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Expression of a neuromodulin-ß-galactosidase fusion protein in primary cultured neurons and its accumulation in growth cones

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Book cover Molecular Mechanisms of Cellular Growth

Part of the book series: Developments in Molecular and Cellular Biochemistry ((DMCB,volume 7))

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Abstract

Cultured embryonic neurons share a number of characteristic morphological and physiological properties with their counterparts in vivo. For example, differentiating hippocampal neurons in culture develop two distinct classes of processes that serve as dendrites and axons. It has also been shown that the microtubule organization and composition in axons differs from those in dendrites, which may contribute to differential transport of macromolecules into axons or dendrites. We have expressed a neuromodulin-ß-galactosidase fusion gene in cultured mesencephalic neurons in order to study the transport of the neurospecific protein neuromodulin into neurite growth cones. When β-galactosidase alone was expressed in neurons, it was found in the cell bodies with diffuse neurite staining. In marked contrast, the neuromodulin-β-galactosidase fusion protein was rapidly transported into neurites and was concentrated in the growth cones. This system may provide a useful model for studying the structural domain(s) of neuromodulin that are required for transport and accumulation of neuromodulin in the growth cones of neurons.

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References

  1. Andreasen TJ, Luetje CW, Heideman W, Storm DR: Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes. Biochem 22: 4615–4618, 1983

    Article  CAS  Google Scholar 

  2. Skene JHP: Axonal growth-associated proteins. Ann Rev Neurosci 12: 127–156, 1989

    Article  PubMed  CAS  Google Scholar 

  3. Liu Y, Storm DR: Regulation of free calmodulin levels by neuromodulin: neuron growth and regeneration. Trends in Pharm Sci 11: 107–111, 1990

    Article  CAS  Google Scholar 

  4. Skene JHP, Willard M: Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems. J Cell Biol 89: 96–103, 1981

    Article  PubMed  CAS  Google Scholar 

  5. Benowitz LI, Lewis ER: Increased transport of 44,000 to 49,000-dalton acidic proteins during regeneration of the goldfish optic nerve: A two-dimensional gel analysis. J Neurosci 3: 2153–2163, 1983

    PubMed  CAS  Google Scholar 

  6. Alexander KA, Cimier BM, Meier KE, Storm DR: Regulation of calmodulin binding to P-57. J Biol Chem 262: 6108–6113, 1987

    PubMed  CAS  Google Scholar 

  7. Akers RF, Routtenberg: Calcium-promoted translocation of protein kinase C to synaptic membranes: relation to the phosphorylation of an endogenous substrate (protein Fl) involved in synaptic activity. J Neurosci 7: 3976–3983, 1987

    PubMed  CAS  Google Scholar 

  8. Oestreicher AB, Van Dongen CJ, Zwiers H, Gispen WH: Affinity-purified anti-B-50 protein antibody: interference with the function of the phosphoprotein B-50 in synaptic plasma membrane. J Neurochem 41: 331–340, 1983

    Article  PubMed  CAS  Google Scholar 

  9. Wakim BT, Alexander KA, Masure HB, Cimier BM, Storm DR, Walsh KA: Amino acid sequence of P-57, a neurospecific calmodulin binding protein. Biochem 26: 7466–7470, 1987

    Article  CAS  Google Scholar 

  10. Cimier BM, Giebelhaus DH, Wakim BT, Storm DR, Moon RT: Characterization of murine cDNAs encoding P-57, a neurospecific calmodulin-binding protein. J Biol Chem 262: 12158–12163, 1987

    Google Scholar 

  11. Basi GS, Jacobson RD, Virag I, Schilling J, Skene JHP: Primary structure and transcriptional regulation of GAP-43, a protein associated with nerve growth. Cell 49: 785–791, 1987

    Article  PubMed  CAS  Google Scholar 

  12. Kosik KS, Orecchio LD, Bruns GAP, MacDonald GP, Cox DR, Neve RL: Human GAP-43: its deduced amino acid sequence and chromosomal localization in mouse and human. Neuron 1: 127–132, 1988

    Article  PubMed  CAS  Google Scholar 

  13. Baizer L, Alkan S, Stocker K, Ciment G: Chicken growth-associated protein (GAP)-43: primary structure and regulated expression of mRNA during embryogenesis. Mol Brain Res 7: 61–68, 1990

    Article  PubMed  CAS  Google Scholar 

  14. LaBate ME, Skene JHP: Selective conservation of GAP-43 structure in vertebrate evolution. Neuron 3: 299–310, 1989

    Article  PubMed  CAS  Google Scholar 

  15. Alexander KA, Wakim BT, Doyle GS, Walsh KA, Storm DR: Identification and characterization of the calmodulin binding domain of neuromodulin, a neurospecific calmodulin binding protein. J Biol Chem 263: 7544–7549, 1988

    PubMed  CAS  Google Scholar 

  16. Zuber MX, Stittmatter SM, Fishman MC: A membrane-targeting signal in the amino terminus of the neuronal protein GAP-43. Nature 341: 345–348, 1989

    Article  PubMed  CAS  Google Scholar 

  17. Apel ED, Byford MF, Au D, Walsh KA, Storm DR: Identification of the protein kinase C phosphorylation site in neuromodulin. Biochem 29: 2330–2335, 1990

    Article  CAS  Google Scholar 

  18. Meiri KF, Johnson MI, Willard M: Distribution and phosphorylation of the growth associated protein GAP-43 in regenerating sympathetic neurons in culture. J Neurosci 8: 2571–2581, 1988

    PubMed  CAS  Google Scholar 

  19. Greene LA, Tischler AS: PC12 pheochromocytoma cultures in neurobiological research. Adv Cell Neurobiol 3: 373–415, 1982

    CAS  Google Scholar 

  20. Casadaban MJ, Martinez-Arias A, Shapira SK, Chou J: β-galactosidase gene fusion for analyzing gene expression in Escherichia coli and yeast. Methods Enzymol 100: 293–308, 1983

    Article  PubMed  CAS  Google Scholar 

  21. Seed B: An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature 329: 840, 1987

    Article  PubMed  CAS  Google Scholar 

  22. Bottenstein JE, Sato GH: Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci USA 70: 514–519, 1979

    Article  Google Scholar 

  23. Caceres A, Banker GA, Binder L: Immunocytochemical localization of tubulin and microtubule-associated protein 2 during the development of hippocampal neurons in culture. J Neurosci 6: 714–722, 1986

    PubMed  CAS  Google Scholar 

  24. Geller A, Freese A: Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli β-galactosidase. Proc Natl Acad Sci USA 87: 1149–1153, 1990

    Article  PubMed  CAS  Google Scholar 

  25. Feigner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M: Lipo-fection: a highly efficient, lipid-mediated DNA-transfec-tion procedure. Proc Natl Acad Sci USA 84: 7413–7417, 1987

    Article  Google Scholar 

  26. Holt CE, Garlick N, Cornel E: Lipofection of cDNAs in the embryonic vertebrate central nervous system. Neuron 4: 203–214, 1990

    Article  PubMed  CAS  Google Scholar 

  27. Picard D, Yamamoto K: Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J 6: 3333–3340, 1987

    PubMed  CAS  Google Scholar 

  28. Skene JHP, Virag I: Post-translational membrane attachment and dynamic fatty acylation of neuronal growth cone protein, GAP-43. J Cell Biol 108: 613–625, 1989

    Article  PubMed  CAS  Google Scholar 

  29. Goslin K, Schreyer DJ, Skene JHP, Banker GA: Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones. Nature 336: 672–674, 1988

    Article  PubMed  CAS  Google Scholar 

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© 1991 Springer Science+Business Media Dordrecht

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Liu, Y., Storm, D.R. (1991). Expression of a neuromodulin-ß-galactosidase fusion protein in primary cultured neurons and its accumulation in growth cones. In: Morgan, H.E. (eds) Molecular Mechanisms of Cellular Growth. Developments in Molecular and Cellular Biochemistry, vol 7. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-3886-8_4

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  • DOI: https://doi.org/10.1007/978-1-4615-3886-8_4

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4613-6733-8

  • Online ISBN: 978-1-4615-3886-8

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