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. 1995 Dec 1;131(5):1275–1290. doi: 10.1083/jcb.131.5.1275

Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts

PMCID: PMC2120637  PMID: 8522589

Abstract

Separate populations of microtubules (MTs) distinguishable by their level of posttranslationally modified tubulin subunits and by their stability in vivo have been described. In polarized 3T3 cells at the edge of an in vitro wound, we have found a striking preferential coalignment of vimentin intermediate filaments (IFs) with detyrosinated MTs (Glu MTs) rather than with the bulk of the MTs, which were tyrosinated MTs (Tyr MTs). Vimentin IFs were not stabilizing the Glu MTs since collapse of the IF network to a perinuclear location, induced by microinjection of monoclonal anti-IF antibody, had no noticeable effect on the array of Glu MTs. To test whether Glu MTs may affect the organization of IFs we regrew MTs in cells that had been treated with nocodazole to depolymerize all the MTs and to collapse IFs; the reextension of IFs into the lamella lagged behind the rapid regrowth of Tyr MTs, but was correlated with the slower reformation of Glu MTs. Similar realignment of IFs with newly formed Glu MTs was observed in serum-starved cells treated with either serum or taxol to induce the formation of Glu MTs. Next, we microinjected affinity purified antibodies specific for Glu tubulin (polyclonal SG and monoclonal 4B8) and specific for Tyr tubulin (polyclonal W2 and monoclonal YL1/2) into 3T3 cells. Both injected SG and 4B8 antibodies labeled the subset of endogenous Glu MTs; W2 and YL1/2 antibodies labeled virtually all of the cytoplasmic MTs. Injection of SG or 4B8 resulted in the collapse of IFs to a perinuclear region. This collapse was comparable to that observed after complete MT depolymerization by nocodazole. Injection of W2, YL1/2, or nonspecific control IgGs did not result in collapse of the IFs. Taken together, these results show that Glu MTs localize IFs in migrating 3T3 fibroblasts and suggest that detyrosination of tubulin acts as a signal for the recruitment of vimentin IFs to MTs.

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Selected References

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  1. Achler C., Filmer D., Merte C., Drenckhahn D. Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epithelium. J Cell Biol. 1989 Jul;109(1):179–189. doi: 10.1083/jcb.109.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baas P. W., Black M. M. Individual microtubules in the axon consist of domains that differ in both composition and stability. J Cell Biol. 1990 Aug;111(2):495–509. doi: 10.1083/jcb.111.2.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ball E. H., Singer S. J. Mitochondria are associated with microtubules and not with intermediate filaments in cultured fibroblasts. Proc Natl Acad Sci U S A. 1982 Jan;79(1):123–126. doi: 10.1073/pnas.79.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blose S. H., Chacko S. Rings of intermediate (100 A) filament bundles in the perinuclear region of vascular endothelial cells. Their mobilization by colcemid and mitosis. J Cell Biol. 1976 Aug;70(2 Pt 1):459–466. doi: 10.1083/jcb.70.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blose S. H., Meltzer D. I., Feramisco J. R. 10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Biol. 1984 Mar;98(3):847–858. doi: 10.1083/jcb.98.3.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Breitling F., Little M. Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL1/34, DM1A and DM1B. J Mol Biol. 1986 May 20;189(2):367–370. doi: 10.1016/0022-2836(86)90517-6. [DOI] [PubMed] [Google Scholar]
  7. Bulinski J. C., Gundersen G. G. Stabilization of post-translational modification of microtubules during cellular morphogenesis. Bioessays. 1991 Jun;13(6):285–293. doi: 10.1002/bies.950130605. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Collot M., Louvard D., Singer S. J. Lysosomes are associated with microtubules and not with intermediate filaments in cultured fibroblasts. Proc Natl Acad Sci U S A. 1984 Feb;81(3):788–792. doi: 10.1073/pnas.81.3.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Couchman J. R., Rees D. A. Organelle-cytoskeleton relationships in fibroblasts: mitochondria, Golgi apparatus, and endoplasmic reticulum in phases of movement and growth. Eur J Cell Biol. 1982 Apr;27(1):47–54. [PubMed] [Google Scholar]
  11. Croop J., Holtzer H. Response of myogenic and fibrogenic cells to cytochalasin B and to colcemid. I. Light microscope observations. J Cell Biol. 1975 May;65(2):271–285. doi: 10.1083/jcb.65.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dabora S. L., Sheetz M. P. The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts. Cell. 1988 Jul 1;54(1):27–35. doi: 10.1016/0092-8674(88)90176-6. [DOI] [PubMed] [Google Scholar]
  13. Forry-Schaudies S., Murray J. M., Toyama Y., Holtzer H. Effects of colcemid and taxol on microtubules and intermediate filaments in chick embryo fibroblasts. Cell Motil Cytoskeleton. 1986;6(3):324–338. doi: 10.1002/cm.970060309. [DOI] [PubMed] [Google Scholar]
  14. Gawlitta W., Osborn M., Weber K. Coiling of intermediate filaments induced by microinjection of a vimentin-specific antibody does not interfere with locomotion and mitosis. Eur J Cell Biol. 1981 Dec;26(1):83–90. [PubMed] [Google Scholar]
  15. 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]
  16. Goldman R. D. The role of three cytoplasmic fibers in BHK-21 cell motility. I. Microtubules and the effects of colchicine. J Cell Biol. 1971 Dec;51(3):752–762. doi: 10.1083/jcb.51.3.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gundersen G. G., Bulinski J. C. Distribution of tyrosinated and nontyrosinated alpha-tubulin during mitosis. J Cell Biol. 1986 Mar;102(3):1118–1126. doi: 10.1083/jcb.102.3.1118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gundersen G. G., Bulinski J. C. Selective stabilization of microtubules oriented toward the direction of cell migration. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5946–5950. doi: 10.1073/pnas.85.16.5946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gundersen G. G., Kalnoski M. H., Bulinski J. C. Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell. 1984 Oct;38(3):779–789. doi: 10.1016/0092-8674(84)90273-3. [DOI] [PubMed] [Google Scholar]
  20. Gundersen G. G., Khawaja S., Bulinski J. C. Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation. J Cell Biol. 1989 Nov;109(5):2275–2288. doi: 10.1083/jcb.109.5.2275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Gundersen G. G., Kim I., Chapin C. J. Induction of stable microtubules in 3T3 fibroblasts by TGF-beta and serum. J Cell Sci. 1994 Mar;107(Pt 3):645–659. doi: 10.1242/jcs.107.3.645. [DOI] [PubMed] [Google Scholar]
  23. Gurland G., Gundersen G. G. Protein phosphatase inhibitors induce the selective breakdown of stable microtubules in fibroblasts and epithelial cells. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8827–8831. doi: 10.1073/pnas.90.19.8827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gyoeva F. K., Gelfand V. I. Coalignment of vimentin intermediate filaments with microtubules depends on kinesin. Nature. 1991 Oct 3;353(6343):445–448. doi: 10.1038/353445a0. [DOI] [PubMed] [Google Scholar]
  25. Heggeness M. H., Simon M., Singer S. J. Association of mitochondria with microtubules in cultured cells. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3863–3866. doi: 10.1073/pnas.75.8.3863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Heimann R., Shelanski M. L., Liem R. K. Microtubule-associated proteins bind specifically to the 70-kDa neurofilament protein. J Biol Chem. 1985 Oct 5;260(22):12160–12166. [PubMed] [Google Scholar]
  27. Heuser J. E., Kirschner M. W. Filament organization revealed in platinum replicas of freeze-dried cytoskeletons. J Cell Biol. 1980 Jul;86(1):212–234. doi: 10.1083/jcb.86.1.212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Ho W. C., Allan V. J., van Meer G., Berger E. G., Kreis T. E. Reclustering of scattered Golgi elements occurs along microtubules. Eur J Cell Biol. 1989 Apr;48(2):250–263. [PubMed] [Google Scholar]
  31. Hollenbeck P. J., Bershadsky A. D., Pletjushkina O. Y., Tint I. S., Vasiliev J. M. Intermediate filament collapse is an ATP-dependent and actin-dependent process. J Cell Sci. 1989 Apr;92(Pt 4):621–631. doi: 10.1242/jcs.92.4.621. [DOI] [PubMed] [Google Scholar]
  32. Houliston E., Maro B. Posttranslational modification of distinct microtubule subpopulations during cell polarization and differentiation in the mouse preimplantation embryo. J Cell Biol. 1989 Feb;108(2):543–551. doi: 10.1083/jcb.108.2.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hynes R. O., Destree A. T. 10 nm filaments in normal and transformed cells. Cell. 1978 Jan;13(1):151–163. doi: 10.1016/0092-8674(78)90146-0. [DOI] [PubMed] [Google Scholar]
  34. Idriss H., Stammers D. K., Ross C. K., Burns R. G. The dynamic instability of microtubules is not modulated by alpha-tubulin tyrosinylation. Cell Motil Cytoskeleton. 1991;20(1):30–37. doi: 10.1002/cm.970200104. [DOI] [PubMed] [Google Scholar]
  35. Kaplan M. P., Chin S. S., Macioce P., Srinawasan J., Hashim G., Liem R. K. Characterization of a panel of neurofilament antibodies recognizing N-terminal epitopes. J Neurosci Res. 1991 Nov;30(3):545–554. doi: 10.1002/jnr.490300312. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]
  39. Klymkowsky M. W. Intermediate filaments in 3T3 cells collapse after intracellular injection of a monoclonal anti-intermediate filament antibody. Nature. 1981 May 21;291(5812):249–251. doi: 10.1038/291249a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Kumar N., Flavin M. Modulation of some parameters of assembly of microtubules in vitro by tyrosinolation of tubulin. Eur J Biochem. 1982 Nov;128(1):215–222. doi: 10.1111/j.1432-1033.1982.tb06954.x. [DOI] [PubMed] [Google Scholar]
  42. L'Hernault S. W., Rosenbaum J. L. Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine. Biochemistry. 1985 Jan 15;24(2):473–478. doi: 10.1021/bi00323a034. [DOI] [PubMed] [Google Scholar]
  43. LeDizet M., Piperno G. Identification of an acetylation site of Chlamydomonas alpha-tubulin. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5720–5724. doi: 10.1073/pnas.84.16.5720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Leterrier J. F., Liem R. K., Shelanski M. L. Interactions between neurofilaments and microtubule-associated proteins: a possible mechanism for intraorganellar bridging. J Cell Biol. 1982 Dec;95(3):982–986. doi: 10.1083/jcb.95.3.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Lieska N., Yang H. Y., Goldman R. D. Purification of the 300K intermediate filament-associated protein and its in vitro recombination with intermediate filaments. J Cell Biol. 1985 Sep;101(3):802–813. doi: 10.1083/jcb.101.3.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Lin J. J., Feramisco J. R. Disruption of the in vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody. Cell. 1981 Apr;24(1):185–193. doi: 10.1016/0092-8674(81)90514-6. [DOI] [PubMed] [Google Scholar]
  47. MacRae T. H., Langdon C. M., Freeman J. A. Spatial distribution of posttranslationally modified tubulins in polarized cells of developing Artemia. Cell Motil Cytoskeleton. 1991;18(3):189–203. doi: 10.1002/cm.970180305. [DOI] [PubMed] [Google Scholar]
  48. Matteoni R., Kreis T. E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol. 1987 Sep;105(3):1253–1265. doi: 10.1083/jcb.105.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Murphy D. B. Assembly-disassembly purification and characterization of microtubule protein without glycerol. Methods Cell Biol. 1982;24:31–49. doi: 10.1016/s0091-679x(08)60646-9. [DOI] [PubMed] [Google Scholar]
  50. Nangaku M., Sato-Yoshitake R., Okada Y., Noda Y., Takemura R., Yamazaki H., Hirokawa N. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell. 1994 Dec 30;79(7):1209–1220. doi: 10.1016/0092-8674(94)90012-4. [DOI] [PubMed] [Google Scholar]
  51. Pepperkok R., Bré M. H., Davoust J., Kreis T. E. Microtubules are stabilized in confluent epithelial cells but not in fibroblasts. J Cell Biol. 1990 Dec;111(6 Pt 2):3003–3012. doi: 10.1083/jcb.111.6.3003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Pruss R. M., Mirsky R., Raff M. C., Thorpe R., Dowding A. J., Anderton B. H. All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell. 1981 Dec;27(3 Pt 2):419–428. doi: 10.1016/0092-8674(81)90383-4. [DOI] [PubMed] [Google Scholar]
  54. Raybin D., Flavin M. Enzyme which specifically adds tyrosine to the alpha chain of tubulin. Biochemistry. 1977 May 17;16(10):2189–2194. doi: 10.1021/bi00629a023. [DOI] [PubMed] [Google Scholar]
  55. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. 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]
  58. Summerhayes I. C., Wong D., Chen L. B. Effect of microtubules and intermediate filaments on mitochondrial distribution. J Cell Sci. 1983 May;61:87–105. doi: 10.1242/jcs.61.1.87. [DOI] [PubMed] [Google Scholar]
  59. Terasaki M., Chen L. B., Fujiwara K. Microtubules and the endoplasmic reticulum are highly interdependent structures. J Cell Biol. 1986 Oct;103(4):1557–1568. doi: 10.1083/jcb.103.4.1557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Thyberg J., Moskalewski S. Microtubules and the organization of the Golgi complex. Exp Cell Res. 1985 Jul;159(1):1–16. doi: 10.1016/s0014-4827(85)80032-x. [DOI] [PubMed] [Google Scholar]
  61. Tint I. S., Hollenbeck P. J., Verkhovsky A. B., Surgucheva I. G., Bershadsky A. D. Evidence that intermediate filament reorganization is induced by ATP-dependent contraction of the actomyosin cortex in permeabilized fibroblasts. J Cell Sci. 1991 Mar;98(Pt 3):375–384. doi: 10.1242/jcs.98.3.375. [DOI] [PubMed] [Google Scholar]
  62. Vale R. D., Hotani H. Formation of membrane networks in vitro by kinesin-driven microtubule movement. J Cell Biol. 1988 Dec;107(6 Pt 1):2233–2241. doi: 10.1083/jcb.107.6.2233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Warn R. M., Harrison A., Planques V., Robert-Nicoud N., Wehland J. Distribution of microtubules containing post-translationally modified alpha-tubulin during Drosophila embryogenesis. Cell Motil Cytoskeleton. 1990;17(1):34–45. doi: 10.1002/cm.970170106. [DOI] [PubMed] [Google Scholar]
  64. 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]
  65. Webster D. R., Wehland J., Weber K., Borisy G. G. Detyrosination of alpha tubulin does not stabilize microtubules in vivo. J Cell Biol. 1990 Jul;111(1):113–122. doi: 10.1083/jcb.111.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. 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]

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