Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Aug 1;111(2):511–522. doi: 10.1083/jcb.111.2.511

A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression

PMCID: PMC2116221  PMID: 2199459

Abstract

A monoclonal antibody raised against mitotic spindles isolated from CHO cells ([CHO1], Sellitto, C., and R. Kuriyama. 1988. J. Cell Biol. 106:431-439) identifies an epitope that resides on polypeptides of 95 and 105 kD and is localized in the spindles of diverse organisms. The antigen is distributed throughout the spindle at metaphase but becomes concentrated in a progressively narrower zone on either side of the spindle midplane as anaphase progresses. Microinjection of CHO1, either as an ascites fluid or as purified IgM, results in mitotic inhibition in a stage-specific and dose-dependent manner. Parallel control injections with nonimmune IgMs do not yield significant mitotic inhibition. Immunofluorescence analysis of injected cells reveals that those which complete mitosis display normal localization of CHO1, whereas arrested cells show no specific localization of the CHO1 antigen within the spindle. Immunoelectron microscopic images of such arrested cells indicate aberrant microtubule organization. The CHO1 antigen in HeLa cell extracts copurifies with taxol-stabilized microtubules. Neither of the polypeptides bearing the antigen is extracted from microtubules by ATP or GTP, but both are approximately 60% extracted with 0.5 M NaCl. Sucrose gradient analysis reveals that the antigens sediment at approximately 11S. The CHO 1 antigen appears to be a novel mitotic MAP whose proper distribution within the spindle is required for mitosis. The properties of the antigen(s) suggest that the corresponding protein(s) are part of the mechanism that holds the antiparallel microtubules of the two interdigitating half spindles together during anaphase.

Full Text

The Full Text of this article is available as a PDF (4.4 MB).

Selected References

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

  1. Arias J. A., Dynan W. S. Promoter-dependent transcription by RNA polymerase II using immobilized enzyme complexes. J Biol Chem. 1989 Feb 25;264(6):3223–3229. [PubMed] [Google Scholar]
  2. Bastmeyer M., Russell D. G. Characterization of Pales spermatocyte spindles, with reference to an MTOC-associated protein. J Cell Sci. 1987 Apr;87(Pt 3):431–438. doi: 10.1242/jcs.87.3.431. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cooke C. A., Heck M. M., Earnshaw W. C. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J Cell Biol. 1987 Nov;105(5):2053–2067. doi: 10.1083/jcb.105.5.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dinsmore J. H., Sloboda R. D. Microinjection of antibodies to a 62 kd mitotic apparatus protein arrests mitosis in dividing sea urchin embryos. Cell. 1989 Apr 7;57(1):127–134. doi: 10.1016/0092-8674(89)90178-5. [DOI] [PubMed] [Google Scholar]
  6. Euteneuer U., Jackson W. T., McIntosh J. R. Polarity of spindle microtubules in Haemanthus endosperm. J Cell Biol. 1982 Sep;94(3):644–653. doi: 10.1083/jcb.94.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Füchtbauer A., Herrmann M., Mandelkow E. M., Jockusch B. M. Disruption of microtubules in living cells and cell models by high affinity antibodies to beta-tubulin. EMBO J. 1985 Nov;4(11):2807–2814. doi: 10.1002/j.1460-2075.1985.tb04007.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hafner M., Petzelt C. Inhibition of mitosis by an antibody to the mitotic calcium transport system. Nature. 1987 Nov 19;330(6145):264–266. doi: 10.1038/330264a0. [DOI] [PubMed] [Google Scholar]
  9. Haimo L. T., Telzer B. R. Dynein-microtubule interactions: ATP-sensitive dynein binding and the structural polarity of mitotic microtubules. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):207–217. doi: 10.1101/sqb.1982.046.01.024. [DOI] [PubMed] [Google Scholar]
  10. Izant J. G., Weatherbee J. A., McIntosh J. R. A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells. J Cell Biol. 1983 Feb;96(2):424–434. doi: 10.1083/jcb.96.2.424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jehanli A., Hough D. A rapid procedure for the isolation of human IgM myeloma proteins. J Immunol Methods. 1981;44(2):199–204. doi: 10.1016/0022-1759(81)90347-1. [DOI] [PubMed] [Google Scholar]
  12. Kiehart D. P., Kaiser D. A., Pollard T. D. Antibody inhibitors of nonmuscle myosin function and assembly. Methods Enzymol. 1986;134:423–453. doi: 10.1016/0076-6879(86)34109-0. [DOI] [PubMed] [Google Scholar]
  13. Kingwell B., Fritzler M. J., Decoteau J., Rattner J. B. Identification and characterization of a protein associated with the stembody using autoimmune sera from patients with systemic sclerosis. Cell Motil Cytoskeleton. 1987;8(4):360–367. doi: 10.1002/cm.970080408. [DOI] [PubMed] [Google Scholar]
  14. Kuriyama R. 225-Kilodalton phosphoprotein associated with mitotic centrosomes in sea urchin eggs. Cell Motil Cytoskeleton. 1989;12(2):90–103. doi: 10.1002/cm.970120204. [DOI] [PubMed] [Google Scholar]
  15. Lydersen B. K., Pettijohn D. E. Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: distribution in a human/hamster hybrid cell. Cell. 1980 Nov;22(2 Pt 2):489–499. doi: 10.1016/0092-8674(80)90359-1. [DOI] [PubMed] [Google Scholar]
  16. McIntosh J. R., Koonce M. P. Mitosis. Science. 1989 Nov 3;246(4930):622–628. doi: 10.1126/science.2683078. [DOI] [PubMed] [Google Scholar]
  17. Mullins J. M., McIntosh J. R. Isolation and initial characterization of the mammalian midbody. J Cell Biol. 1982 Sep;94(3):654–661. doi: 10.1083/jcb.94.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Neighbors B. W., Williams R. C., Jr, McIntosh J. R. Localization of kinesin in cultured cells. J Cell Biol. 1988 Apr;106(4):1193–1204. doi: 10.1083/jcb.106.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Olmsted J. B. Microtubule-associated proteins. Annu Rev Cell Biol. 1986;2:421–457. doi: 10.1146/annurev.cb.02.110186.002225. [DOI] [PubMed] [Google Scholar]
  20. Riabowol K., Draetta G., Brizuela L., Vandre D., Beach D. The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells. Cell. 1989 May 5;57(3):393–401. doi: 10.1016/0092-8674(89)90914-8. [DOI] [PubMed] [Google Scholar]
  21. Saxton W. M., McIntosh J. R. Interzone microtubule behavior in late anaphase and telophase spindles. J Cell Biol. 1987 Aug;105(2):875–886. doi: 10.1083/jcb.105.2.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Scholey J. M., Neighbors B., McIntosh J. R., Salmon E. D. Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs. J Biol Chem. 1984 May 25;259(10):6516–6525. [PubMed] [Google Scholar]
  23. Schollmeyer J. E. Calpain II involvement in mitosis. Science. 1988 May 13;240(4854):911–913. doi: 10.1126/science.2834825. [DOI] [PubMed] [Google Scholar]
  24. Sellitto C., Kuriyama R. Distribution of a matrix component of the midbody during the cell cycle in Chinese hamster ovary cells. J Cell Biol. 1988 Feb;106(2):431–439. doi: 10.1083/jcb.106.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Silver R. B. Mitosis in sand dollar embryos is inhibited by antibodies directed against the calcium transport enzyme of muscle. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4302–4306. doi: 10.1073/pnas.83.12.4302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vandre D. D., Davis F. M., Rao P. N., Borisy G. G. Phosphoproteins are components of mitotic microtubule organizing centers. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4439–4443. doi: 10.1073/pnas.81.14.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]

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

RESOURCES