Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Nov 1;115(3):705–716. doi: 10.1083/jcb.115.3.705

Striated microtubule-associated fibers: identification of assemblin, a novel 34-kD protein that forms paracrystals of 2-nm filaments in vitro

PMCID: PMC2289178  PMID: 1918160

Abstract

Microtubule-associated fibers from the basal apparatus of the green flagellate alga Spermatozopsis similis exhibit a complex cross- striation pattern with 28-nm periodicity and consist of 2-nm filaments arranged in several layers. Fibers enriched by mechanical disintegration and high salt extraction (2 M NaCl) of isolated basal apparatuses are soluble in 2 M urea. Dialysis of solubilized fibers against 150 mM KCl yields paracrystals which closely resemble the native fibers in filament arrangement and striation pattern. Paracrystals purified through several cycles of disassembly and reassembly are greatly enriched (greater than 90%) in a single protein of 34 kD (assemblin) as shown by SDS-PAGE. A rabbit polyclonal antibody raised against assemblin labels the striated fibers as shown by indirect immunofluorescence of isolated cytoskeletons or methanol permeabilized cells and immunogold EM. Two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE) resolves assemblin into at least four isoforms (a-d) with pI's of 5.45, 5.55, 5.75, and 5.85. The two more acidic isoforms are phosphoproteins as shown by in vivo 32PO4- labeling and autoradiography. Amino acid analysis of assemblin shows a high content of helix-forming residues (leucine) and a relatively low content of glycine. We conclude that assemblin may be representative of a class of proteins that form fine filaments alongside microtubules.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

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

  1. Aebi U., Fowler W. E., Rew P., Sun T. T. The fibrillar substructure of keratin filaments unraveled. J Cell Biol. 1983 Oct;97(4):1131–1143. doi: 10.1083/jcb.97.4.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aggarwal A., Adam R. D., Nash T. E. Characterization of a 29.4-kilodalton structural protein of Giardia lamblia and localization to the ventral disk [corrected]. Infect Immun. 1989 Apr;57(4):1305–1310. doi: 10.1128/iai.57.4.1305-1310.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker D. A., Holberton D. V., Marshall J. Sequence of a giardin subunit cDNA from Giardia lamblia. Nucleic Acids Res. 1988 Jul 25;16(14B):7177–7177. doi: 10.1093/nar/16.14.7177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Becherer P. R., Mertz L. F., Baenziger N. L. Regulation of prostaglandin synthesis mediated by thrombin and B2 bradykinin receptors in a fibrosarcoma cell line. Cell. 1982 Aug;30(1):243–251. doi: 10.1016/0092-8674(82)90030-7. [DOI] [PubMed] [Google Scholar]
  5. Chang X. J., Piperno G. Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits. J Cell Biol. 1987 Jun;104(6):1563–1568. doi: 10.1083/jcb.104.6.1563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crossley R., Holberton D. V. Characterization of proteins from the cytoskeleton of Giardia lamblia. J Cell Sci. 1983 Jan;59:81–103. doi: 10.1242/jcs.59.1.81. [DOI] [PubMed] [Google Scholar]
  7. Crossley R., Holberton D. V. Selective extraction with Sarkosyl and repolymerization in vitro of cytoskeleton proteins from Giardia. J Cell Sci. 1983 Jul;62:419–438. doi: 10.1242/jcs.62.1.419. [DOI] [PubMed] [Google Scholar]
  8. Crossley R., Holberton D. Assembly of 2.5 nm filaments from giardin, a protein associated with cytoskeletal microtubules in Giardia. J Cell Sci. 1985 Oct;78:205–231. doi: 10.1242/jcs.78.1.205. [DOI] [PubMed] [Google Scholar]
  9. Dingle A. D., Larson D. E. Structure and protein composition of the striated flagellar rootlets of some protists. Biosystems. 1981;14(3-4):345–358. doi: 10.1016/0303-2647(81)90041-1. [DOI] [PubMed] [Google Scholar]
  10. Geisler N., Weber K. Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain. EMBO J. 1988 Jan;7(1):15–20. doi: 10.1002/j.1460-2075.1988.tb02778.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goodenough U. W., Weiss R. L. Interrelationships between microtubules, a striated fiber, and the gametic mating structure of Chlamydomonas reinhardi. J Cell Biol. 1978 Feb;76(2):430–438. doi: 10.1083/jcb.76.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hagestedt T., Lichtenberg B., Wille H., Mandelkow E. M., Mandelkow E. Tau protein becomes long and stiff upon phosphorylation: correlation between paracrystalline structure and degree of phosphorylation. J Cell Biol. 1989 Oct;109(4 Pt 1):1643–1651. doi: 10.1083/jcb.109.4.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holberton D. V. Arrangement of subunits in microribbons from Giardia. J Cell Sci. 1981 Feb;47:167–185. doi: 10.1242/jcs.47.1.167. [DOI] [PubMed] [Google Scholar]
  14. Holberton D. V., Ward A. P. Isolation of the cytoskeleton from Giardia. Tubulin and a low-molecular-weight protein associated with microribbon structures. J Cell Sci. 1981 Feb;47:139–166. doi: 10.1242/jcs.47.1.139. [DOI] [PubMed] [Google Scholar]
  15. Holberton D., Baker D. A., Marshall J. Segmented alpha-helical coiled-coil structure of the protein giardin from the Giardia cytoskeleton. J Mol Biol. 1988 Dec 5;204(3):789–795. doi: 10.1016/0022-2836(88)90370-1. [DOI] [PubMed] [Google Scholar]
  16. Honts J. E., Williams N. E. Tetrins: polypeptides that form bundled filaments in Tetrahymena. J Cell Sci. 1990 Jun;96(Pt 2):293–302. doi: 10.1242/jcs.96.2.293. [DOI] [PubMed] [Google Scholar]
  17. Huang B., Mengersen A., Lee V. D. Molecular cloning of cDNA for caltractin, a basal body-associated Ca2+-binding protein: homology in its protein sequence with calmodulin and the yeast CDC31 gene product. J Cell Biol. 1988 Jul;107(1):133–140. doi: 10.1083/jcb.107.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huang B., Watterson D. M., Lee V. D., Schibler M. J. Purification and characterization of a basal body-associated Ca2+-binding protein. J Cell Biol. 1988 Jul;107(1):121–131. doi: 10.1083/jcb.107.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hyams J. S., Borisy G. G. Flagellar coordination in Chlamydomonas reinhardtii: isolation and reactivation of the flagellar apparatus. Science. 1975 Sep 12;189(4206):891–893. doi: 10.1126/science.1098148. [DOI] [PubMed] [Google Scholar]
  20. Inagaki M., Nishi Y., Nishizawa K., Matsuyama M., Sato C. Site-specific phosphorylation induces disassembly of vimentin filaments in vitro. Nature. 1987 Aug 13;328(6131):649–652. doi: 10.1038/328649a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Larson D. E., Dingle A. D. Isolation, ultrastructure, and protein composition of the flagellar rootlet of Naegleria gruberi. J Cell Biol. 1981 Jun;89(3):424–432. doi: 10.1083/jcb.89.3.424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Linck R. W., Langevin G. L. Structure and chemical composition of insoluble filamentous components of sperm flagellar microtubules. J Cell Sci. 1982 Dec;58:1–22. doi: 10.1242/jcs.58.1.1. [DOI] [PubMed] [Google Scholar]
  24. McFadden G. I., Schulze D., Surek B., Salisbury J. L., Melkonian M. Basal body reorientation mediated by a Ca2+-modulated contractile protein. J Cell Biol. 1987 Aug;105(2):903–912. doi: 10.1083/jcb.105.2.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Melkonian M. Ultrastructural aspects of basal body associated fibrous structures in green algae: a critical review. Biosystems. 1980;12(1-2):85–104. doi: 10.1016/0303-2647(80)90040-4. [DOI] [PubMed] [Google Scholar]
  26. O'Connor C. M., Gard D. L., Lazarides E. Phosphorylation of intermediate filament proteins by cAMP-dependent protein kinases. Cell. 1981 Jan;23(1):135–143. doi: 10.1016/0092-8674(81)90278-6. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Peattie D. A., Alonso R. A., Hein A., Caulfield J. P. Ultrastructural localization of giardins to the edges of disk microribbons of Giarida lamblia and the nucleotide and deduced protein sequence of alpha giardin. J Cell Biol. 1989 Nov;109(5):2323–2335. doi: 10.1083/jcb.109.5.2323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Peter M., Nakagawa J., Dorée M., Labbé J. C., Nigg E. A. In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell. 1990 May 18;61(4):591–602. doi: 10.1016/0092-8674(90)90471-p. [DOI] [PubMed] [Google Scholar]
  30. Ris H. The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections. J Cell Biol. 1985 May;100(5):1474–1487. doi: 10.1083/jcb.100.5.1474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Roberts T. M. Fine (2-5-nm) filaments: new types of cytoskeletal structures. Cell Motil Cytoskeleton. 1987;8(2):130–142. doi: 10.1002/cm.970080205. [DOI] [PubMed] [Google Scholar]
  32. Rubin R. W., Cunningham W. P. Partial purification and phosphotungstate solubilization of basal bodies and kinetodesmal fibers from Tetrahymena pyriformis. J Cell Biol. 1973 Jun;57(3):601–612. doi: 10.1083/jcb.57.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Salisbury J. L., Baron A. T., Sanders M. A. The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells. J Cell Biol. 1988 Aug;107(2):635–641. doi: 10.1083/jcb.107.2.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Salisbury J. L., Baron A., Surek B., Melkonian M. Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle. J Cell Biol. 1984 Sep;99(3):962–970. doi: 10.1083/jcb.99.3.962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Salisbury J. L., Floyd G. L. Calcium-induced contraction of the rhizoplast of a quadriflagellate green alga. Science. 1978 Dec 1;202(4371):975–977. doi: 10.1126/science.202.4371.975. [DOI] [PubMed] [Google Scholar]
  36. Salisbury J. L., Sanders M. A., Harpst L. Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii. J Cell Biol. 1987 Oct;105(4):1799–1805. doi: 10.1083/jcb.105.4.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sanders M. A., Salisbury J. L. Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii. J Cell Biol. 1989 May;108(5):1751–1760. doi: 10.1083/jcb.108.5.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sepsenwol S., Ris H., Roberts T. M. A unique cytoskeleton associated with crawling in the amoeboid sperm of the nematode, Ascaris suum. J Cell Biol. 1989 Jan;108(1):55–66. doi: 10.1083/jcb.108.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Steffen W., Linck R. W. Relationship between tektins and intermediate filament proteins: an immunological study. Cell Motil Cytoskeleton. 1989;14(3):359–371. doi: 10.1002/cm.970140306. [DOI] [PubMed] [Google Scholar]
  40. Stephens R. E. The basal apparatus. Mass isolation from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets. J Cell Biol. 1975 Feb;64(2):408–420. doi: 10.1083/jcb.64.2.408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stewart M., Quinlan R. A., Moir R. D. Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure. J Cell Biol. 1989 Jul;109(1):225–234. doi: 10.1083/jcb.109.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wang K., Ramirez-Mitchell R., Palter D. Titin is an extraordinarily long, flexible, and slender myofibrillar protein. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3685–3689. doi: 10.1073/pnas.81.12.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Williams N. E., Vaudaux P. E., Skriver L. Cytoskeletal proteins of the cell surface in Tetrahymena I. Identification and localization of major proteins. Exp Cell Res. 1979 Oct 15;123(2):311–320. doi: 10.1016/0014-4827(79)90473-7. [DOI] [PubMed] [Google Scholar]
  44. Wright R. L., Salisbury J., Jarvik J. W. A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation. J Cell Biol. 1985 Nov;101(5 Pt 1):1903–1912. doi: 10.1083/jcb.101.5.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES