Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1983 Oct 1;97(4):1249–1254. doi: 10.1083/jcb.97.4.1249

Microinjection of fluorescent tubulin into dividing sea urchin cells

PMCID: PMC2112626  PMID: 6684663

Abstract

To follow the dynamics of microtubule (MT) assembly and disassembly during mitosis in living cells, tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)aminofluorescein and microinjected into fertilized eggs of the sea urchin Lytechinus variegatus. The changing distribution of the fluorescent protein probe is visualized in a fluorescence microscope coupled to an image intensification video system. Cells that have been injected with fluorescent tubulin show fluorescent linear polymers that assemble very rapidly and radiate from the spindle poles, coincident with the position of the astral fibers. No fluorescent polymer is apparent in other areas of the cytoplasm. When fluorescent tubulin is injected near the completion of anaphase, little incorporation of fluorescent tubulin into polymer is apparent, suggesting that new polymerization does not occur past a critical point in anaphase. These results demonstrate that MT polymerization is very rapid in vivo and that the assembly is both temporally and spatially regulated within the injected cells. Furthermore, the microinjected tubulin is stable within the sea urchin cytoplasm for at least 1 h since it can be reutilized in successive daughter cell spindles. Control experiments indicate that the observed fluorescence is dependent on MT assembly. The fluorescence is greatly diminished upon treatment of the cells with cold or colchicine agents known to cause the depolymerization of assembled MT. In addition, cells injected with fluorescent bovine serum albumin or assembly-incompetent fluorescent tubulin do not exhibit fluorescence localized in the spindle but rather appear diffusely fluorescent throughout the cytoplasm.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

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

  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. Burns R. G., Starling D. The in vitro assembly of tubulins from sea-urchin eggs and rat brain: use of heterologous seeds. J Cell Sci. 1974 Mar;14(2):411–419. doi: 10.1242/jcs.14.2.411. [DOI] [PubMed] [Google Scholar]
  3. Burridge K., Feramisco J. R. Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin. Cell. 1980 Mar;19(3):587–595. doi: 10.1016/s0092-8674(80)80035-3. [DOI] [PubMed] [Google Scholar]
  4. Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence. J Cell Biol. 1978 Mar;76(3):781–786. doi: 10.1083/jcb.76.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Feramisco J. R., Blose S. H. Distribution of fluorescently labeled alpha-actinin in living and fixed fibroblasts. J Cell Biol. 1980 Aug;86(2):608–615. doi: 10.1083/jcb.86.2.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gawlitta W., Stockem W., Wehland J., Weber K. Organization and spatial arrangement of fluorescein-labeled native actin microinjected into normal locomoting and experimentally influenced Amoeba proteus. Cell Tissue Res. 1980;206(2):181–191. doi: 10.1007/BF00232762. [DOI] [PubMed] [Google Scholar]
  8. Glacy S. D. Pattern and time course of rhodamine-actin incorporation in cardiac myocytes. J Cell Biol. 1983 Apr;96(4):1164–1167. doi: 10.1083/jcb.96.4.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hard R., Allen R. D. Behaviour of kinetochore fibres in Haemanthus katherinae during anaphase movements of chromosomes. J Cell Sci. 1977;27:47–56. doi: 10.1242/jcs.27.1.47. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Inoué S., Borisy G. G., Kiehart D. P. Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin. J Cell Biol. 1974 Jul;62(1):175–184. doi: 10.1083/jcb.62.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Inoué S., Ritter H., Jr Dynamics of mitotic spindle organization and function. Soc Gen Physiol Ser. 1975;30:3–30. [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Keller T. C., 3rd, Rebhun L. I. Strongylocentrotus purpuratus spindle tubulin. I. Characteristics of its polymerization and depolymerization in vitro. J Cell Biol. 1982 Jun;93(3):788–796. doi: 10.1083/jcb.93.3.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kiehart D. P. Microinjection of echinoderm eggs: apparatus and procedures. Methods Cell Biol. 1982;25(Pt B):13–31. doi: 10.1016/s0091-679x(08)61418-1. [DOI] [PubMed] [Google Scholar]
  17. Kirschner M. W. Implications of treadmilling for the stability and polarity of actin and tubulin polymers in vivo. J Cell Biol. 1980 Jul;86(1):330–334. doi: 10.1083/jcb.86.1.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kreis T. E., Winterhalter K. H., Birchmeier W. In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3814–3818. doi: 10.1073/pnas.76.8.3814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Margolis R. L., Wilson L. Microtubule treadmills--possible molecular machinery. Nature. 1981 Oct 29;293(5835):705–711. doi: 10.1038/293705a0. [DOI] [PubMed] [Google Scholar]
  20. Margolis R. L., Wilson L. Opposite end assembly and disassembly of microtubules at steady state in vitro. Cell. 1978 Jan;13(1):1–8. doi: 10.1016/0092-8674(78)90132-0. [DOI] [PubMed] [Google Scholar]
  21. McIntosh J. R., Landis S. C. The distribution of spindle microtubules during mitosis in cultured human cells. J Cell Biol. 1971 May 1;49(2):468–497. doi: 10.1083/jcb.49.2.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rebhun L. I., Rosenbaum J., Lefebvre P., Smith G. Reversible restoration of the birefringence of cold-treated, isolated mitotic apparatus of surf clam eggs with chick brain tubulin. Nature. 1974 May 10;249(453):113–115. doi: 10.1038/249113a0. [DOI] [PubMed] [Google Scholar]
  23. Salmon E. D. Spindle microtubules: thermodynamics of in vivo assembly and role in chromosome movement. Ann N Y Acad Sci. 1975 Jun 30;253:383–406. doi: 10.1111/j.1749-6632.1975.tb19216.x. [DOI] [PubMed] [Google Scholar]
  24. Sheir-Neiss G., Lai M. H., Morris N. R. Identification of a gene for beta-tubulin in Aspergillus nidulans. Cell. 1978 Oct;15(2):639–647. doi: 10.1016/0092-8674(78)90032-6. [DOI] [PubMed] [Google Scholar]
  25. Sloboda R. D., Dentler W. L., Rosenbaum J. L. Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry. 1976 Oct 5;15(20):4497–4505. doi: 10.1021/bi00665a026. [DOI] [PubMed] [Google Scholar]
  26. Snell W. J., Dentler W. L., Haimo L. T., Binder L. I., Rosenbaum J. L. Assembly of chick brain tubulin onto isolated basal bodies of Chlamydomonas reinhardi. Science. 1974 Jul 26;185(4148):357–360. doi: 10.1126/science.185.4148.357. [DOI] [PubMed] [Google Scholar]
  27. Snyder J. A., Hamilton B. T., Mullins J. M. Loss of mitotic centrosomal microtubule initiation capacity at the metaphase-anaphase transition. Eur J Cell Biol. 1982 Jun;27(2):191–199. [PubMed] [Google Scholar]
  28. Stephens R. E. Primary structural differences among tubulin subunits from flagella, cilia, and the cytoplasm. Biochemistry. 1978 Jul 11;17(14):2882–2891. doi: 10.1021/bi00607a029. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Wadsworth P., Sloboda R. D. Characterization of tubulin modified with the sulfhydryl-reactive fluorochrome monobromo(trimethylammonio)bimane. Biochemistry. 1982 Jan 5;21(1):21–28. doi: 10.1021/bi00530a004. [DOI] [PubMed] [Google Scholar]
  31. Water R. D., Kleinsmith L. J. Identification of alpha and beta tubulin in yeast. Biochem Biophys Res Commun. 1976 Jun 7;70(3):704–708. doi: 10.1016/0006-291x(76)90649-5. [DOI] [PubMed] [Google Scholar]
  32. Weber K., Pollack R., Bibring T. Antibody against tuberlin: the specific visualization of cytoplasmic microtubules in tissue culture cells. Proc Natl Acad Sci U S A. 1975 Feb;72(2):459–463. doi: 10.1073/pnas.72.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Williams R. C., Jr, Detrich H. W., 3rd Separation of tubulin from microtubule-associated proteins on phosphocellulose. Accompanying alterations in concentrations of buffer components. Biochemistry. 1979 Jun 12;18(12):2499–2503. doi: 10.1021/bi00579a010. [DOI] [PubMed] [Google Scholar]
  35. Willingham M. C., Pastan I. The visualization of fluorescent proteins in living cells by video intensification microscopy (VIM). Cell. 1978 Mar;13(3):501–507. doi: 10.1016/0092-8674(78)90323-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES