Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Nov 1;101(5):1858–1870. doi: 10.1083/jcb.101.5.1858

Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein

PMCID: PMC2113957  PMID: 2932452

Abstract

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three- dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button- like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule- associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75- kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)

Full Text

The Full Text of this article is available as a PDF (2.8 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. Bloom G. S., Luca F. C., Vallee R. B. Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules. J Cell Biol. 1984 Jan;98(1):331–340. doi: 10.1083/jcb.98.1.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brinkley B. R., Cartwright J., Jr Ultrastructural analysis of mitotic spindle elongation in mammalian cells in vitro. Direct microtubule counts. J Cell Biol. 1971 Aug;50(2):416–431. doi: 10.1083/jcb.50.2.416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cande W. Z. Inhibition of spindle elongation in permeabilized mitotic cells by erythro-9-[3-(2-hydroxynonyl)] adenine. Nature. 1982 Feb 25;295(5851):700–701. doi: 10.1038/295700a0. [DOI] [PubMed] [Google Scholar]
  5. Cande W. Z. Nucleotide requirements for anaphase chromosome movements in permeabilized mitotic cells: anaphase B but not anaphase A requires ATP. Cell. 1982 Jan;28(1):15–22. doi: 10.1016/0092-8674(82)90370-1. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Euteneuer U., McIntosh J. R. Polarity of some motility-related microtubules. Proc Natl Acad Sci U S A. 1981 Jan;78(1):372–376. doi: 10.1073/pnas.78.1.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Euteneuer U., McIntosh J. R. Structural polarity of kinetochore microtubules in PtK1 cells. J Cell Biol. 1981 May;89(2):338–345. doi: 10.1083/jcb.89.2.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goodenough U. W., Heuser J. E. Substructure of the outer dynein arm. J Cell Biol. 1982 Dec;95(3):798–815. doi: 10.1083/jcb.95.3.798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Heuser J. E., Salpeter S. R. Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell Biol. 1979 Jul;82(1):150–173. doi: 10.1083/jcb.82.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hirokawa N., Bloom G. S., Vallee R. B. Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta'-iminodipropionitrile-intoxicated axon as a model system. J Cell Biol. 1985 Jul;101(1):227–239. doi: 10.1083/jcb.101.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hirokawa N. Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J Cell Biol. 1982 Jul;94(1):129–142. doi: 10.1083/jcb.94.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hirokawa N., Heuser J. E. Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J Cell Biol. 1981 Nov;91(2 Pt 1):399–409. doi: 10.1083/jcb.91.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hisanaga S., Sakai H. Cytoplasmic dynein of the sea urchin egg. II. Purification, characterization and interactions with microtubules and Ca-calmodulin. J Biochem. 1983 Jan;93(1):87–98. doi: 10.1093/oxfordjournals.jbchem.a134182. [DOI] [PubMed] [Google Scholar]
  16. Hollenbeck P. J., Suprynowicz F., Cande W. Z. Cytoplasmic dynein-like ATPase cross-links microtubules in an ATP-sensitive manner. J Cell Biol. 1984 Oct;99(4 Pt 1):1251–1258. doi: 10.1083/jcb.99.4.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Inoué S., Sato H. Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement. J Gen Physiol. 1967 Jul;50(6 Suppl):259–292. [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. MAZIA D., CHAFFEE R. R., IVERSON R. M. Adenosine triphosphatase in the mitotic apparatus. Proc Natl Acad Sci U S A. 1961 Jun 15;47:788–790. doi: 10.1073/pnas.47.6.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McDonald K. L., Edwards M. K., McIntosh J. R. Cross-sectional structure of the central mitotic spindle of Diatoma vulgare. Evidence for specific interactions between antiparallel microtubules. J Cell Biol. 1979 Nov;83(2 Pt 1):443–461. doi: 10.1083/jcb.83.2.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McDonald K., Pickett-Heaps J. D., McIntosh J. R., Tippit D. H. On the mechanism of anaphase spindle elongation in Diatoma vulgare. J Cell Biol. 1977 Aug;74(2):377–388. doi: 10.1083/jcb.74.2.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miller K. R., Prescott C. S., Jacobs T. L., Lassignal N. L. Artifacts associated with quick-freezing and freeze-drying. J Ultrastruct Res. 1983 Feb;82(2):123–133. doi: 10.1016/s0022-5320(83)90047-3. [DOI] [PubMed] [Google Scholar]
  24. Pickett-Heaps J. D., Tippit D. H., Porter K. R. Rethinking mitosis. Cell. 1982 Jul;29(3):729–744. doi: 10.1016/0092-8674(82)90435-4. [DOI] [PubMed] [Google Scholar]
  25. Pratt M. M., Otter T., Salmon E. D. Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis). J Cell Biol. 1980 Sep;86(3):738–745. doi: 10.1083/jcb.86.3.738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pratt M. M. The identification of a dynein ATPase in unfertilized sea urchin eggs. Dev Biol. 1980 Feb;74(2):364–378. doi: 10.1016/0012-1606(80)90438-8. [DOI] [PubMed] [Google Scholar]
  27. Salmon E. D. Mitotic spindles isolated from sea urchin eggs with EGTA lysis buffers. Methods Cell Biol. 1982;25(Pt B):69–105. doi: 10.1016/s0091-679x(08)61421-1. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Schultheiss R., Mandelkow E. Three-dimensional reconstruction of tubulin sheets and re-investigation of microtubule surface lattice. J Mol Biol. 1983 Oct 25;170(2):471–496. doi: 10.1016/s0022-2836(83)80158-2. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Warner F. D., Mitchell D. R. Polarity of dynein-microtubule interactions in vitro: cross-bridging between parallel and antiparallel microtubules. J Cell Biol. 1981 Apr;89(1):35–44. doi: 10.1083/jcb.89.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES