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Ultrastructural immunocytochemical localization of B-50/GAP43, a protein kinase C substrate, in isolated presynaptic nerve terminals and neuronal growth cones

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Journal of Neurocytology

Summary

Accumulating evidence indicates that the neuron-specific B-50/GAP43, a substrate for protein kinase C, plays a role in neuronal differentiation and neuritogenesis during nervous tissue development and axonal regeneration. An ultrastructural immunocytochemical study on the localization of B-50 in presynaptic terminals (synaptosomes) and neuronal growth cones was carried out by means of cryoultramicrotomy with affinity-purified B-50 antibodies. Detection was accomplished with colloidal gold, conjugated either to protein-A or goat anti-rabbit immunoglobulins. In synaptosomes, isolated from the frontal cortex of 6-week-old rats, and in neuronal growth cones, isolated from forebrains of 5-day-old rats, the majority of B-50 is detected at the surrounding neuronal plasma membrane. In both neuronal growth cones and synaptosomes, a relatively small fraction of B-50 in the cytoplasm was not evidently associated with internal membranes. Our results indicate that B-50 is mainly located at the cytoplasmic face of the synaptosomal and neuronal growth cone plasma membrane. The similar B-50 localization in neuronal growth cones and synaptosomes suggests that, both in extending axons and mature synaptic terminals, B-50 may exert identical functions as a protein kinase C substrate at the plasma membrane.

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References

  • AKERS, R. F. & ROUTTENBERG, A. (1987) Calcium-promoted translocation of protein kinase C to synaptic membranes: relation to the phosphorylation of an endogenous substrate (protein F1) involved in synaptic plasticity.Journal of Neuroscience 7, 3976–83.

    Google Scholar 

  • ALEXANDER, K. A., CIMLER, B. M., MEIER, K. E. & STORM, D. R. (1987) Regulation of calmodulin binding to P-57.Journal of Biological Chemistry 262, 6108–13.

    Google Scholar 

  • ALOYO, V. J., ZWIERS, H. & GISPEN, W. H. (1983) Phosphorylation of B-50 protein by calcium-activated, phospholipid-dependent protein kinase and B-50 protein kinase.Journal of Neurochemistry 41, 649–53.

    Google Scholar 

  • BASI, G. S., JACOBSON, R. D., VIRAG, I., SCHILLING, J. & SKENE, P. (1987) Primary structure and transcriptional regulation of GAP43, a protein associated with nerve growth.Cell 49, 785–91.

    Google Scholar 

  • BENOWITZ, L. I. & ROUTTENBERG, A. (1987) A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity.Trends in Neurosciences 10, 527–32.

    Google Scholar 

  • BIENZ, K., EGGER, D. & PASAMONTES, L. (1986) Electron microscopic immunocytochemistry. Silver enhancement of colloidal gold marker allows double labeling with the same primary antibody.Journal of Histochemistry and Cytochemistry 10, 1337–42.

    Google Scholar 

  • CIMLER, B. M., GIEBELHAUS, D. H., WAKIM, B. T., STORM, D. R. & MOON, R. T. (1987) Characterization of murine cDNAs encoding P-57, a neural-specific calmodulin-binding protein.Journal of Biological Chemistry 262, 12158–63.

    Google Scholar 

  • DE CAMILLI, P., HARRIS, S. M., HUTTNER, S. M. & GREENGARD, P. (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes.Journal of Cell Biology 96, 1355–73.

    Google Scholar 

  • DE GRAAN, P. N. E., DEKKER, L. V., DE WIT, M., SCHRAMA, L. H. & GISPEN, W. H. (1988) Modulation of B-50 phosphorylation and polyphosphoinositide metabolism in synaptic plasma membranes by protein kinase C, phorbol diesters and ACTH.Journal of Receptor Research 8, 1–4.

    Google Scholar 

  • DE GRAAN, P. N. E., VAN HOOF, C. O. M., TILLY, B. C., OESTREICHER, A. B., SCHOTMAN, P. & GISPEN, W. H. (1985) Phosphoprotein B-50 in neuronal growth cones from fetal rat brain.Neuroscience Letters 61, 235–41.

    Google Scholar 

  • DEKKER, L. V., DE GRAAN, P. N. E., VERSTEEG, D. H. G., OESTREICHER, A. B. & GISPEN, W. H. (1989) Phosphorylation of B-50 (GAP43) is correlated with neurotransmitter release in rat hippocampal slices.Journal of Neurochemistry 52, 24–30.

    Google Scholar 

  • DUNKLEY, P. R., HEATH, J. W., HARRISON, S. M., JARVIE, P. E., GLENFIELD, P. J. & ROSTAS, J. A. P. (1988) A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellar fractions.Brain Research 441, 59–71.

    Google Scholar 

  • DUNKLEY, P. R., ROSTAS, J. A. P., HEATH, J. W. & POWIS, D. A. (1987) The preparation and use of synaptosomes for studying secretion of catecholamines. InThe Secretory Process, Vol. 3 (edited by POISNER, A. & TRIFARO, J. M.), pp. 315–34. Amsterdam: Elsevier.

    Google Scholar 

  • EICHBERG, J., DE GRAAN, P. N. E., SCHRAMA, L. H. & GISPEN, W. H. (1986) Dioctanoylglycerol and phorbol diesters enhance phosphorylation of phosphoprotein B-50 in native synaptic plasma membranes.Biochemical and Biophysical Research Communications 136, 1007–12.

    Google Scholar 

  • GISPEN, W. H., DE GRAAN, P. N. E., CHAN, S. Y. & ROUTTENBERG, A. (1986) Comparison between the neural acidic phosphoproteins B-50 and F1.Progress in Brain Research 69, 383–6.

    Google Scholar 

  • GISPEN, W. H., LEUNISSEN, J. L. M., OESTREICHER, A. B., VERKLEIJ, A. J. & ZWIERS, H. (1985) Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism.Brain Research 328, 381–5.

    Google Scholar 

  • GOLDENRING, J. R., LASHER, R. S., VALLANO, M. L., UEDA, T., NAITO, S., STERNBERGER, N. H., STERNBERGER, L. A. & DE LORENZO, R. J. (1986) Association of synapsin I with neuronal cytoskeleton.Journal of Biological Chemistry 261, 8495–504.

    Google Scholar 

  • GORDON-WEEKS, P. R. (1987) The cytoskeleton of isolated, neuronal growth cones.Neuroscience 21, 977–89.

    Google Scholar 

  • GORDON-WEEKS, P. R. & LOCKERBIE, R. O. (1984) Isolation and partial characterization of neuronal growth cones from neonatal rat forebrain.Neuroscience 13, 119–36.

    Google Scholar 

  • GORGELS, TH. G. M. F., OESTREICHER, A. B., DE KORT, E. J. M. & GISPEN, W. H. (1987) Immunocytochemical distribution of the protein kinase C substrate B-50 (GAP-43) in developing rat pyramidal tract.Neuroscience Letters 83, 59–64.

    Google Scholar 

  • GORGELS, TH. G. M. F., VAN LOOKEREN CAMPAGNE, M., OESTREICHER, A. B., GRIBNAU, A. A. M. & GISPEN, W. H. (1989) Ultrastructural localization of B-50/GAP43 in developing and mature pyramidal tract in the rat: predominant localization at the cytoplasmic side of the plasma membrane.Journal of Neuroscience (in press).

  • HESSELMANS, L. F. G. M., JENNEKENS, F. G. I., VAN DEN OORD, C. J. M., OESTREICHER, A. B., VELDMAN, H. & GISPEN, W. H. (1989) A light and electron microscopical study of B-50 (GAP43) in human intramuscular nerve and neuromuscular junctions during development.Journal of the Neurological Sciences 89, 301–11.

    Google Scholar 

  • HOLGATE, C., JACKSON, P., COWEN, P. & BIRD, C. (1983) Immunogold silver staining: new method for immunostaining with enhanced sensitivity.Journal of Histochemistry and Cytochemistry 31, 938–44.

    Google Scholar 

  • KARNS, L. R., FREEMAN, J. A. & FISHMAN, M. C. (1987) Cloning of complementary DNA for GAP43, a neuronal growth-related protein.Science 236, 597–600.

    Google Scholar 

  • MEIRI, K. F., PFENNINGER, K. H. & WILLARD, M. B. (1986) Growth associated protein, GAP43, a polypeptide that is induced when neurons extend axons, is a component of neuronal growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in neuronal growth cones.Proceedings of the National Academy of Sciences USA 83, 3537–41.

    Google Scholar 

  • MEIRI, K. F., WILLARD, M. & JOHNSON, M. I. (1988) Distribution and phosphorylation of the growth-associated protein GAP43 in regenerating sympathetic neurons in culture.Journal of Neuroscience 8, 2571–81.

    Google Scholar 

  • NEVE, R. L., PERRONE-BIZZOZERO, N. I., FINKLESTEIN, S., ZWIERS, H., BIRD, E., KURNIT, D. M. & BENOWITZ, L. I. (1987) The neuronal growth-associated protein GAP43 (B-50, F1): neuronal specificity, developmental regulation and regional distribution of the human and rat mRNA.Molecular Brain Research 2, 177–83.

    Google Scholar 

  • NIELANDER, H. B., SCHRAMA, L. H., VAN ROZEN, A. J., KASPERAITIS, M., OESTREICHER, A. B., DE GRAAN, P. N. E., GISPEN, W. H. & SCHOTMAN, P. (1987) Primary structure of the neuron-specific phosphoprotein B-50 is identical to growth-associated protein GAP43.Neuroscience Research Communications 1, 163–72.

    Google Scholar 

  • NORDEN, J. J., COSTELLO, B. & FREEMAN, J. A. (1987) The growth-associated protein, GAP43, is associated with synaptic vesicles and with plasma membrane in presynaptic terminals.Society for Neuroscience Abstracts 13, 1480.

    Google Scholar 

  • OESTREICHER, A. B. & GISPEN, W. H. (1986) Comparison of the immunocytochemical distribution of the phosphoprotein B-50 in the cerebellum and hippocampus of immature and adult rat brain.Brain Research 375, 267–9.

    Google Scholar 

  • OESTREICHER, A. B., VAN DONGEN, C. J., ZWIERS, H. & GISPEN, W. H. (1983) Affinity-purified anti-B-50 protein antibody: interference with the function of the phosphoprotein B-50 in synaptic plasma membranes.Journal of Neurochemistry 41, 331–40.

    Google Scholar 

  • PETRUCCI, T. C. & MORROW, J. S. (1987) Synapsin I: An actin-bundling protein under phosphorylation control.Journal of Cell Biology 105, 1355–63.

    Google Scholar 

  • ROSENTHAL, A., CHAN, S. Y., HENZEL, W., HASKELL, C., KUANG, W. J., CHEN, E., WILCOX, J. N., ULLRICH, A., GOEDDEL, D. V. & ROUTTENBERG, A. (1987) Primary structure and mRNA localization of protein F1, a growth-related protein kinase C substrate associated with synaptic plasticity.EMBO Journal 6, 3641–6.

    Google Scholar 

  • SCHIEBLER, W., JAHN, R., DOUCET, J. P., ROTHLEIN, J. & GREENGARD, P. (1986) Characterization of synapsin I binding to small synaptic vesicles.Journal of Biological Chemistry 261, 8383–90.

    Google Scholar 

  • SKENE, J. H. P., JACOBSON, R. D., SNIPES, G. J., MCGUIRE, C. B., NORDEN, J. J. & FREEMAN, J. A. (1986) A protein induced during nerve growth (GAP43) is a major component of growth-cone membranes.Science 233, 783–6.

    Google Scholar 

  • SKENE, J. H. P. & VIRAG, I. (1989) Post-translational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP43.Journal of Cell Biology 108, 613–24.

    Google Scholar 

  • TOKUYASU, K. T. (1984) Immunocryoultramicroscopy. InImmunolabeling for Electronmicroscopy (edited by POLAK, J. M. & VARNDELL, I. M.), pp. 71–82. Amsterdam: Elsevier.

    Google Scholar 

  • TOKUYASU, K. T. (1986) Cryosections for immunohistochemistry. InProceedings 11th International Congress Electron Microscopy (edited by IMURA, T., MARUSE, S. & SUZUKI, T.), Vol. III, pp. 1977–8, Kyoto, Japan.

  • VAN BERGEN EN HENEGOUWEN, P. M. P. & LEUNISSEN, J. L. M. (1986) Controlled growth of colloidal gold particles and implications for labelling efficiency.Histochemistry 85, 81–7.

    Google Scholar 

  • VAN HOOFF, C. O. M. (1988) The growth associated phosphoprotein B-50 in neuronal differentiation. PhD Thesis, University of Utrecht, Utrecht, The Netherlands.

    Google Scholar 

  • VAN HOOFF, C. O. M., DE GRAAN, P. N. E., BOONSTRA, J., OESTREICHER, A. B., SCMIDT-MICHELS, M. H. & GISPEN, W. H. (1986) Nerve growth factor enhances the level of the protein kinase C substrate B-50 in pheochromocytoma PC12 cell.Biochemical and Biophysical Research Communications 139, 644–51.

    Google Scholar 

  • VAN HOOFF, C. O. M., DE GRAAN, P. N. E., OESTREICHER, A. B. & GISPEN, W. H. (1988) B-50 phosphorylation and polyphosphoinositide metabolism in nerve growth cone membranes.Journal of Neuroscience 8, 1789–95.

    Google Scholar 

  • VAN HOOFF, C. O. M., HOLTHUIS, J., OESTREICHER, A. B., BOONSTRA, J., DE GRAAN, P. N. E. & GISPEN, W. H. (1989) Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells.Journal of Cell Biology 108, 1115–25.

    Google Scholar 

  • VERHAAGEN, J., OESTREICHER, A. B., EDWARDS, P. M., VELDMAN, H., JENNEKENS, F. G. I. & GISPEN, W. H. (1988) Light and electron microscopical study of phosphoprotein B-50 following denervation and reinnervation of the rat soleus muscle.Journal of Neuroscience 8, 1759–66.

    Google Scholar 

  • ZWIERS, H., OESTREICHER, A. B., BISBY, M. A., DE GRAAN, P. N. E. & GISPEN, W. H. (1987) Protein kinase C substrate B-50 in adult and developing rat brain is identical to axonally-transported GAP43 in regenerating peripheral rat nerve. InAxonal Transport (edited by SMITH, R. A. & BISBY, M.), pp. 421–33. New York: Alan R. Liss.

    Google Scholar 

  • ZWIERS, H., SCHOTMAN, P. & GISPEN, W. H. (1980) Purification and some characteristics of an ACTH-sensitive protein kinase and its substrate protein in rat brain.Journal of Neurochemistry 34, 1689–99.

    Google Scholar 

  • ZWIERS, H., TONNAER, J., WIEGANT, V. M., SCHOTMAN, P. & GISPEN, W. H. (1979) ACTH-sensitive protein kinase from rat brain membranes.Journal of Neurochemistry 33, 247–56.

    Google Scholar 

  • ZWIERS, H., VERHAAGEN, J., VAN DONGEN, C., DE GRAAN, P. N. E. & GISPEN, W. H. (1985) Resolution of rat brain phosphoprotein B-50 into multiple forms by two dimensional electrophoresis: evidence for multisite phosphorylation.Journal of Neurochemistry 44, 1083–90.

    Google Scholar 

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Authors and Affiliations

  1. Division of Molecular Neurobiology, Rudolf Magnus Institute and Institute of Molecular Biology and Medical Biotechnology, University of Utrecht, 3584 CH, Utrecht, The Netherlands

    M. Van Lookeren Campagne, A. Beate Oestreicher & W. H. Gispen

  2. Department of Molecular Cell Biology, University of Utrecht, 3584 CH, Utrecht, The Netherlands

    P. M. P. Van Bergen En Henegouwen

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  1. M. Van Lookeren Campagne
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  2. A. Beate Oestreicher
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  3. P. M. P. Van Bergen En Henegouwen
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  4. W. H. Gispen
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Van Lookeren Campagne, M., Beate Oestreicher, A., Van Bergen En Henegouwen, P.M.P. et al. Ultrastructural immunocytochemical localization of B-50/GAP43, a protein kinase C substrate, in isolated presynaptic nerve terminals and neuronal growth cones. J Neurocytol 18, 479–489 (1989). https://doi.org/10.1007/BF01474544

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