Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Feb 2;132(4):643–655. doi: 10.1083/jcb.132.4.643

Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells

PMCID: PMC2199861  PMID: 8647895

Abstract

The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens- epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host- specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.

Full Text

The Full Text of this article is available as a PDF (4.7 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. Albers K., Fuchs E. The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines. J Cell Biol. 1987 Aug;105(2):791–806. doi: 10.1083/jcb.105.2.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brunkener M., Georgatos S. D. Membrane-binding properties of filensin, a cytoskeletal protein of the lens fiber cells. J Cell Sci. 1992 Nov;103(Pt 3):709–718. doi: 10.1242/jcs.103.3.709. [DOI] [PubMed] [Google Scholar]
  4. Ching G. Y., Liem R. K. Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments. J Cell Biol. 1993 Sep;122(6):1323–1335. doi: 10.1083/jcb.122.6.1323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Colucci-Guyon E., Portier M. M., Dunia I., Paulin D., Pournin S., Babinet C. Mice lacking vimentin develop and reproduce without an obvious phenotype. Cell. 1994 Nov 18;79(4):679–694. doi: 10.1016/0092-8674(94)90553-3. [DOI] [PubMed] [Google Scholar]
  6. Engel A., Eichner R., Aebi U. Polymorphism of reconstituted human epidermal keratin filaments: determination of their mass-per-length and width by scanning transmission electron microscopy (STEM). J Ultrastruct Res. 1985 Mar;90(3):323–335. doi: 10.1016/s0022-5320(85)80010-1. [DOI] [PubMed] [Google Scholar]
  7. Engel A. Molecular weight determination by scanning transmission electron microscopy. Ultramicroscopy. 1978;3(3):273–281. doi: 10.1016/s0304-3991(78)80037-0. [DOI] [PubMed] [Google Scholar]
  8. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. FitzGerald P. G., Graham D. Ultrastructural localization of alpha A-crystallin to the bovine lens fiber cell cytoskeleton. Curr Eye Res. 1991 May;10(5):417–436. doi: 10.3109/02713689109001750. [DOI] [PubMed] [Google Scholar]
  10. Fowler W. E., Aebi U. Preparation of single molecules and supramolecular complexes for high-resolution metal shadowing. J Ultrastruct Res. 1983 Jun;83(3):319–334. doi: 10.1016/s0022-5320(83)90139-9. [DOI] [PubMed] [Google Scholar]
  11. Fuchs E., Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem. 1994;63:345–382. doi: 10.1146/annurev.bi.63.070194.002021. [DOI] [PubMed] [Google Scholar]
  12. Georgatos S. D. Dynamics of intermediate filaments. Recent progress and unanswered questions. FEBS Lett. 1993 Mar 1;318(2):101–107. doi: 10.1016/0014-5793(93)80001-b. [DOI] [PubMed] [Google Scholar]
  13. Georgatos S. D., Maison C. Integration of intermediate filaments into cellular organelles. Int Rev Cytol. 1996;164:91–138. doi: 10.1016/s0074-7696(08)62385-2. [DOI] [PubMed] [Google Scholar]
  14. Gounari F., Merdes A., Quinlan R., Hess J., FitzGerald P. G., Ouzounis C. A., Georgatos S. D. Bovine filensin possesses primary and secondary structure similarity to intermediate filament proteins. J Cell Biol. 1993 May;121(4):847–853. doi: 10.1083/jcb.121.4.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heins S., Aebi U. Making heads and tails of intermediate filament assembly, dynamics and networks. Curr Opin Cell Biol. 1994 Feb;6(1):25–33. doi: 10.1016/0955-0674(94)90112-0. [DOI] [PubMed] [Google Scholar]
  16. Heins S., Wong P. C., Müller S., Goldie K., Cleveland D. W., Aebi U. The rod domain of NF-L determines neurofilament architecture, whereas the end domains specify filament assembly and network formation. J Cell Biol. 1993 Dec;123(6 Pt 1):1517–1533. doi: 10.1083/jcb.123.6.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Henderson D., Geisler N., Weber K. A periodic ultrastructure in intermediate filaments. J Mol Biol. 1982 Feb 25;155(2):173–176. doi: 10.1016/0022-2836(82)90444-2. [DOI] [PubMed] [Google Scholar]
  18. Hisanaga S., Hirokawa N. Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing. J Mol Biol. 1988 Jul 20;202(2):297–305. doi: 10.1016/0022-2836(88)90459-7. [DOI] [PubMed] [Google Scholar]
  19. Ireland M., Maisel H. Phosphorylation of chick lens proteins. Curr Eye Res. 1984 Jul;3(7):961–968. doi: 10.3109/02713688409167214. [DOI] [PubMed] [Google Scholar]
  20. Kaufmann E., Weber K., Geisler N. Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues. J Mol Biol. 1985 Oct 20;185(4):733–742. doi: 10.1016/0022-2836(85)90058-0. [DOI] [PubMed] [Google Scholar]
  21. Kouklis P. D., Hatzfeld M., Brunkener M., Weber K., Georgatos S. D. In vitro assembly properties of vimentin mutagenized at the beta-site tail motif. J Cell Sci. 1993 Nov;106(Pt 3):919–928. doi: 10.1242/jcs.106.3.919. [DOI] [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. Lee M. K., Xu Z., Wong P. C., Cleveland D. W. Neurofilaments are obligate heteropolymers in vivo. J Cell Biol. 1993 Sep;122(6):1337–1350. doi: 10.1083/jcb.122.6.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Masaki S., Tamai K., Shoji R., Watanabe T. Defect of a fiber cell-specific 94-kDa protein in the lens of inherited microphthalmic mutant mouse Elo. Biochem Biophys Res Commun. 1991 Sep 30;179(3):1175–1180. doi: 10.1016/0006-291x(91)91695-9. [DOI] [PubMed] [Google Scholar]
  25. Merdes A., Brunkener M., Horstmann H., Georgatos S. D. Filensin: a new vimentin-binding, polymerization-competent, and membrane-associated protein of the lens fiber cell. J Cell Biol. 1991 Oct;115(2):397–410. doi: 10.1083/jcb.115.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Merdes A., Gounari F., Georgatos S. D. The 47-kD lens-specific protein phakinin is a tailless intermediate filament protein and an assembly partner of filensin. J Cell Biol. 1993 Dec;123(6 Pt 1):1507–1516. doi: 10.1083/jcb.123.6.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Milam L., Erickson H. P. Visualization of a 21-nm axial periodicity in shadowed keratin filaments and neurofilaments. J Cell Biol. 1982 Sep;94(3):592–596. doi: 10.1083/jcb.94.3.592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nakamura Y., Takeda M., Aimoto S., Hariguchi S., Kitajima S., Nishimura T. Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain. Eur J Biochem. 1993 Mar 1;212(2):565–571. doi: 10.1111/j.1432-1033.1993.tb17694.x. [DOI] [PubMed] [Google Scholar]
  29. Nicholl I. D., Quinlan R. A. Chaperone activity of alpha-crystallins modulates intermediate filament assembly. EMBO J. 1994 Feb 15;13(4):945–953. doi: 10.1002/j.1460-2075.1994.tb06339.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Papamarcaki T., Kouklis P. D., Kreis T. E., Georgatos S. D. The "lamin B-fold". Anti-idiotypic antibodies reveal a structural complementarity between nuclear lamin B and cytoplasmic intermediate filament epitopes. J Biol Chem. 1991 Nov 5;266(31):21247–21251. [PubMed] [Google Scholar]
  31. Sarria A. J., Nordeen S. K., Evans R. M. Regulated expression of vimentin cDNA in cells in the presence and absence of a preexisting vimentin filament network. J Cell Biol. 1990 Aug;111(2):553–565. doi: 10.1083/jcb.111.2.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sauk J. J., Krumweide M., Cocking-Johnson D., White J. G. Reconstitution of cytokeratin filaments in vitro: further evidence for the role of nonhelical peptides in filament assembly. J Cell Biol. 1984 Nov;99(5):1590–1597. doi: 10.1083/jcb.99.5.1590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES