formationin vitro.Journal of Cell Biology 53, 164–76.

    Google Scholar 

  • Dennerll, T. J., Joshi, H. C., Steel, V. L., Buxbaum, R. E. &Heidemann, S. R. (1988) Tension and compression in the cytoskeleton of PC12 neurites II: quantitative measurements.Journal of Cell Biology 107, 665–74.

    Google Scholar 

  • Ferreira, A. &Cáceres, A. (1989) The expression of acetylated microtubules during axonal and dendritic growth in cerebellar macroneurons which developin vitro.Developmental Brain Research 49, 205–213.

    Google Scholar 

  • Gordon-Weeks, P. R. (1987) The cytoskeletons of isolated, neuronal growth cones.Neuroscience 21, 977–87.

    Google Scholar 

  • Gordon-Weeks, P. R. (1989) Growth at the growth cone.Trends in Neurosciences 12, 238–240.

    Google Scholar 

  • Gordon-Weeks, P. R. (1991) The growth cone cytoskeleton.Comments on Developmental Neurobiology (in press).

  • Gordon-Weeks, P. R. &Lang, R. D. A. (1988) The α-tubulin of the growth cone is predominantly in the tyrosinated form.Developmental Brain Research 42, 156–60.

    Google Scholar 

  • Gordon-Weeks, P. R., Mansfield, S. G. &Curran, I. (1989) Direct visualisation of the soluble pool of tubulin in the neuronal growth cone: immunofluorescence studies following taxol polymerisation.Developmental Brain Research 49, 305–10.

    Google Scholar 

  • Goslin, K. &Banker, G. (1989) Experimental observations on the development of polarity by hippocampal neurons in culture.Journal of Cell Biology 108, 1507–16.

    Google Scholar 

  • Gundersen, G. G., Kalnoski, M. H. &Bulinski, J. C. (1984) Distinct populations of microtubules: tyrosinated and nontyrosinated alpha-tubulins are distributed differentlyin vivo.Cell 38, 779–89.

    Google Scholar 

  • Gundersen, G. G., Khawaja, S. &Bulinski, J. C. (1987) Post-polymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules.Journal of Cell Biology 105, 251–64.

    Google Scholar 

  • Khawaja, S., Gundersen, G. F. &Bulinski, J. C. (1988) Enhanced stability of microtubules enriched in detyrosinated alpha-tubulin is not a direct function of detyrosination level.Journal of Cell Biology 106, 141–49.

    Google Scholar 

  • Kilmartin, J. V., Wright, B. &Milstein, D. (1982) Rat monoclonal antitubulin antibodies derived by using a new non-secretory rat cell line.Journal of Cell Biology 93, 576–82.

    Google Scholar 

  • Kolani, S., Nishida, E., Kumagai, H. &Sakai, H. (1985) Calmodulin inhibits interaction of actin with MAP2 and tau, two major microtubule-associated proteins.Journal of Biological Chemistry 260, 10779–83.

    Google Scholar 

  • Kreis, T. E. (1987) Microtubules containing detyrosinated tubulin are less dynamic.EMBO Journal 6, 2597–606.

    Google Scholar 

  • Kumar, N. &Flavin, M. (1981) Preferential action of a brain detyrosinolated carboxypeptidase on polymerised tubulin.Journal of Biological Chemistry 256, 7678–86.

    Google Scholar 

  • Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.

    Google Scholar 

  • Letourneau, P. C. (1983) Differences in the organization of actin in the growth cones compared with neurites of cultured neurons from chick embryos.Journal of Cell Biology 97, 963–72.

    Google Scholar 

  • Letourneau, P. C. &Ressler, A. H. (1984) Inhibition of neurite initiation and growth by taxol.Journal of Cell Biology 98, 1355–62.

    Google Scholar 

  • Letourneau, P. C. &Shattuck, T. A. (1989) Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones.Development 105, 505–519.

    Google Scholar 

  • Letourneau, P. C., Shattuck, T. A. &Ressler, A. H. (1987) ‘Pull and push’ in neurite elongation, observations on the effects of different concentrations of cytochalasin B and taxol.Cell Motility and the Cytoskeleton 8, 193–209.

    Google Scholar 

  • Levi, G., Aloisi, F., Ciotti, M. T. &Gallo, V. (1984) Autoradiographic localisation and depolarisation-induced release of acidic amino acids in differentiating cerebellar granule cell cultures.Brain Research 290, 77–86.

    Google Scholar 

  • Lewis, S. A. &Cowan, N. J. (1990) Tubulin genes, structure, expression and regulation. InMicrotubule Proteins (edited byAvila, J.) pp. 37–66. Boca Raton, FL: CRC Press.

    Google Scholar 

  • Lim, S.-S., Sammak, P. J. &Borisy, G. G. (1989) Progressive and spatially differentiated stability of microtubules in developing neuronal cells.Journal of Cell Biology 109, 253–63.

    Google Scholar 

  • Mansfield, S. G. &Gordon-Weeks, P. R. (1990) Post-translational modification of tubulin in rat cerebral cortical neurons extending neurites in culture: effects of taxol.Journal of Physiology 426, 118P.

    Google Scholar 

  • Mitchison, T. &Kirschner, M. (1988) Cytoskeletal dynamics and nerve growth.Neuron 1, 761–72.

    Google Scholar 

  • Okabe, S. &Hirokawa, N. (1990) Turnover of fluorescently labelled tubulin and actin in the axon.Nature 343, 479–82.

    Google Scholar 

  • Parnass, J. &Horwitz, S. B. (1981) Taxol binds to polymerized tubulinin vitro.Journal of Cell Biology 91, 479–87.

    Google Scholar 

  • Piperno, G. &Fuller, M. T. (1985) Monoclonal antibodies specific for an acetylated form of α-tubulin recognize the antigen in cilia and flagella from a variety of organisms.Journal of Cell Biology 101, 2085–94.

    Google Scholar 

  • Piperno, G., Ledizet, M. &Chang, X. (1987) Microtubules containing acetylated α-tubulin in mammalian cells in culture.Journal of Cell Biology 104, 289–302.

    Google Scholar 

  • Pratt, L. F., Okamura, S. &Cleveland, D. W. (1987) A divergent testis-specific alpha tubulin isotype that does not contain a coded C-terminal tyrosine.Molecular Cell Biology 7, 552–5.

    Google Scholar 

  • Ramón, Y., Cajal, S. (1890) A quelle époque apparaissent les expensions des cellules nerveuses de la möelle épinière du poulet?Anatomischer Anzeiger 5, 609–13, 631–9.

    Google Scholar 

  • Raybin, D. &Flavin, M. (1977) Enzyme which specifically adds tyrosine to the α-chain of tubulin.Biochemistry 16, 2189–94.

    Google Scholar 

  • Robson, S. J. &Burgoyne, R. D. (1989) Differential localisation of tyrosinated, detyrosinated, and acetylated α-tubulins in neurites and growth cones of dorsal root ganglion neurons.Cell Motility and the Cytoskeleton 12, 273–82.

    Google Scholar 

  • Sargent, P. B. (1989) What distinguishes axons from dendrites? Neurons know more than we do.Trends in Neurosciences 12, 203–5.

    Google Scholar 

  • Satillaro, W. (1986) Interaction of microtubule-associated protein 2 with actin filaments.Biochemistry 25, 2003–9.

    Google Scholar 

  • Schiff, P. B. &Horwitz, S. B. (1980) Taxol stabilizes microtubules in mouse fibroblast cells.Proceedings of the National Academy of Science (USA) 77, 1561–5.

    Google Scholar 

  • Schiff, P. B., Fant, J. &Horwitz, S. B. (1979) Promotion of microtubule assemblyin vitro by taxol.Nature 277, 665–7.

    Google Scholar 

  • Schulze, E., Asai, D. J., Bulinski, J. C. &Kirschner, M. (1987) Post-translational modification and microtubule stability.Journal of Cell Biology 105, 2167–77.

    Google Scholar 

  • Seldon, S. &Pollard, T. D. (1983) Phosphorylation of microtubule-associated proteins regulates their interaction with actin filaments.Journal of Biological Chemistry 258, 7064–71.

    Google Scholar 

  • Serrano, L. &Avila, J. (1990) Structure and function of tubulin regions. InMicrotubule Proteins (edited byAvila, J.) pp. 67–88. Florida: CRC Press.

    Google Scholar 

  • Thompson, W. C. (1982) The cyclic tyrosination/detyrosination of alpha tubulin.Methods in Cell Biology 24, 235–55.

    Google Scholar 

  • Wehland, J., Schroder, H. C. &Weber, K. (1984) Amino acid sequence requirements in the epitope recognised by the α-tubulin-specific rat monoclonal antibody YL 1/2.EMBO Journal 3, 1295–1300.

    Google Scholar 

  • Yamada, K. M., Spooner, B. S. &Wessells, N. K. (1970) Axon growth: roles of microfilaments and microtubules.Proceedings of the National Academy of Sciences (USA) 66, 1206–12.

    Google Scholar 

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Mansfield, S.G., Gordon-Weeks, P.R. Dynamic post-translational modification of tubulin in rat cerebral cortical neurons extending neurites in culture: Effects of taxol. J Neurocytol 20, 654–666 (1991). https://doi.org/10.1007/BF01187067

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