Summary
Primary cultures of dissociated embryonic day 18 rat cerebral cortices were labelled by immunofluorescence with antibodies directed either against phosphorylated and non-phosphorylated MAP 1B (antibody 81) or against phosphorylated MAP 1B (antibody 150). Both antibodies stain cortical neurons, including their neurites and growth cones, in early (18 h) cultures, whereas only antibody 81 stained glial cells. By 4 days in culture, phosphorylated MAP 1B is largely restricted to axonal processes and growth cones, where it is often distributed in a gradient that is highest distally. In axonal processes and growth cones after 18 h and 4 days in culture, the phosphorylated form of MAP 1B is present both in a soluble form and bound to microtubules. Growth cones isolated from postnatal day 5 rat forebrain were labelledin vitro with32P-orthophosphate and detergent soluble and insoluble (cytoskeleton) fractions prepared. SDS-PAGE analysis revealed several major phosphoproteins in isolated growth cone cytoskeletons, including MAP 1B. Phosphorylated MAP 1B was also present in the detergent soluble fraction of growth cones. Immunoblotting and immunoprecipitation with MAP 1B antibodies confirmed the identification of MAP 1B and that the protein is phosphorylated in growth cones. These data show that MAP 1B, in particular the phosphorylated isoform, is present in growth cones and suggest that phosphorylation of MAP 1B may play an important role in neurite elongation.
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Aletta, J. M., Lewis, S. A., Cowan, N. J. &Greene, L. A. (1988) Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule associated protein 1.2 (MAP 1.2).Journal of Cell Biology 106, 1573–81.
Asai, D. J., Thompson, W. C., Wilson, L., Dresden, C. F., Schulman, H. &Purich, D. L. (1985). Microtubule associated proteins (MAPs): a monoclonal antibody to MAP 1 decorates microtubulesin vitro but stains stress fibres and not microtubulesin vivo.Proceedings of the National Academy of Science (USA) 82, 1434–8.
Baas, P. W., White, L. A. &Heidemann, S. R. (1987) Microtubule polarity reversal accompanies regrowth of amputated neurites.Proceedings of the National Academy of Science (USA) 84, 5272–6.
Bamburg, J. R., Bray, D. &Chapman, K. (1986) Assembly of microtubules at the tip of growing axons.Nature 321, 788–90.
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.
Bloom, G. S., Luca, F. C. &Vallee, R. B. (1985a) Identification of high molecular weight microtubule associated proteins in anterior pituitary tissue and cells using taxol dependent purification combined with microtubule associated protein specific antibodies.Biochemistry 24, 4185–91.
Bloom, G. S., Luca, F. C. &Vallee, R. B. (1985b) Microtubule associated protein 1B: identification of a major component of the neuronal cytoskeleton.Proceedings of the National Academy of Science (USA) 82, 5404–8.
Bloom, G. S., Schoenfield, T. A. &Vallee, R. B. (1984) Widespread distribution of the major polypeptide component of MAP 1 (Microtubule associated protein 1) in the nervous system.Journal of Cell Biology 98, 320–30.
Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye-binding.Analytical Biochemistry 72, 248–54.
Brugg, B. &Matus, A. (1988) PC12 cells express juvenile microtubule-associated proteins during nerve factorinduced neurite outgrowth.Journal of Cell Biology 107, 643–50.
Calvert, R. &Anderton, B. H. (1985) A microtubule associated protein MAP 1 which is expressed at elevated levels during development of rat cerebellum.EMBO Journal 4, 1171–6.
Daniels, M. (1972) Colchicine inhibition of nerve fiber formationin vitro.Journal of Cell Biology 53, 164–76.
Diaz-Nido, J., Armas-Potela, R., Martinez, A., Rocha, M. &Avila, J. (1991) Role of microtubules in neurite outgrowth. InThe Nerve Growth Cone (edited byKater, S. B., Letourneau, P. C. &Macagno, E. R.) New York: Raven Press, in press.
Diaz-Nido, J. &Avila, J. (1989) Characterization of proteins immunologically related to brain microtubule associated protein MAP 1B in non-neuronal cells.Journal of Cell Science 92, 607–20.
Diaz-Nido, J., Serrano, L., Hernandez, M. A. &Avila, J. (1990) Phosphorylation of microtubule proteins in rat brain at different developmental stages: comparison with that found in neural cultures.Journal of Neurochemistry 54, 211–22.
Diaz-Nido, J., Serrano, L., Mendez, E. &Avila, J. (1988) A casein kinase II-related activity is involved in phosphorylation of microtubule associated protein MAP 1B during neuroblastoma cell differentiation.Journal of Cell Biology 106, 2057–65.
Dodd, J. &Jessell, T. M. (1988) Axonal guidance and the patterning of neuronal projections in vertebrates.Science 242, 692–9.
Drubin, D. G., Feinstein, S., Shooter, E. &Kirschner, M. (1985) Nerve growth factor induced outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly promoting factors.Journal of Cell Biology 101, 1799–1807.
Fairbanks, G., Steck, N. C. &Wallach, D. F. (1981) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane.Biochemistry 10, 2606–17.
Fischer, I. &Romano-Clarke, G. (1990). Changes in microtubule-associated protein MAP 1B phosphorylation during rat brain development.Journal of Neurochemistry 55, 328–33.
Fried, R. C. &Blaustein, M. P. (1978) Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes).Journal of Cell Biology 78, 685–700.
Gard, D. L. &Kirschner, M. W. (1985) A polymerdependent increase in phosphorylation of β-tubulin accompanies differentiation of a neuroblastoma cell line.Journal of Cell Biology 100, 764–74.
Garner, C. C., Garner, A., Huber, G., Kozak, C. &Matus, A. (1990) Molecular cloning of MAP 1 (MAP 1A) and MAP 5 (MAP 1B): identification of distinct genes and their differential expression in developing brain.Journal of Neurochemistry 55, 146–54.
Garner, C. C., Matus, A., Anderton, B. &Calvert, R. (1989) Microtubule-associated proteins MAP 5 and MAP 1x: closely related components of the neuronal cytoskeleton with different cytoplasmic distribution in the developing brain.Molecular Brain Research 5, 85–92.
Girault, J. A., Hemmings, H. C. Jr., Williams, K. R., Nairn, A. C. &Greengard, P. (1989) Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein by casein kinase II.Journal of Biological Chemisfry 264, 21748–59.
Girault, J. A., Hemmings, H. C. Jr., Zorn, S. H., Gustafson, E. L. &Greengard, P. (1990) Characterization in mammalian brain of a DARPP-32 serine kinase identical to casein kinase II.Journal of Neurochemistry 55, 1772–83.
Gogstad, G. O. &Krutnes, M. -B. (1982) Measurement of protein in cell suspensions using the Coomassie Brilliant Blue dye-binding assay.Analytical Biochemistry 126, 355–9.
Gordon-Weeks, P. R. (1987) The cytoskeletons of isolated, neuronal growth cones.Neuroscience 21, 977–89.
Gordon-Weeks, P. R. (1991) Growth cones: the mechanism of neurite advance.BioEssays 13, 235–9.
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.
Gordon-Weeks, P. R. &Lockerbie, R. O. (1984) Isolation and partial characterization of neuronal growth cones from neonatal rat forebrain.Neuroscience 13, 119–36.
Gordon-Weeks, P. R. &Mansfield, S. G. (1991) The assembly of microtubules in growth cones: the role of microtubule-associated proteins. InThe Nerve Growth Cone (edited byKater, S. B., Letourneau, P. C. &Macagno, E. R.). New York: Raven Press, in press.
Gordon-Weeks, P. R., Mansfield, S. G. &Curran, I. (1989) Direct visualization of the soluble pool of tubulin in the neuronal growth cone: immunofluorescence studies following taxol polymerisation.Developmental Brain Research 49, 305–10.
Goslin, K., Schreyer, D. J., Skene, J. H. P. &Banker, G. A. (1988) Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones.Nature 336, 672–4.
Greene, L. A., Liem, R. K. H. &Shelanski, M. L. (1983) Regulation of a high molecular weight microtubule associated protein in PC12 cells by nerve growth factor.Journal of Cell Biology 96, 76–83.
Gundersen, G. G., Kalnoski, M. H. &Bulinski, J. C. (1984) Distinct populations of microtubules: tyrosinated and nontyrosinated alpha-tubulins are distributed differently in vivo.Cell 38, 779–89.
Hasegawa, M., Arai, T. &Ihara, Y. (1990) Immunochemical evidence that fragments of phosphorylated MAP5 (MAP 1B) are bound to neurofibrillary tangles in Alzheimer's disease.Neuron 4, 909–18.
Hoshi, M., Nishida, E., Inagaki, M., Gotoh, Y. &Sakai, H. (1990) Activation of a serine/-threonine kinase that phosphorylates microtubule-associated protein 1Bin vitro by growth factors and phorbol esters in quiescent rat fibroblastic cells.European Journal of Biochemistry 193, 513–19.
Iimoto, D. S., Masliah, E., Deteresa, R., Terry, R. D. &Saitoh, T. (1989) Aberrant casein kinase II in Alzheimer's disease.Brain Research 507, 273–80.
Kilmartin, J. V., Wright, B. &Milstein, C. (1982) Rat monoclonal antitubulin antibodies derived using a new non-secreting rat cell line.Journal of Cell Biology 93, 576–82.
Kuznetsov, S. A., &Gelfand, V. I. (1987) 18kDa microtubule associated protein: identification as a new light chain (LC-3) of microtubule associated protein 1 (MAP 1).FEBS Letters 212, 145–8.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.
Letourneau, P. C. &Ressler, A. H. (1984) Inhibition of neurite initiation and growth by taxol.Journal of Cell Biology 98, 1355–62.
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.
Lindwall, G. &Cole, R. D. (1984) Phosphorylation affects the ability of tau protein to promote microtubule assembly.Journal of Cell Biological Chemistry 259, 5301–5.
Lockerbie, R. O. (1987) The neuronal growth cone: a review of its locomotory, navigational and target recognition capabilities.Neuroscience 20, 719–29.
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.
Mansfield, S. G. &Gordon-Weeks, P. R. (1991) Dynamic post-translational modification of tubulin in rat cerebral cortical neurons extending neurites in culture: effects of taxol.Journal of Neurocytology 20, 654–66.
Matus, A. (1988) Microtubule-associated proteins: their potential role in determining neuronal morphology.Annual Review of Neuroscience 11, 29–44.
Meiri, K. &Gordon-Weeks, P. R. (1990) GAP-43 in growth cones is associated with areas of membrane that are tightly bound to substrate, and is a component of a membrane skeleton subcellular fraction.Journal of Neuroscience 10, 256–66.
Murthy, A. &Flavin, M. (1983) Microtubule assembly using the microtubule associated protein MAP 2 prepared in defined states of phosphorylation with protein kinase and phosphatase.European Journal of Biochemistry 137, 37–46.
Noble, M., Lewis, S. A. &Cowan, N. J. (1989) The microtubule binding domain of microtubule associated protein MAP 1B contains a repeated sequence motif unrelated to that of MAP 2 and tau.Journal of Cell Biology 109, 3367–76.
Olmsted, J. B. (1986) Microtubule-associated proteins.Annual Review of Neuroscience 11, 29–44.
Pfenninger, K. H., Ellis, L., Johnson, M. P., Friedman, L. B., &Somlo, S. (1983) Nerve growth cones isolated from fetal rat brain. Subcellular fractionation and characterization.Cell 35, 573–84.
Pisano, M. R., Hegazy, M. G., Reimann, E. M. &Lokas, L. A. (1988) Phosphorylation of protein B-50 (GAP-43) from adult rat brain cortex by casein kinase II.Biochemical and Biophysical Research Communications 155, 1207–1212.
Ramón Y Cajal, S. (1890) A quelle époque apparaissent les expensions des cellules nerveuses de la môlle épinière du populet?Anatomischer Anzeiger 21, 609–13, 631–9.
Read, S. M. &Northcote, D. H. (1981) Minimisation of variation in the response to different proteins of the Coomassie Blue G dye-binding assay for protein.Analytical Biochemistry 116, 53–64.
Riederer, B., Cohen, R. &Matus, A. (1986) MAP 5: a novel microtubule associated protein under strong developmental regulation.Journal of Neurocytology 15, 763–75.
Riederer, B. M., Guadano-Ferraz, A. &Innocenti, G. M. (1990) Difference in distribution of microtubule-associated proteins 5a and 5b during the development of cerebral cortex and corpus callosum in cats: dependence on phosphorylation.Developmental Brain Research 56, 235–43.
Sargent, P. B. (1990) What distinguishes axons from dendrites? Neurons know more than we do.Trends in Neuroscience 12, 203–5.
Sato-Yoshitake, R., Shiomura, Y., Miyasaka, H. &Hirokawa, N. (1989) Microtubule associated protein 1B: molecular structure, localization and phosphorylation-dependent expression in developing neurons.Neuron 3, 229–38.
Schiff, P. B., Fant, J. &Howitz, S. B. (1979) Promotion of microtubule assemblyin vitro by taxol.Nature 277, 665–7.
Schoenfeld, J. A., Mckerracher, L., Obar, R. &Vallee, R. B. (1989) MAP 1A and MAP 1B are structurally related microtubule associated proteins with distinct developmental patterns in the CNS.Journal of Neuroscience 9, 1712–30.
Serrano, L., Hernandez, M. A., Diaz-Nido, J. &Avila, J. (1989) Association of casein kinase II with microtubules.Experimental Cell Research 181, 263–72.
Tsao, H., Aletta, J. M. &Greene, L. A. (1990) Nerve Growth Factor and Fibroblast Growth Factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins.Journal of Biological Chemistry 265, 15471–80.
Viereck, C. &Matus, A. (1990) The expression of phosphorylated and non-phosphorylated forms of MAP 5 in the amphibian CNS.Brain Research 508, 257–64.
Viereck, C., Tucker, R. P. &Matus, A. (1989) The adult rat olfactory system expresses microtubule-associated proteins found in the developing brain.Journal of Neuroscience 9, 3547–57.
Wiche, G., Oberkanins, C. &Himmler, A. (1991) Molecular structure and function of microtubule-associated proteins.International Review of Cytology 124, 217–73.
Yamada, K. M., Spooner, B. S. &Wessells, N. K. (1970) Axon growth: roles of microfilaments and microtubules.Proceedings of the National Academy of Science (USA) 66, 1206–12.
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Mansfield, S.G., Diaz-Nido, J., Gordon-Weeks, P.R. et al. The distribution and phosphorylation of the microtubule-associated protein MAP 1B in growth cones. J Neurocytol 20, 1007–1022 (1991). https://doi.org/10.1007/BF01187918
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DOI: https://doi.org/10.1007/BF01187918