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. 1990 Aug 1;111(2):495–509. doi: 10.1083/jcb.111.2.495

Individual microtubules in the axon consist of domains that differ in both composition and stability

PMCID: PMC2116207  PMID: 2199458

Abstract

We have explored the composition and stability properties of individual microtubules (MTs) in the axons of cultured sympathetic neurons. Using morphometric means to quantify the MT mass remaining in axons after various times in 2 micrograms/ml nocodazole, we observed that approximately 48% of the MT mass in the axon is labile, depolymerizing with a t1/2 of approximately 5 min, whereas the remaining 52% of the MT mass is stable, depolymerizing with a t1/2 of approximately 240 min. Immunofluorescence analyses show that the labile MTs in the axon are rich in tyrosinated alpha-tubulin, whereas the stable MTs contain little or no tyrosinated alpha-tubulin and are instead rich in posttranslationally detyrosinated and acetylated alpha-tubulin. These results were confirmed quantitatively by immunoelectron microscopic analyses of the distribution of tyrosinated alpha-tubulin among axonal MTs. Individual MT profiles were typically either uniformly labeled for tyrosinated alpha-tubulin all along their length, or were completely unlabeled. Roughly 48% of the MT mass was tyrosinated, approximately 52% was detyrosinated, and approximately 85% of the tyrosinated MTs were depleted within 15 min of nocodazole treatment. Thus, the proportion of MT profiles that were either tyrosinated or detyrosinated corresponded precisely with the proportion of MTs that were either labile or stable respectively. We also observed MT profiles that were densely labeled for tyrosinated alpha-tubulin at one end but completely unlabeled at the other end. In all of these latter cases, the tyrosinated, and therefore labile domain, was situated at the plus end of the MT, whereas the detyrosinated, and therefore stable domain was situated at the minus end of the MT, and in each case there was an abrupt transition between the two domains. Based on the frequency with which these latter MT profiles were observed, we estimate that minimally 40% of the MTs in the axon are composite, consisting of a stable detyrosinated domain in direct continuity with a labile tyrosinated domain. The extreme drug sensitivity of the labile domains suggests that they are very dynamic, turning over rapidly within the axon. The direct continuity between the labile and stable domains indicates that labile MTs assemble directly from stable MTs. We propose that stable MTs act as MT nucleating structures that spatially regulate MT dynamics in the axon.

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Selected References

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  1. Baas P. W., Black M. M., Banker G. A. Changes in microtubule polarity orientation during the development of hippocampal neurons in culture. J Cell Biol. 1989 Dec;109(6 Pt 1):3085–3094. doi: 10.1083/jcb.109.6.3085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baas P. W., Black M. M. Compartmentation of alpha-tubulin in neurons: identification of a somatodendritic-specific variant of alpha-tubulin. Neuroscience. 1989;30(3):795–803. doi: 10.1016/0306-4522(89)90170-x. [DOI] [PubMed] [Google Scholar]
  3. Baas P. W., Heidemann S. R. Microtubule reassembly from nucleating fragments during the regrowth of amputated neurites. J Cell Biol. 1986 Sep;103(3):917–927. doi: 10.1083/jcb.103.3.917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baas P. W., White L. A., Heidemann S. R. Microtubule polarity reversal accompanies regrowth of amputated neurites. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5272–5276. doi: 10.1073/pnas.84.15.5272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bamburg J. R., Bray D., Chapman K. Assembly of microtubules at the tip of growing axons. Nature. 1986 Jun 19;321(6072):788–790. doi: 10.1038/321788a0. [DOI] [PubMed] [Google Scholar]
  6. Bartlett W. P., Banker G. A. An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. I. Cells which develop without intercellular contacts. J Neurosci. 1984 Aug;4(8):1944–1953. doi: 10.1523/JNEUROSCI.04-08-01944.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bergen L. G., Borisy G. G. Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly. J Cell Biol. 1980 Jan;84(1):141–150. doi: 10.1083/jcb.84.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Binder L. I., Dentler W. L., Rosenbaum J. L. Assembly of chick brain tubulin onto flagellar microtubules from Chlamydomonas and sea urchin sperm. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1122–1126. doi: 10.1073/pnas.72.3.1122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Black M. M., Baas P. W., Humphries S. Dynamics of alpha-tubulin deacetylation in intact neurons. J Neurosci. 1989 Jan;9(1):358–368. doi: 10.1523/JNEUROSCI.09-01-00358.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Black M. M., Cochran J. M., Kurdyla J. T. Solubility properties of neuronal tubulin: evidence for labile and stable microtubules. Brain Res. 1984 Mar 19;295(2):255–263. doi: 10.1016/0006-8993(84)90974-0. [DOI] [PubMed] [Google Scholar]
  11. Black M. M., Greene L. A. Changes in the colchicine susceptibility of microtubules associated with neurite outgrowth: studies with nerve growth factor-responsive PC12 pheochromocytoma cells. J Cell Biol. 1982 Nov;95(2 Pt 1):379–386. doi: 10.1083/jcb.95.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Black M. M., Keyser P. Acetylation of alpha-tubulin in cultured neurons and the induction of alpha-tubulin acetylation in PC12 cells by treatment with nerve growth factor. J Neurosci. 1987 Jun;7(6):1833–1842. doi: 10.1523/JNEUROSCI.07-06-01833.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Black M. M., Keyser P., Sobel E. Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons. J Neurosci. 1986 Apr;6(4):1004–1012. doi: 10.1523/JNEUROSCI.06-04-01004.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Bray D., Bunge M. B. Serial analysis of microtubules in cultured rat sensory axons. J Neurocytol. 1981 Aug;10(4):589–605. doi: 10.1007/BF01262592. [DOI] [PubMed] [Google Scholar]
  16. Brinkley B. R. Microtubule organizing centers. Annu Rev Cell Biol. 1985;1:145–172. doi: 10.1146/annurev.cb.01.110185.001045. [DOI] [PubMed] [Google Scholar]
  17. Bruckenstein D. A., Higgins D. Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. II. Serum promotes dendritic growth. Dev Biol. 1988 Aug;128(2):337–348. doi: 10.1016/0012-1606(88)90296-5. [DOI] [PubMed] [Google Scholar]
  18. Bulinski J. C., Richards J. E., Piperno G. Posttranslational modifications of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells. J Cell Biol. 1988 Apr;106(4):1213–1220. doi: 10.1083/jcb.106.4.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Burton P. R., Paige J. L. Polarity of axoplasmic microtubules in the olfactory nerve of the frog. Proc Natl Acad Sci U S A. 1981 May;78(5):3269–3273. doi: 10.1073/pnas.78.5.3269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Chalfie M., Thomson J. N. Organization of neuronal microtubules in the nematode Caenorhabditis elegans. J Cell Biol. 1979 Jul;82(1):278–289. doi: 10.1083/jcb.82.1.278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Daniels M. P. Fine structural changes in neurons and nerve fibers associated with colchicine inhibition of nerve fiber formation in vitro. J Cell Biol. 1973 Aug;58(2):463–470. doi: 10.1083/jcb.58.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Euteneuer U., McIntosh J. R. Structural polarity of kinetochore microtubules in PtK1 cells. J Cell Biol. 1981 May;89(2):338–345. doi: 10.1083/jcb.89.2.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Geuens G., Gundersen G. G., Nuydens R., Cornelissen F., Bulinski J. C., DeBrabander M. Ultrastructural colocalization of tyrosinated and detyrosinated alpha-tubulin in interphase and mitotic cells. J Cell Biol. 1986 Nov;103(5):1883–1893. doi: 10.1083/jcb.103.5.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gundersen G. G., Bulinski J. C. Microtubule arrays in differentiated cells contain elevated levels of a post-translationally modified form of tubulin. Eur J Cell Biol. 1986 Dec;42(2):288–294. [PubMed] [Google Scholar]
  25. Gundersen G. G., Khawaja S., Bulinski J. C. Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules. J Cell Biol. 1987 Jul;105(1):251–264. doi: 10.1083/jcb.105.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Haimo L. T., Telzer B. R., Rosenbaum J. L. Dynein binds to and crossbridges cytoplasmic microtubules. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5759–5763. doi: 10.1073/pnas.76.11.5759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Heidemann S. R., Hamborg M. A., Thomas S. J., Song B., Lindley S., Chu D. Spatial organization of axonal microtubules. J Cell Biol. 1984 Oct;99(4 Pt 1):1289–1295. doi: 10.1083/jcb.99.4.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Heidemann S. R., Landers J. M., Hamborg M. A. Polarity orientation of axonal microtubules. J Cell Biol. 1981 Dec;91(3 Pt 1):661–665. doi: 10.1083/jcb.91.3.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Heidemann S. R., McIntosh J. R. Visualization of the structural polarity of microtubules. Nature. 1980 Jul 31;286(5772):517–519. doi: 10.1038/286517a0. [DOI] [PubMed] [Google Scholar]
  30. Joshi H. C., Baas P., Chu D. T., Heidemann S. R. The cytoskeleton of neurites after microtubule depolymerization. Exp Cell Res. 1986 Mar;163(1):233–245. doi: 10.1016/0014-4827(86)90576-8. [DOI] [PubMed] [Google Scholar]
  31. Joshi H. C., Cleveland D. W. Differential utilization of beta-tubulin isotypes in differentiating neurites. J Cell Biol. 1989 Aug;109(2):663–673. doi: 10.1083/jcb.109.2.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Khawaja S., Gundersen G. G., Bulinski J. C. Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level. J Cell Biol. 1988 Jan;106(1):141–149. doi: 10.1083/jcb.106.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kilmartin J. V., Wright B., Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol. 1982 Jun;93(3):576–582. doi: 10.1083/jcb.93.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kirschner M. W. Microtubule assembly and nucleation. Int Rev Cytol. 1978;54:1–71. doi: 10.1016/s0074-7696(08)60164-3. [DOI] [PubMed] [Google Scholar]
  35. Kirschner M., Mitchison T. Beyond self-assembly: from microtubules to morphogenesis. Cell. 1986 May 9;45(3):329–342. doi: 10.1016/0092-8674(86)90318-1. [DOI] [PubMed] [Google Scholar]
  36. Kreis T. E. Microtubules containing detyrosinated tubulin are less dynamic. EMBO J. 1987 Sep;6(9):2597–2606. doi: 10.1002/j.1460-2075.1987.tb02550.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lim S. S., Sammak P. J., Borisy G. G. Progressive and spatially differentiated stability of microtubules in developing neuronal cells. J Cell Biol. 1989 Jul;109(1):253–263. doi: 10.1083/jcb.109.1.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lyser K. M. An electron-microscopic study of centrioles in differentiating motor neuroblasts. J Embryol Exp Morphol. 1968 Nov;20(3):343–354. [PubMed] [Google Scholar]
  39. Meininger V., Binet S. Characteristics of microtubules at the different stages of neuronal differentiation and maturation. Int Rev Cytol. 1989;114:21–79. doi: 10.1016/s0074-7696(08)60858-x. [DOI] [PubMed] [Google Scholar]
  40. Mitchison T., Kirschner M. Cytoskeletal dynamics and nerve growth. Neuron. 1988 Nov;1(9):761–772. doi: 10.1016/0896-6273(88)90124-9. [DOI] [PubMed] [Google Scholar]
  41. Morris J. R., Lasek R. J. Monomer-polymer equilibria in the axon: direct measurement of tubulin and actin as polymer and monomer in axoplasm. J Cell Biol. 1984 Jun;98(6):2064–2076. doi: 10.1083/jcb.98.6.2064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Morris J. R., Lasek R. J. Stable polymers of the axonal cytoskeleton: the axoplasmic ghost. J Cell Biol. 1982 Jan;92(1):192–198. doi: 10.1083/jcb.92.1.192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Moya F., Bunge M. B., Bunge R. P. Schwann cells proliferate but fail to differentiate in defined medium. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6902–6906. doi: 10.1073/pnas.77.11.6902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Okabe S., Hirokawa N. Turnover of fluorescently labelled tubulin and actin in the axon. Nature. 1990 Feb 1;343(6257):479–482. doi: 10.1038/343479a0. [DOI] [PubMed] [Google Scholar]
  46. Peng I., Binder L. I., Black M. M. Biochemical and immunological analyses of cytoskeletal domains of neurons. J Cell Biol. 1986 Jan;102(1):252–262. doi: 10.1083/jcb.102.1.252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Piperno G., Fuller M. T. Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol. 1985 Dec;101(6):2085–2094. doi: 10.1083/jcb.101.6.2085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Piperno G., LeDizet M., Chang X. J. Microtubules containing acetylated alpha-tubulin in mammalian cells in culture. J Cell Biol. 1987 Feb;104(2):289–302. doi: 10.1083/jcb.104.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Robson S. J., Burgoyne R. D. Differential localisation of tyrosinated, detyrosinated, and acetylated alpha-tubulins in neurites and growth cones of dorsal root ganglion neurons. Cell Motil Cytoskeleton. 1989;12(4):273–282. doi: 10.1002/cm.970120408. [DOI] [PubMed] [Google Scholar]
  50. Sahenk Z., Brady S. T. Axonal tubulin and microtubules: morphologic evidence for stable regions on axonal microtubules. Cell Motil Cytoskeleton. 1987;8(2):155–164. doi: 10.1002/cm.970080207. [DOI] [PubMed] [Google Scholar]
  51. Schliwa M., van Blerkom J. Structural interaction of cytoskeletal components. J Cell Biol. 1981 Jul;90(1):222–235. doi: 10.1083/jcb.90.1.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schulze E., Asai D. J., Bulinski J. C., Kirschner M. Posttranslational modification and microtubule stability. J Cell Biol. 1987 Nov;105(5):2167–2177. doi: 10.1083/jcb.105.5.2167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sharp G. A., Weber K., Osborn M. Centriole number and process formation in established neuroblastoma cells and primary dorsal root ganglion neurones. Eur J Cell Biol. 1982 Nov;29(1):97–103. [PubMed] [Google Scholar]
  54. Sherwin T., Gull K. Visualization of detyrosination along single microtubules reveals novel mechanisms of assembly during cytoskeletal duplication in trypanosomes. Cell. 1989 Apr 21;57(2):211–221. doi: 10.1016/0092-8674(89)90959-8. [DOI] [PubMed] [Google Scholar]
  55. Webster D. R., Gundersen G. G., Bulinski J. C., Borisy G. G. Differential turnover of tyrosinated and detyrosinated microtubules. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9040–9044. doi: 10.1073/pnas.84.24.9040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wehland J., Weber K. Turnover of the carboxy-terminal tyrosine of alpha-tubulin and means of reaching elevated levels of detyrosination in living cells. J Cell Sci. 1987 Sep;88(Pt 2):185–203. doi: 10.1242/jcs.88.2.185. [DOI] [PubMed] [Google Scholar]
  57. Wehland J., Willingham M. C. A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. II. Effects on cell movement, organization of microtubules, and intermediate filaments, and arrangement of Golgi elements. J Cell Biol. 1983 Nov;97(5 Pt 1):1476–1490. doi: 10.1083/jcb.97.5.1476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. White L. A., Baas P. W., Heidemann S. R. Microtubule stability in severed axons. J Neurocytol. 1987 Dec;16(6):775–784. doi: 10.1007/BF01611985. [DOI] [PubMed] [Google Scholar]
  59. Yamada K. M., Spooner B. S., Wessells N. K. Axon growth: roles of microfilaments and microtubules. Proc Natl Acad Sci U S A. 1970 Aug;66(4):1206–1212. doi: 10.1073/pnas.66.4.1206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yamada K. M., Spooner B. S., Wessells N. K. Ultrastructure and function of growth cones and axons of cultured nerve cells. J Cell Biol. 1971 Jun;49(3):614–635. doi: 10.1083/jcb.49.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]

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