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. 1984 Jan 1;98(1):253–266. doi: 10.1083/jcb.98.1.253

Purification and characterization of oocyte cytoplasmic tubulin and meiotic spindle tubulin of the surf clam Spisula solidissima

PMCID: PMC2113012  PMID: 6538572

Abstract

Assembly-competent tubulin was purified from the cytoplasm of unfertilized and parthogenetically activated oocytes, and from isolated meiotic spindles of the surf clam, Spisula solidissima. At 22 degrees C or 37 degrees C, Spisula tubulin assembled into 48-51-nm macrotubules during the first cycle of polymerization and 25-nm microtubules during the third and subsequent cycles of assembly. Macrotubules were formed from sheets of 26-27 protofilaments helically arranged at a 36 degree angle relative to the long axis of the polymer and were composed of alpha and beta tubulins and several other proteins ranging in molecular weight from 30,000 to 270,000. Third cycle microtubules contained 14-15 protofilaments in cross-section and were composed of greater than 95% alpha and beta tubulins. After three cycles of polymerization at 37 degrees C, unfertilized and activated oocyte tubulin self-assembled into microtubules at a critical concentration (Ccr) of 0.09 mg/ml. At the physiological temperature of 22 degrees C, unfertilized oocyte tubulin assembled into microtubules at a Ccr of 0.36 mg/ml, activated oocyte tubulin assembled at a Ccr of 0.42 mg/ml, and isolated meiotic spindle tubulin assembled at a Ccr of 0.33 mg/ml. The isoelectric points of tubulin from both unfertilized oocytes and isolated meiotic spindles were 5.8 for alpha tubulin and 5.6 for beta tubulin. In addition, one dimensional peptide maps of oocyte and spindle alpha and beta tubulins were very similar, if not identical. These results indicate that unfertilized oocyte tubulin and tubulin isolated from the first meiotic spindle are indistinguishable on the basis of assembly properties, isoelectric focusing, and one dimensional peptide mapping. These results suggest that the transition of tubulin from the quiescent oocyte state to that competent to form spindle microtubules in vivo does not require special modification of tubulin but may involve changes in the availability of microtubule organizing centers or assembly-promoting microtubule-associated proteins.

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Selected References

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  1. Amos L., Klug A. Arrangement of subunits in flagellar microtubules. J Cell Sci. 1974 May;14(3):523–549. doi: 10.1242/jcs.14.3.523. [DOI] [PubMed] [Google Scholar]
  2. Asnes C. F., Wilson L. Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: evidence for a protease. Arch Biochem Biophys. 1981 Mar;207(1):75–80. doi: 10.1016/0003-9861(81)90010-2. [DOI] [PubMed] [Google Scholar]
  3. Begg D. A., Rodewald R., Rebhun L. I. The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments. J Cell Biol. 1978 Dec;79(3):846–852. doi: 10.1083/jcb.79.3.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  5. Binder L. I., Rosenbaum J. L. The in vitro assembly of flagellar outer doublet tubulin. J Cell Biol. 1978 Nov;79(2 Pt 1):500–515. doi: 10.1083/jcb.79.2.500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown D. L., Bouck G. B. Microtubule biogenesis and cell shape in Ochromonas. 3. Effects of herbicidal mitotic inhibitor isopropyl N-phenylcarbamate on shape and flagellum regeneration. J Cell Biol. 1974 May;61(2):514–536. doi: 10.1083/jcb.61.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bryan J. B., Nagle B. W., Doenges K. H. Inhibition of tubulin assembly by RNA and other polyanions: evidence for a required protein. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3570–3574. doi: 10.1073/pnas.72.9.3570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burnside B., Kozak C., Kafatos F. C. Tubulin determination by an isotope dilution-vinblastine precipitation method. The tubulin content of Spisula eggs and embryos. J Cell Biol. 1973 Dec;59(3):755–762. doi: 10.1083/jcb.59.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burton P. R., Fernandez H. L. Delineation by lanthanum staining of filamentous elements associated with the surfaces of axonal microtubules. J Cell Sci. 1973 Mar;12(2):567–583. doi: 10.1242/jcs.12.2.567. [DOI] [PubMed] [Google Scholar]
  10. Burton P. R., Hinkley R. E., Pierson G. B. Tannic acid-stained microtubules with 12, 13, and 15 protofilaments. J Cell Biol. 1975 Apr;65(1):227–233. doi: 10.1083/jcb.65.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Caron J. M., Berlin R. D. Interaction of microtubule proteins with phospholipid vesicles. J Cell Biol. 1979 Jun;81(3):665–671. doi: 10.1083/jcb.81.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chalfie M., Thomson J. N. Structural and functional diversity in the neuronal microtubules of Caenorhabditis elegans. J Cell Biol. 1982 Apr;93(1):15–23. doi: 10.1083/jcb.93.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dentler W. L., Granett S., Rosenbaum J. L. Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules. J Cell Biol. 1975 Apr;65(1):237–241. doi: 10.1083/jcb.65.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Erickson H. P. Microtubule surface lattice and subunit structure and observations on reassembly. J Cell Biol. 1974 Jan;60(1):153–167. doi: 10.1083/jcb.60.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  16. Gaskin F., Cantor C. R., Shelanski M. L. Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules. J Mol Biol. 1974 Nov 15;89(4):737–755. doi: 10.1016/0022-2836(74)90048-5. [DOI] [PubMed] [Google Scholar]
  17. Gaskin F., Kress Y. Zinc ion-induced assembly of tubulin. J Biol Chem. 1977 Oct 10;252(19):6918–6924. [PubMed] [Google Scholar]
  18. Hinkley R. E., Jr Macrotubules induced by halothane: in vitro assembly. J Cell Sci. 1978 Aug;32:99–108. doi: 10.1242/jcs.32.1.99. [DOI] [PubMed] [Google Scholar]
  19. Hinkley R. E., Jr Microtubule-macrotubule transformations induced by volatile anesthetics. Mechanism of macrotubule assembly. J Ultrastruct Res. 1976 Dec;57(3):237–250. doi: 10.1016/s0022-5320(76)80113-x. [DOI] [PubMed] [Google Scholar]
  20. Hyams J. S., Borisy G. G. Nucleation of microtubules in vitro by isolated spindle pole bodies of the yeast Saccharomyces cerevisiae. J Cell Biol. 1978 Aug;78(2):401–414. doi: 10.1083/jcb.78.2.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Johnson K. A., Borisy G. G. The equilibrium assembly of microtubules in vitro. Soc Gen Physiol Ser. 1975;30:119–141. [PubMed] [Google Scholar]
  22. Keller T. C., 3rd, Jemiolo D. K., Burgess W. H., Rebhun L. I. Strongylocentrotus purpuratus spindle tubulin. II. Characteristics of its sensitivity to Ca++ and the effects of calmodulin isolated from bovine brain and S. purpuratus eggs. J Cell Biol. 1982 Jun;93(3):797–803. doi: 10.1083/jcb.93.3.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Kilmartin J. V. Purification of yeast tubulin by self-assembly in vitro. Biochemistry. 1981 Jun 9;20(12):3629–3633. doi: 10.1021/bi00515a050. [DOI] [PubMed] [Google Scholar]
  25. Kim H., Binder L. I., Rosenbaum J. L. The periodic association of MAP2 with brain microtubules in vitro. J Cell Biol. 1979 Feb;80(2):266–276. doi: 10.1083/jcb.80.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klausner R. D., Kumar N., Weinstein J. N., Blumenthal R., Flavin M. Interaction of tubulin with phospholipid vesicles. I. Association with vesicles at the phase transition. J Biol Chem. 1981 Jun 10;256(11):5879–5885. [PubMed] [Google Scholar]
  27. Kuriyama R. In vitro polymerization of marine egg tubulin into microtubules. J Biochem. 1977 Apr;81(4):1115–1125. doi: 10.1093/oxfordjournals.jbchem.a131536. [DOI] [PubMed] [Google Scholar]
  28. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Langford G. M. Arrangement of subunits in microtubules with 14 profilaments. J Cell Biol. 1980 Nov;87(2 Pt 1):521–526. doi: 10.1083/jcb.87.2.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Langford G. M. In vitro assembly of dogfish brain tubulin and the induction of coiled ribbon polymers by calcium. Exp Cell Res. 1978 Jan;111(1):139–151. doi: 10.1016/0014-4827(78)90244-6. [DOI] [PubMed] [Google Scholar]
  32. Larsson H., Wallin M., Edström A. Induction of a sheet polymer of tubulin by Zn2+. Exp Cell Res. 1976 Jun;100(1):104–110. doi: 10.1016/0014-4827(76)90332-3. [DOI] [PubMed] [Google Scholar]
  33. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  34. Murphy D. B., Borisy G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2696–2700. doi: 10.1073/pnas.72.7.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Murphy D. B. Identification of microtubule-associated proteins in the meiotic spindle of surf clam oocytes. J Cell Biol. 1980 Feb;84(2):235–245. doi: 10.1083/jcb.84.2.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nagano T., Suzuki F. Microtubules with 15 subunits in cockroach epidermal cells. J Cell Biol. 1975 Jan;64(1):242–245. doi: 10.1083/jcb.64.1.242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  38. Olmsted J. B., Borisy G. G. Ionic and nucleotide requirements for microtubule polymerization in vitro. Biochemistry. 1975 Jul;14(13):2996–3005. doi: 10.1021/bi00684a032. [DOI] [PubMed] [Google Scholar]
  39. Pepper D. A., Brinkley B. R. Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells. Inhibition of site-specific nucleation by tubulin antibody. J Cell Biol. 1979 Aug;82(2):585–591. doi: 10.1083/jcb.82.2.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pierson G. B., Burton P. R., Himes R. H. Alterations in number of protofilaments in microtubules assembled in vitro. J Cell Biol. 1978 Jan;76(1):223–228. doi: 10.1083/jcb.76.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rebhun L. I., Sawada N. Augmentation and dispersion of the in vivo mitotic apparatus of living marine eggs. Protoplasma. 1969;68(1):1–22. doi: 10.1007/BF01247894. [DOI] [PubMed] [Google Scholar]
  42. Scheele R. B., Bergen L. G., Borisy G. G. Control of the structural fidelity of microtubules by initiation sites. J Mol Biol. 1982 Jan 25;154(3):485–500. doi: 10.1016/s0022-2836(82)80008-9. [DOI] [PubMed] [Google Scholar]
  43. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stephens R. E. Chemical differences distinguish ciliary membrane and axonemal tubulins. Biochemistry. 1981 Aug 4;20(16):4716–4723. doi: 10.1021/bi00519a030. [DOI] [PubMed] [Google Scholar]
  45. Suprenant K. A., Rebhun L. I. Assembly of unfertilized sea urchin egg tubulin at physiological temperatures. J Biol Chem. 1983 Apr 10;258(7):4518–4525. [PubMed] [Google Scholar]
  46. Telzer B. R., Rosenbaum J. L. Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells. J Cell Biol. 1979 Jun;81(3):484–497. doi: 10.1083/jcb.81.3.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tilney L. G., Bryan J., Bush D. J., Fujiwara K., Mooseker M. S., Murphy D. B., Snyder D. H. Microtubules: evidence for 13 protofilaments. J Cell Biol. 1973 Nov;59(2 Pt 1):267–275. doi: 10.1083/jcb.59.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tilney L. G., Byers B. Studies on the microtubules in heliozoa. V. Factors controlling the organization of microtubules in the Axonemal pattern in Echinosphaerium (Actinosphaerium) nucleofilum. J Cell Biol. 1969 Oct;43(1):148–165. doi: 10.1083/jcb.43.1.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tyson G. E., Bulger R. E. Vinblastine-induced paracrystals and unusually large microtubules (macrotubules) in rat renal cells. Z Zellforsch Mikrosk Anat. 1973 Aug 14;141(4):443–458. doi: 10.1007/BF00307116. [DOI] [PubMed] [Google Scholar]
  50. VENABLE J. H., COGGESHALL R. A SIMPLIFIED LEAD CITRATE STAIN FOR USE IN ELECTRON MICROSCOPY. J Cell Biol. 1965 May;25:407–408. doi: 10.1083/jcb.25.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Vallee R. Structure and phosphorylation of microtubule-associated protein 2 (MAP 2). Proc Natl Acad Sci U S A. 1980 Jun;77(6):3206–3210. doi: 10.1073/pnas.77.6.3206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Warfield R. K., Bouck G. B. On macrotubule structure. J Mol Biol. 1975 Mar 25;93(1):117–120. doi: 10.1016/0022-2836(75)90365-4. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Weisenberg R. C. Changes in the organization of tubulin during meiosis in the eggs of the surf clam, Spisula solidissima. J Cell Biol. 1972 Aug;54(2):266–278. doi: 10.1083/jcb.54.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Weisenberg R. C., Rosenfeld A. C. In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs. J Cell Biol. 1975 Jan;64(1):146–158. doi: 10.1083/jcb.64.1.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. Zackroff R. V., Rosenfeld A. C., Weisenberg R. C. Effects of RNase and RNA on in vitro aster assembly. J Supramol Struct. 1976;5(4):577(429)589(441)–577(429)589(441). doi: 10.1002/jss.400050412. [DOI] [PubMed] [Google Scholar]

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