Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 May 1;89(2):323–337. doi: 10.1083/jcb.89.2.323

Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly

PMCID: PMC2111703  PMID: 7251656

Abstract

B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication- derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

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

  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. Bell C. W., Fronk E., Gibbons I. R. Polypeptide subunits of dynein 1 from sea urchin sperm flagella. J Supramol Struct. 1979;11(3):311–317. doi: 10.1002/jss.400110305. [DOI] [PubMed] [Google Scholar]
  3. Berkowitz S. A., Katagiri J., Binder H. K., Williams R. C., Jr Separation and characterization of microtubule proteins from calf brain. Biochemistry. 1977 Dec 13;16(25):5610–5617. doi: 10.1021/bi00644a035. [DOI] [PubMed] [Google Scholar]
  4. Bibring T., Baxandall J., Denslow S., Walker B. Heterogeneity of the alpha subunit of tubulin and the variability of tubulin within a single organism. J Cell Biol. 1976 May;69(2):301–312. doi: 10.1083/jcb.69.2.301. [DOI] [PMC free article] [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. Bryan J. Biochemical properties of microtubules. Fed Proc. 1974 Feb;33(2):152–157. [PubMed] [Google Scholar]
  7. CASPAR D. L., KLUG A. Physical principles in the construction of regular viruses. Cold Spring Harb Symp Quant Biol. 1962;27:1–24. doi: 10.1101/sqb.1962.027.001.005. [DOI] [PubMed] [Google Scholar]
  8. Cleveland D. W., Hwo S. Y., Kirschner M. W. Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. J Mol Biol. 1977 Oct 25;116(2):227–247. doi: 10.1016/0022-2836(77)90214-5. [DOI] [PubMed] [Google Scholar]
  9. Cleveland D. W., Hwo S. Y., Kirschner M. W. Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin. J Mol Biol. 1977 Oct 25;116(2):207–225. doi: 10.1016/0022-2836(77)90213-3. [DOI] [PubMed] [Google Scholar]
  10. Crepeau R. H., McEwen B., Edelstein S. J. Differences in alpha and beta polypeptide chains of tubulin resolved by electron microscopy with image reconstruction. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5006–5010. doi: 10.1073/pnas.75.10.5006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Farrell K. W., Morse A., Wilson L. Characterization of the in vitro reassembly of tubulin derived from stable Strongylocentrotus purpuratus outer doublet microtubules. Biochemistry. 1979 Mar 6;18(5):905–911. doi: 10.1021/bi00572a027. [DOI] [PubMed] [Google Scholar]
  14. Farrell K. W., Wilson L. Microtubule reassembly in vitro of Strongylocentrotus purpuratus sperm tail outer doublet tubulin. J Mol Biol. 1978 May 25;121(3):393–410. doi: 10.1016/0022-2836(78)90371-6. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Gibbons I. R. Chemical dissection of cilia. Arch Biol (Liege) 1965;76(2):317–352. [PubMed] [Google Scholar]
  17. Gibbons I. R., Fronk E. A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella. J Biol Chem. 1979 Jan 10;254(1):187–196. [PubMed] [Google Scholar]
  18. Haimo L. T., Telzer B. R., Rosenbaum J. L. Dynein binds to and crossbridges cytoplasmic microtubules. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5759–5763. doi: 10.1073/pnas.76.11.5759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kirschner M. W. Microtubule assembly and nucleation. Int Rev Cytol. 1978;54:1–71. doi: 10.1016/s0074-7696(08)60164-3. [DOI] [PubMed] [Google Scholar]
  20. Kuriyama R. In vitro polymerization of flagellar and ciliary outer fiber tubulin into microtubules. J Biochem. 1976 Jul;80(1):153–165. doi: 10.1093/oxfordjournals.jbchem.a131247. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Ledbetter M. C., Porter K. R. Morphology of Microtubules of Plant Cell. Science. 1964 May 15;144(3620):872–874. doi: 10.1126/science.144.3620.872. [DOI] [PubMed] [Google Scholar]
  26. Lee J. C., Frigon R. P., Timasheff S. N. The chemical characterization of calf brain microtubule protein subunits. J Biol Chem. 1973 Oct 25;248(20):7253–7262. [PubMed] [Google Scholar]
  27. Lee J. C., Timasheff S. N. In vitro reconstitution of calf brain microtubules: effects of solution variables. Biochemistry. 1977 Apr 19;16(8):1754–1764. doi: 10.1021/bi00627a037. [DOI] [PubMed] [Google Scholar]
  28. Lee J. C., Tweedy N., Timasheff S. N. In vitro reconstitution of calf brain microtubules: effects of macromolecules. Biochemistry. 1978 Jul 11;17(14):2783–2790. doi: 10.1021/bi00607a013. [DOI] [PubMed] [Google Scholar]
  29. Linck R. W., Amos L. A. The hands of helical lattices in flagellar doublet microtubules. J Cell Sci. 1974 May;14(3):551–559. doi: 10.1242/jcs.14.3.551. [DOI] [PubMed] [Google Scholar]
  30. Linck R. W. Chemical and structural differences between cilia and flagella from the lamellibranch mollusc, Aequipecten irradians. J Cell Sci. 1973 May;12(3):951–981. doi: 10.1242/jcs.12.3.951. [DOI] [PubMed] [Google Scholar]
  31. Linck R. W. Flagellar doublet microtubules: fractionation of minor components and alpha-tubulin from specific regions of the A-tubule. J Cell Sci. 1976 Mar;20(2):405–439. doi: 10.1242/jcs.20.2.405. [DOI] [PubMed] [Google Scholar]
  32. Linck R. W., Olson G. E., Langevin G. L. Arrangement of tubulin subunits and microtubule-associated proteins in the central-pair microtubule apparatus of squid (Loligo pealei) sperm flagella. J Cell Biol. 1981 May;89(2):309–322. doi: 10.1083/jcb.89.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Little M. Identification of a second beta chain in pig brain tubulin. FEBS Lett. 1979 Dec 1;108(1):283–286. doi: 10.1016/0014-5793(79)81229-6. [DOI] [PubMed] [Google Scholar]
  34. Ludueńa R. F., Shooter E. M., Wilson L. Structure of the tubulin dimer. J Biol Chem. 1977 Oct 25;252(20):7006–7014. [PubMed] [Google Scholar]
  35. Mandelkow E. M., Mandelkow E. Junctions between microtubule walls. J Mol Biol. 1979 Mar 25;129(1):135–148. doi: 10.1016/0022-2836(79)90064-0. [DOI] [PubMed] [Google Scholar]
  36. Mandelkow E. M., Mandelkow E., Schultheiss R. Correlation between structural polarity and polar assembly of brain tubulin. J Mol Biol. 1979 Nov 25;135(1):293–299. doi: 10.1016/0022-2836(79)90354-1. [DOI] [PubMed] [Google Scholar]
  37. Mandelkow E. M., Mandelkow E., Unwin N., Cohen C. Tubulin hoops. Nature. 1977 Feb 17;265(5595):655–657. doi: 10.1038/265655a0. [DOI] [PubMed] [Google Scholar]
  38. Mandelkow E., Thomas J., Cohen C. Microtubule structure at low resolution by x-ray diffraction. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3370–3374. doi: 10.1073/pnas.74.8.3370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. McEwen B., Edelstein S. J. Evidence for a mixed lattice in microtubules reassembled in vitro. J Mol Biol. 1980 May 15;139(2):123–145. doi: 10.1016/0022-2836(80)90300-9. [DOI] [PubMed] [Google Scholar]
  40. Meza I., Huang B., Bryan J. Chemical heterogeneity of protofilaments forming the outer doublets from sea urchin flagella. Exp Cell Res. 1972 Oct;74(2):535–540. doi: 10.1016/0014-4827(72)90413-2. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Murphy D. B., Johnson K. A., Borisy G. G. Role of tubulin-associated proteins in microtubule nucleation and elongation. J Mol Biol. 1977 Nov 25;117(1):33–52. doi: 10.1016/0022-2836(77)90021-3. [DOI] [PubMed] [Google Scholar]
  43. Olmsted J. B., Borisy G. G. Characterization of microtubule assembly in porcine brain extracts by viscometry. Biochemistry. 1973 Oct 9;12(21):4282–4289. doi: 10.1021/bi00745a037. [DOI] [PubMed] [Google Scholar]
  44. Olmsted J. B., Marcum J. M., Johnson K. A., Allen C., Borisy G. G. Microtuble assembly: some possible regulatory mechanisms. J Supramol Struct. 1974;2(2-4):429–450. doi: 10.1002/jss.400020230. [DOI] [PubMed] [Google Scholar]
  45. Olson G. E., Linck R. W. Observations of the structural components of flagellar axonemes and central pair microtubules from rat sperm. J Ultrastruct Res. 1977 Oct;61(1):21–43. doi: 10.1016/s0022-5320(77)90004-1. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Pierson G. B., Burton P. R., Himes R. H. Wall substructure of microtubules polymerized in vitro from tubulin of crayfish nerve cord and fixed with tannic acid. J Cell Sci. 1979 Oct;39:89–99. doi: 10.1242/jcs.39.1.89. [DOI] [PubMed] [Google Scholar]
  48. Pollard T. D., Weihing R. R. Actin and myosin and cell movement. CRC Crit Rev Biochem. 1974 Jan;2(1):1–65. doi: 10.3109/10409237409105443. [DOI] [PubMed] [Google Scholar]
  49. Sandoval I. V., Weber K. Different tubulin polymers are produced by microtubule-associated proteins MAP2 and tau in the presence of guanosine 5'-(alpha, beta-methylene)triphosphate. J Biol Chem. 1980 Oct 10;255(19):8952–8954. [PubMed] [Google Scholar]
  50. Sandoval I. V., Weber K. Polymerization of tubulin in the presence of colchicine or podophyllotoxin. Formation of a ribbon structure induced by guanylyl-5'-methylene diphosphonate. J Mol Biol. 1979 Oct 15;134(1):159–172. doi: 10.1016/0022-2836(79)90418-2. [DOI] [PubMed] [Google Scholar]
  51. Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]
  52. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stephens R. E., Edds K. T. Microtubules: structure, chemistry, and function. Physiol Rev. 1976 Oct;56(4):709–777. doi: 10.1152/physrev.1976.56.4.709. [DOI] [PubMed] [Google Scholar]
  54. Stephens R. E. Structural chemistry of the axoneme: evidence for chemically and functionally unique tubulin dimers in outer fibers. Soc Gen Physiol Ser. 1975;30:181–206. [PubMed] [Google Scholar]
  55. Stephens R. E. Thermal fractionation of outer fiber doublet microtubules into A- and B-subfiber components. A- and B-tubulin. J Mol Biol. 1970 Feb 14;47(3):353–363. doi: 10.1016/0022-2836(70)90307-4. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. Ullmann A., Goldberg M. E., Perrin D., Monod J. On the determination of molecular weight of proteins and protein subunits in the presence of 6 M guanidine hydrochloride. Biochemistry. 1968 Jan;7(1):261–265. doi: 10.1021/bi00841a031. [DOI] [PubMed] [Google Scholar]
  58. Warner F. D., Satir P. The substructure of ciliary microtubules. J Cell Sci. 1973 Jan;12(1):313–326. doi: 10.1242/jcs.12.1.313. [DOI] [PubMed] [Google Scholar]
  59. Webb B. C., Wilson L. Cold-stable microtubules from brain. Biochemistry. 1980 Apr 29;19(9):1993–2001. doi: 10.1021/bi00550a041. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. Witman G. B., Carlson K., Berliner J., Rosenbaum J. L. Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. J Cell Biol. 1972 Sep;54(3):507–539. doi: 10.1083/jcb.54.3.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Witman G. B., Carlson K., Rosenbaum J. L. Chlamydomonas flagella. II. The distribution of tubulins 1 and 2 in the outer doublet microtubules. J Cell Biol. 1972 Sep;54(3):540–555. doi: 10.1083/jcb.54.3.540. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES