Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Oct 1;105(4):1721–1730. doi: 10.1083/jcb.105.4.1721

An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin

PMCID: PMC2114672  PMID: 3117804

Abstract

Rhodamine-labeled monoclonal antibodies, which react with tyrosinated alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) and label microtubules in vivo (Wehland, J., M. C. Willingham, and I. Sandoval, 1983, J. Cell Biol., 97:1467-1475) were microinjected into syncytial stage Drosophila embryos. At 1 mg/ml antibody concentration, the microtubule arrays of the surface caps became labeled by YL 1/2 but normal development was found to continue. The results are compared with the data from fixed material particularly with regard to interphase microtubules, centrosome separation, and spindle and midbody formation. At 5 mg/ml antibody concentration the microtubules took up larger quantities of antibodies and clumped around the nuclei. Nuclei with clumped microtubules lost their position in the surface layer and moved into the interior. As a result, the F-actin cap meshwork associated with such nuclei either failed to form or subsided. It is concluded that microtubule activity is required to maintain the nuclei in the surface layer and organize the F-actin meshwork of the caps.

Full Text

The Full Text of this article is available as a PDF (3.1 MB).

Selected References

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

  1. Edgar B. A., Kiehle C. P., Schubiger G. Cell cycle control by the nucleo-cytoplasmic ratio in early Drosophila development. Cell. 1986 Jan 31;44(2):365–372. doi: 10.1016/0092-8674(86)90771-3. [DOI] [PubMed] [Google Scholar]
  2. Foe V. E., Alberts B. M. Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J Cell Sci. 1983 May;61:31–70. doi: 10.1242/jcs.61.1.31. [DOI] [PubMed] [Google Scholar]
  3. Freeman M., Nüsslein-Volhard C., Glover D. M. The dissociation of nuclear and centrosomal division in gnu, a mutation causing giant nuclei in Drosophila. Cell. 1986 Aug 1;46(3):457–468. doi: 10.1016/0092-8674(86)90666-5. [DOI] [PubMed] [Google Scholar]
  4. Fujiwara K., Pollard T. D. Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining. J Cell Biol. 1978 Apr;77(1):182–195. doi: 10.1083/jcb.77.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fuller G. M., Brinkley B. R., Boughter J. M. Immunofluorescence of mitotic spindles by using monospecific antibody against bovine brain tubulin. Science. 1975 Mar 14;187(4180):948–950. doi: 10.1126/science.1096300. [DOI] [PubMed] [Google Scholar]
  6. Hamaguchi Y., Toriyama M., Sakai H., Hiramoto Y. Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage. J Cell Biol. 1985 Apr;100(4):1262–1272. doi: 10.1083/jcb.100.4.1262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harris P., Osborn M., Weber K. Distribution of tubulin-containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage. J Cell Biol. 1980 Mar;84(3):668–679. doi: 10.1083/jcb.84.3.668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kalfayan L., Wensink P. C. Developmental regulation of Drosophila alpha-tubulin genes. Cell. 1982 May;29(1):91–98. doi: 10.1016/0092-8674(82)90093-9. [DOI] [PubMed] [Google Scholar]
  9. Karr T. L., Alberts B. M. Organization of the cytoskeleton in early Drosophila embryos. J Cell Biol. 1986 Apr;102(4):1494–1509. doi: 10.1083/jcb.102.4.1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Loyd J. E., Raff E. C., Raff R. A. Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster. Dev Biol. 1981 Sep;86(2):272–284. doi: 10.1016/0012-1606(81)90185-8. [DOI] [PubMed] [Google Scholar]
  14. Mischke D., Pardue M. L. Organization and expression of alpha-tubulin genes in Drosophila melanogaster. One member of the alpha-tubulin multigene family is transcribed in both oogenesis and later embryonic development. J Mol Biol. 1982 Apr 15;156(3):449–466. doi: 10.1016/0022-2836(82)90260-1. [DOI] [PubMed] [Google Scholar]
  15. Mitchison T., Evans L., Schulze E., Kirschner M. Sites of microtubule assembly and disassembly in the mitotic spindle. Cell. 1986 May 23;45(4):515–527. doi: 10.1016/0092-8674(86)90283-7. [DOI] [PubMed] [Google Scholar]
  16. Natzle J. E., McCarthy B. J. Regulation of Drosophila alpha- and beta-tubulin genes during development. Dev Biol. 1984 Jul;104(1):187–198. doi: 10.1016/0012-1606(84)90047-2. [DOI] [PubMed] [Google Scholar]
  17. Niki Y. Developmental analysis of the grandchildless (gs(1)N26) mutation in Drosophila melanogaster: abnormal cleavage patterns and defects in pole cell formation. Dev Biol. 1984 May;103(1):182–189. doi: 10.1016/0012-1606(84)90019-8. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Saxton W. M., Stemple D. L., Leslie R. J., Salmon E. D., Zavortink M., McIntosh J. R. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Solomon F., Magendantz M. Cytochalasin separates microtubule disassembly from loss of asymmetric morphology. J Cell Biol. 1981 Apr;89(1):157–161. doi: 10.1083/jcb.89.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Stafstrom J. P., Staehelin L. A. Dynamics of the nuclear envelope and of nuclear pore complexes during mitosis in the Drosophila embryo. Eur J Cell Biol. 1984 May;34(1):179–189. [PubMed] [Google Scholar]
  25. Standart N. M., Bray S. J., George E. L., Hunt T., Ruderman J. V. The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs. J Cell Biol. 1985 Jun;100(6):1968–1976. doi: 10.1083/jcb.100.6.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Theurkauf W. E., Baum H., Bo J., Wensink P. C. Tissue-specific and constitutive alpha-tubulin genes of Drosophila melanogaster code for structurally distinct proteins. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8477–8481. doi: 10.1073/pnas.83.22.8477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tomasek J. J., Hay E. D. Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels. J Cell Biol. 1984 Aug;99(2):536–549. doi: 10.1083/jcb.99.2.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Warn R. M., Magrath R. Observations by a novel method of surface changes during the syncytial blastoderm stage of the Drosophila embryo. Dev Biol. 1982 Feb;89(2):540–548. doi: 10.1016/0012-1606(82)90344-x. [DOI] [PubMed] [Google Scholar]
  31. Warn R. M., Magrath R., Webb S. Distribution of F-actin during cleavage of the Drosophila syncytial blastoderm. J Cell Biol. 1984 Jan;98(1):156–162. doi: 10.1083/jcb.98.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Warn R. M., Smith L., Warn A. Three distinct distributions of F-actin occur during the divisions of polar surface caps to produce pole cells in Drosophila embryos. J Cell Biol. 1985 Apr;100(4):1010–1015. doi: 10.1083/jcb.100.4.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Warn R. M. The cytoskeleton of the early Drosophila embryo. J Cell Sci Suppl. 1986;5:311–328. doi: 10.1242/jcs.1986.supplement_5.20. [DOI] [PubMed] [Google Scholar]
  34. Warn R. M., Warn A. Microtubule arrays present during the syncytial and cellular blastoderm stages of the early Drosophila embryo. Exp Cell Res. 1986 Mar;163(1):201–210. doi: 10.1016/0014-4827(86)90573-2. [DOI] [PubMed] [Google Scholar]
  35. Weber K., Bibring T., Osborn M. Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence. Cytoplasmic microtubules, vinblastine-induced paracrystals, and mitotic figures. Exp Cell Res. 1975 Oct 1;95(1):111–120. doi: 10.1016/0014-4827(75)90615-1. [DOI] [PubMed] [Google Scholar]
  36. Wehland J., Schröder H. C., Weber K. Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2. EMBO J. 1984 Jun;3(6):1295–1300. doi: 10.1002/j.1460-2075.1984.tb01965.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Wehland J., Willingham M. C., Sandoval I. V. A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo. J Cell Biol. 1983 Nov;97(5 Pt 1):1467–1475. doi: 10.1083/jcb.97.5.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES