Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Jun;86(11):4127–4131. doi: 10.1073/pnas.86.11.4127

Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons.

S Okabe 1, N Hirokawa 1
PMCID: PMC287402  PMID: 2657741

Abstract

We studied the mechanism of compartmentation of microtubule-associated protein 2 (MAP2) in the dendrites and cell bodies by using microinjection of biotin-labeled MAP2 into mature spinal cord neurons in culture. MAP2 molecules microinjected into the nerve cell body were distributed not only throughout the cytoplasm of the cell body and dendrites, but also in the axon as far as a few millimeters from the cell body within 24 hr after injection. However, when injected cells were incubated for more than 3 days, the amount of biotin-labeled MAP2 in the axon decreased remarkably compared with that in the dendrites. This indicates that there is no sorting mechanism in the cell body for the transport of MAP2 selectively into the dendrites but that the turnover rate of MAP2 in the axons differs from that in the dendrites. To further characterize the mechanism of MAP2 compartmentation, we performed immunoelectron microscopy of injected cells and detergent extraction of microinjected cells prior to immunocytochemistry with anti-biotin. The results strongly suggest that a large part of axonal MAP2 is not associated with cytoskeleton and that this weak association of MAP2 favors selective loss of MAP2 from the axon.

Full text

PDF
4128

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Borisy G. G., Marcum J. M., Olmsted J. B., Murphy D. B., Johnson K. A. Purification of tubulin and associated high molecular weight proteins from porcine brain and characterization of microtubule assembly in vitro. Ann N Y Acad Sci. 1975 Jun 30;253:107–132. doi: 10.1111/j.1749-6632.1975.tb19196.x. [DOI] [PubMed] [Google Scholar]
  2. Brady S. T., Tytell M., Lasek R. J. Axonal tubulin and axonal microtubules: biochemical evidence for cold stability. J Cell Biol. 1984 Nov;99(5):1716–1724. doi: 10.1083/jcb.99.5.1716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Caceres A., Banker G., Steward O., Binder L., Payne M. MAP2 is localized to the dendrites of hippocampal neurons which develop in culture. Brain Res. 1984 Apr;315(2):314–318. doi: 10.1016/0165-3806(84)90167-6. [DOI] [PubMed] [Google Scholar]
  4. Cambray-Deakin M. A., Burgoyne R. D. Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum. J Cell Biol. 1987 Jun;104(6):1569–1574. doi: 10.1083/jcb.104.6.1569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cáceres A., Banker G. A., Binder L. Immunocytochemical localization of tubulin and microtubule-associated protein 2 during the development of hippocampal neurons in culture. J Neurosci. 1986 Mar;6(3):714–722. doi: 10.1523/JNEUROSCI.06-03-00714.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Camilli P., Miller P. E., Navone F., Theurkauf W. E., Vallee R. B. Distribution of microtubule-associated protein 2 in the nervous system of the rat studied by immunofluorescence. Neuroscience. 1984 Apr;11(4):817–846. [PubMed] [Google Scholar]
  7. Gorbsky G. J., Sammak P. J., Borisy G. G. Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends. J Cell Biol. 1987 Jan;104(1):9–18. doi: 10.1083/jcb.104.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Graessmann A., Graessmann M., Mueller C. Microinjection of early SV40 DNA fragments and T antigen. Methods Enzymol. 1980;65(1):816–825. doi: 10.1016/s0076-6879(80)65076-9. [DOI] [PubMed] [Google Scholar]
  9. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  10. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hatanaka H. Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line. Brain Res. 1981 Oct 19;222(2):225–233. doi: 10.1016/0006-8993(81)91029-5. [DOI] [PubMed] [Google Scholar]
  12. Hirokawa N. 270K microtubule-associated protein cross-reacting with anti-MAP2 IgG in the crayfish peripheral nerve axon. J Cell Biol. 1986 Jul;103(1):33–39. doi: 10.1083/jcb.103.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hirokawa N., Bloom G. S., Vallee R. B. Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta'-iminodipropionitrile-intoxicated axon as a model system. J Cell Biol. 1985 Jul;101(1):227–239. doi: 10.1083/jcb.101.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hirokawa N. Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J Cell Biol. 1982 Jul;94(1):129–142. doi: 10.1083/jcb.94.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hirokawa N., Glicksman M. A., Willard M. B. Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. J Cell Biol. 1984 Apr;98(4):1523–1536. doi: 10.1083/jcb.98.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hirokawa N., Hisanaga S., Shiomura Y. MAP2 is a component of crossbridges between microtubules and neurofilaments in the neuronal cytoskeleton: quick-freeze, deep-etch immunoelectron microscopy and reconstitution studies. J Neurosci. 1988 Aug;8(8):2769–2779. doi: 10.1523/JNEUROSCI.08-08-02769.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hollenbeck P. J., Bray D. Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth. J Cell Biol. 1987 Dec;105(6 Pt 1):2827–2835. doi: 10.1083/jcb.105.6.2827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim H., Jensen C. G., Rebhun L. I. The binding of MAP-2 and tau on brain microtubules in vitro: implications for microtubule structure. Ann N Y Acad Sci. 1986;466:218–239. doi: 10.1111/j.1749-6632.1986.tb38396.x. [DOI] [PubMed] [Google Scholar]
  19. Kosik K. S., Finch E. A. MAP2 and tau segregate into dendritic and axonal domains after the elaboration of morphologically distinct neurites: an immunocytochemical study of cultured rat cerebrum. J Neurosci. 1987 Oct;7(10):3142–3153. doi: 10.1523/JNEUROSCI.07-10-03142.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kristofferson D., Mitchison T., Kirschner M. Direct observation of steady-state microtubule dynamics. J Cell Biol. 1986 Mar;102(3):1007–1019. doi: 10.1083/jcb.102.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Letourneau P. C., Ressler A. H. Inhibition of neurite initiation and growth by taxol. J Cell Biol. 1984 Apr;98(4):1355–1362. doi: 10.1083/jcb.98.4.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matus A., Bernhardt R., Hugh-Jones T. High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain. Proc Natl Acad Sci U S A. 1981 May;78(5):3010–3014. doi: 10.1073/pnas.78.5.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Neale E. A., Macdonald R. L., Nelson P. G. Intracellular horseradish peroxidase injection for correlation of light and electron microscopic anatomy with synaptic physiology of cultured mouse spinal cord neurons. Brain Res. 1978 Aug 25;152(2):265–282. doi: 10.1016/0006-8993(78)90255-x. [DOI] [PubMed] [Google Scholar]
  25. Okabe S., Hirokawa N. Microtubule dynamics in nerve cells: analysis using microinjection of biotinylated tubulin into PC12 cells. J Cell Biol. 1988 Aug;107(2):651–664. doi: 10.1083/jcb.107.2.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Papasozomenos S. C., Binder L. I., Bender P. K., Payne M. R. Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons. J Cell Biol. 1985 Jan;100(1):74–85. doi: 10.1083/jcb.100.1.74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ransom B. R., Neale E., Henkart M., Bullock P. N., Nelson P. G. Mouse spinal cord in cell culture. I. Morphology and intrinsic neuronal electrophysiologic properties. J Neurophysiol. 1977 Sep;40(5):1132–1150. doi: 10.1152/jn.1977.40.5.1132. [DOI] [PubMed] [Google Scholar]
  28. Schulze E., Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. doi: 10.1083/jcb.102.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shiomura Y., Hirokawa N. Colocalization of microtubule-associated protein 1A and microtubule-associated protein 2 on neuronal microtubules in situ revealed with double-label immunoelectron microscopy. J Cell Biol. 1987 Jun;104(6):1575–1578. doi: 10.1083/jcb.104.6.1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Solomon F. Specification of cell morphology by endogenous determinants. J Cell Biol. 1981 Sep;90(3):547–553. doi: 10.1083/jcb.90.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Soltys B. J., Borisy G. G. Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes. J Cell Biol. 1985 May;100(5):1682–1689. doi: 10.1083/jcb.100.5.1682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vallee R. B. A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs). J Cell Biol. 1982 Feb;92(2):435–442. doi: 10.1083/jcb.92.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vallee R. B., DiBartolomeis M. J., Theurkauf W. E. A protein kinase bound to the projection portion of MAP 2 (microtubule-associated protein 2). J Cell Biol. 1981 Sep;90(3):568–576. doi: 10.1083/jcb.90.3.568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Webster R. E., Henderson D., Osborn M., Weber K. Three-dimensional electron microscopical visualization of the cytoskeleton of animal cells: immunoferritin identification of actin- and tubulin-containing structures. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5511–5515. doi: 10.1073/pnas.75.11.5511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES