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. 1984 Dec 1;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175

Tubulin dynamics in cultured mammalian cells

PMCID: PMC2113582  PMID: 6501419

Abstract

Bovine neurotubulin has been labeled with dichlorotriazinyl- aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cell-cycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (approximately 3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF- tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells.

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Selected References

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  1. 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]
  2. Brinkley B. R., Cox S. M., Pepper D. A., Wible L., Brenner S. L., Pardue R. L. Tubulin assembly sites and the organization of cytoplasmic microtubules in cultured mammalian cells. J Cell Biol. 1981 Sep;90(3):554–562. doi: 10.1083/jcb.90.3.554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bryan J. A quantitative analysis of microtubule elongation. J Cell Biol. 1976 Dec;71(3):749–767. doi: 10.1083/jcb.71.3.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. De Brabander M. J., Van de Veire R. M., Aerts F. E., Borgers M., Janssen P. A. The effects of methyl (5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl) carbamate, (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. Cancer Res. 1976 Mar;36(3):905–916. [PubMed] [Google Scholar]
  5. De Brabander M., Geuens G., Nuydens R., Willebrords R., De Mey J. Taxol induces the assembly of free microtubules in living cells and blocks the organizing capacity of the centrosomes and kinetochores. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5608–5612. doi: 10.1073/pnas.78.9.5608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Giloh H., Sedat J. W. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science. 1982 Sep 24;217(4566):1252–1255. doi: 10.1126/science.7112126. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Hiller G., Weber K. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues. Cell. 1978 Aug;14(4):795–804. doi: 10.1016/0092-8674(78)90335-5. [DOI] [PubMed] [Google Scholar]
  9. Inoué S., Fuseler J., Salmon E. D., Ellis G. W. Functional organization of mitotic microtubules. Physical chemistry of the in vivo equilibrium system. Biophys J. 1975 Jul;15(7):725–744. doi: 10.1016/S0006-3495(75)85850-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Inoué S., Sato H. Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement. J Gen Physiol. 1967 Jul;50(6 Suppl):259–292. [PMC free article] [PubMed] [Google Scholar]
  11. Karr T. L., Kristofferson D., Purich D. L. Mechanism of microtubule depolymerization. Correlation of rapid induced disassembly experiments with a kinetic model for endwise depolymerization. J Biol Chem. 1980 Sep 25;255(18):8560–8566. [PubMed] [Google Scholar]
  12. Keith C. H., Feramisco J. R., Shelanski M. Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts. J Cell Biol. 1981 Jan;88(1):234–240. doi: 10.1083/jcb.88.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Kreis T. E., Birchmeier W. Microinjection of fluorescently labeled proteins into living cells with emphasis on cytoskeletal proteins. Int Rev Cytol. 1982;75:209–214. doi: 10.1016/s0074-7696(08)61005-0. [DOI] [PubMed] [Google Scholar]
  15. Kreis T. E., Geiger B., Schlessinger J. Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery. Cell. 1982 Jul;29(3):835–845. doi: 10.1016/0092-8674(82)90445-7. [DOI] [PubMed] [Google Scholar]
  16. Leslie R. J., Saxton W. M., Mitchison T. J., Neighbors B., Salmon E. D., McIntosh J. R. Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching. J Cell Biol. 1984 Dec;99(6):2146–2156. doi: 10.1083/jcb.99.6.2146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McIntosh J. R., Cande W. Z., Snyder J. A. Structure and physiology of the mammalian mitotic spindle. Soc Gen Physiol Ser. 1975;30:31–76. [PubMed] [Google Scholar]
  18. Pickett-Heaps J. D., Tippit D. H., Porter K. R. Rethinking mitosis. Cell. 1982 Jul;29(3):729–744. doi: 10.1016/0092-8674(82)90435-4. [DOI] [PubMed] [Google Scholar]
  19. Rieder C. L. The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells. Chromosoma. 1981;84(1):145–158. doi: 10.1007/BF00293368. [DOI] [PubMed] [Google Scholar]
  20. Salmon E. D., Leslie R. J., Saxton W. M., Karow M. L., McIntosh J. R. Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. J Cell Biol. 1984 Dec;99(6):2165–2174. doi: 10.1083/jcb.99.6.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Salmon E. D., McKeel M., Hays T. Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine. J Cell Biol. 1984 Sep;99(3):1066–1075. doi: 10.1083/jcb.99.3.1066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Salmon E. D., Saxton W. M., Leslie R. J., Karow M. L., McIntosh J. R. Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching. J Cell Biol. 1984 Dec;99(6):2157–2164. doi: 10.1083/jcb.99.6.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stacey D. W., Allfrey V. G. Evidence for the autophagy of microinjected proteins in HeLA cells. J Cell Biol. 1977 Dec;75(3):807–817. doi: 10.1083/jcb.75.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Taylor D. L., Wang Y. L. Fluorescently labelled molecules as probes of the structure and function of living cells. Nature. 1980 Apr 3;284(5755):405–410. doi: 10.1038/284405a0. [DOI] [PubMed] [Google Scholar]
  25. Taylor D. L., Wang Y. L. Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells. Proc Natl Acad Sci U S A. 1978 Feb;75(2):857–861. doi: 10.1073/pnas.75.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wadsworth P., Sloboda R. D. Microinjection of fluorescent tubulin into dividing sea urchin cells. J Cell Biol. 1983 Oct;97(4):1249–1254. doi: 10.1083/jcb.97.4.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wang K., Feramisco J. R., Ash J. F. Fluorescent localization of contractile proteins in tissue culture cells. Methods Enzymol. 1982;85(Pt B):514–562. doi: 10.1016/0076-6879(82)85050-7. [DOI] [PubMed] [Google Scholar]
  28. Wang Y. L., Heiple J. M., Taylor D. L. Fluorescent analog cytochemistry of contractile proteins. Methods Cell Biol. 1982;25(Pt B):1–11. [PubMed] [Google Scholar]
  29. Wang Y. L., Taylor D. L. Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin. J Histochem Cytochem. 1980 Nov;28(11):1198–1206. doi: 10.1177/28.11.6107318. [DOI] [PubMed] [Google Scholar]
  30. Wegner A. Head to tail polymerization of actin. J Mol Biol. 1976 Nov;108(1):139–150. doi: 10.1016/s0022-2836(76)80100-3. [DOI] [PubMed] [Google Scholar]
  31. Zavortink M., Welsh M. J., McIntosh J. R. The distribution of calmodulin in living mitotic cells. Exp Cell Res. 1983 Dec;149(2):375–385. doi: 10.1016/0014-4827(83)90350-6. [DOI] [PubMed] [Google Scholar]
  32. Zieve G. W., Turnbull D., Mullins J. M., McIntosh J. R. Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole. Nocodazole accumulated mitotic cells. Exp Cell Res. 1980 Apr;126(2):397–405. doi: 10.1016/0014-4827(80)90279-7. [DOI] [PubMed] [Google Scholar]

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