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. 1985 Aug 1;101(2):524–530. doi: 10.1083/jcb.101.2.524

Identification of molecular components of the centrosphere in the mitotic spindle of sea urchin eggs

PMCID: PMC2113690  PMID: 3894377

Abstract

Monoclonal antibodies were prepared to identify molecular components specific to the mitotic apparatus of sea urchin eggs. The mitotic apparatus or asters induced within unfertilized eggs by taxol treatment were isolated from Strongylocentrotus purpuratus and used for immunization of mice. After fusion with spleen cells, the supernatant of hybridomas were screened in two stages by indirect immunofluorescence staining, first on isolated sea urchin mitotic spindles in 96-well microtiter plates to identify rapidly potential positive hybridomas, and second, on whole mitotic eggs on coverslips to distinguish between spindle-specific staining and adventitious contamination. Two hybridomas, SU4 and SU5, secreted antibodies reactive to microtubule-containing structures in eggs during the course of development. They preferentially stained the centrosphere both in isolated mitotic apparatus and in whole metaphase eggs, which was further confirmed by staining the isolated centrospheres with these antibodies. SU4 recognized a major 190-kD polypeptide on immunoblots as well as a species at 180 and 20 kD, whereas hybridoma SU5 stained a species at 50 kD. Thus, these polypeptides may be components of the centrosphere.

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Selected References

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  1. Berns M. W., Richardson S. M. Continuation of mitosis after selective laser microbeam destruction of the centriolar region. J Cell Biol. 1977 Dec;75(3):977–982. doi: 10.1083/jcb.75.3.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brenner S., Branch A., Meredith S., Berns M. W. The absence of centrioles from spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division in vitro. J Cell Biol. 1977 Feb;72(2):368–379. doi: 10.1083/jcb.72.2.368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Calarco-Gillam P. D., Siebert M. C., Hubble R., Mitchison T., Kirschner M. Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material. Cell. 1983 Dec;35(3 Pt 2):621–629. doi: 10.1016/0092-8674(83)90094-6. [DOI] [PubMed] [Google Scholar]
  4. Connolly J. A., Kalnins V. I. Visualization of centrioles and basal bodies by fluorescent staining with nonimmune rabbit sera. J Cell Biol. 1978 Nov;79(2 Pt 1):526–532. doi: 10.1083/jcb.79.2.526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gould R. R., Borisy G. G. The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation. J Cell Biol. 1977 Jun;73(3):601–615. doi: 10.1083/jcb.73.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Harris P. The role of membranes in the ogranization of the mitotic apparatus. Exp Cell Res. 1975 Sep;94(2):409–425. doi: 10.1016/0014-4827(75)90507-8. [DOI] [PubMed] [Google Scholar]
  8. KANE R. E. THE MITOTIC APPARATUS. PHYSICAL-CHEMICAL FACTORS CONTROLLING STABILITY. J Cell Biol. 1965 Apr;25:SUPPL–SUPPL:144. doi: 10.1083/jcb.25.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kasamatsu H., Nehorayan A. Intracellular localization of viral polypeptides during simian virus 40 infection. J Virol. 1979 Nov;32(2):648–660. doi: 10.1128/jvi.32.2.648-660.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  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. Kuriyama R., Borisy G. G. Cytasters induced within unfertilized sea-urchin eggs. J Cell Sci. 1983 May;61:175–189. doi: 10.1242/jcs.61.1.175. [DOI] [PubMed] [Google Scholar]
  13. Kuriyama R., Keryer G., Borisy G. G. The mitotic spindle of Chinese hamster ovary cells isolated in taxol-containing medium. J Cell Sci. 1984 Mar;66:265–275. doi: 10.1242/jcs.66.1.265. [DOI] [PubMed] [Google Scholar]
  14. Köhler G., Milstein C. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol. 1976 Jul;6(7):511–519. doi: 10.1002/eji.1830060713. [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. Nenci I., Marchetti E. Concerning the localization of steroids in centrioles and basal bodies by immunofluorescence. J Cell Biol. 1978 Feb;76(2):255–260. doi: 10.1083/jcb.76.2.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nigg E. A., Walter G., Singer S. J. On the nature of crossreactions observed with antibodies directed to defined epitopes. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5939–5943. doi: 10.1073/pnas.79.19.5939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Oliver J. M., Osborne W. R., Pfeiffer J. R., Child F. M., Berlin R. D. Purine nucleoside phosphorylase is associated with centrioles and basal bodies. J Cell Biol. 1981 Dec;91(3 Pt 1):837–847. doi: 10.1083/jcb.91.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rattner J. B., Berns M. W. Distribution of microtubules during centriole separation in rat kangaroo (Potorous) cells. Cytobios. 1976;15(57):37–43. [PubMed] [Google Scholar]
  20. 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]
  21. Robbins E., Jentzsch G., Micali A. The centriole cycle in synchronized HeLa cells. J Cell Biol. 1968 Feb;36(2):329–339. doi: 10.1083/jcb.36.2.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Salmon E. D., Segall R. R. Calcium-labile mitotic spindles isolated from sea urchin eggs (Lytechinus variegatus). J Cell Biol. 1980 Aug;86(2):355–365. doi: 10.1083/jcb.86.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schatten G., Schatten H., Bestor T. H., Balczon R. Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs. J Cell Biol. 1982 Aug;94(2):455–465. doi: 10.1083/jcb.94.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sherline P., Mascardo R. N. Epidermal growth factor induces rapid centrosomal separation in HeLa and 3T3 cells. J Cell Biol. 1982 May;93(2):507–512. doi: 10.1083/jcb.93.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Szollosi D., Calarco P., Donahue R. P. Absence of centrioles in the first and second meiotic spindles of mouse oocytes. J Cell Sci. 1972 Sep;11(2):521–541. doi: 10.1242/jcs.11.2.521. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Turksen K., Aubin J. E., Kalnins V. I. Identification of a centriole-associated protein by antibodies present in normal rabbit sera. Nature. 1982 Aug 19;298(5876):763–765. doi: 10.1038/298763a0. [DOI] [PubMed] [Google Scholar]
  28. Vallee R. B., Bloom G. S. Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule-associated proteins with monoclonal antibodies. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6259–6263. doi: 10.1073/pnas.80.20.6259. [DOI] [PMC free article] [PubMed] [Google Scholar]

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