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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Jan;86(2):549–553. doi: 10.1073/pnas.86.2.549

Dynamic aspects of intermediate filament networks in BHK-21 cells.

K L Vikstrom 1, G G Borisy 1, R D Goldman 1
PMCID: PMC286509  PMID: 2643116

Abstract

A procedure was developed for the conjugation of vimentin with biotin. Biotinylated vimentin was then microinjected into BHK-21 cells and the fate of the labeled protein was determined at various times postinjection by indirect immunofluorescence. Microinjected vimentin could be traced through a specific sequence of morphological changes ultimately resulting in the formation of a filamentous network. The injected protein was first detected in spots dispersed throughout the cytoplasm. Subsequently, these spots appeared to cluster near the nucleus where they merged into a diffuse "cap." This cap coincided with a concentration of endogenous intermediate filaments and eventually gave rise to a filamentous network that was coincident with the endogenous intermediate filament network as determined by double-label immunofluorescence. The results indicate that the incorporation of exogenous vimentin into a filamentous network is initiated in a perinuclear region and progresses in a polarized fashion toward the cell surface.

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Selected References

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  1. 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]
  2. Blikstad I., Lazarides E. Vimentin filaments are assembled from a soluble precursor in avian erythroid cells. J Cell Biol. 1983 Jun;96(6):1803–1808. doi: 10.1083/jcb.96.6.1803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Eckert B. S., Daley R. A., Parysek L. M. Assembly of keratin onto PtK1 cytoskeletons: evidence for an intermediate filament organizing center. J Cell Biol. 1982 Feb;92(2):575–578. doi: 10.1083/jcb.92.2.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Franke W. W., Schmid E., Grund C., Geiger B. Intermediate filament proteins in nonfilamentous structures: transient disintegration and inclusion of subunit proteins in granular aggregates. Cell. 1982 Aug;30(1):103–113. doi: 10.1016/0092-8674(82)90016-2. [DOI] [PubMed] [Google Scholar]
  6. Franke W. W., Schmid E., Mittnacht S., Grund C., Jorcano J. L. Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells. Cell. 1984 Apr;36(4):813–825. doi: 10.1016/0092-8674(84)90031-x. [DOI] [PubMed] [Google Scholar]
  7. Goldman R. D., Follett E. A. Birefringent filamentous organelle in BHK-21 cells and its possible role in cell spreading and motility. Science. 1970 Jul 17;169(3942):286–288. doi: 10.1126/science.169.3942.286. [DOI] [PubMed] [Google Scholar]
  8. Goldman R. D., Goldman A. E., Green K. J., Jones J. C., Jones S. M., Yang H. Y. Intermediate filament networks: organization and possible functions of a diverse group of cytoskeletal elements. J Cell Sci Suppl. 1986;5:69–97. doi: 10.1242/jcs.1986.supplement_5.5. [DOI] [PubMed] [Google Scholar]
  9. Goldman R., Goldman A., Green K., Jones J., Lieska N., Yang H. Y. Intermediate filaments: possible functions as cytoskeletal connecting links between the nucleus and the cell surface. Ann N Y Acad Sci. 1985;455:1–17. doi: 10.1111/j.1749-6632.1985.tb50400.x. [DOI] [PubMed] [Google Scholar]
  10. Graessmann M., Graessman A. "Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication. Proc Natl Acad Sci U S A. 1976 Feb;73(2):366–370. doi: 10.1073/pnas.73.2.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Horwitz B., Kupfer H., Eshhar Z., Geiger B. Reorganization of arrays of prekeratin filaments during mitosis. Immunofluorescence microscopy with multiclonal and monoclonal prekeratin antibodies. Exp Cell Res. 1981 Aug;134(2):281–290. doi: 10.1016/0014-4827(81)90427-4. [DOI] [PubMed] [Google Scholar]
  12. Ip W., Hartzer M. K., Pang Y. Y., Robson R. M. Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments. J Mol Biol. 1985 Jun 5;183(3):365–375. doi: 10.1016/0022-2836(85)90007-5. [DOI] [PubMed] [Google Scholar]
  13. Jones J. C., Goldman A. E., Yang H. Y., Goldman R. D. The organizational fate of intermediate filament networks in two epithelial cell types during mitosis. J Cell Biol. 1985 Jan;100(1):93–102. doi: 10.1083/jcb.100.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Keith C. H., Feramisco J. R., Shelanski M. Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts. J Cell Biol. 1981 Jan;88(1):234–240. doi: 10.1083/jcb.88.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klymkowsky M. W., Miller R. H., Lane E. B. Morphology, behavior, and interaction of cultured epithelial cells after the antibody-induced disruption of keratin filament organization. J Cell Biol. 1983 Feb;96(2):494–509. doi: 10.1083/jcb.96.2.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kreis T. E., Birchmeier W. Microinjection of fluorescently labeled proteins into living cells with emphasis on cytoskeletal proteins. Int Rev Cytol. 1982;75:209–214. doi: 10.1016/s0074-7696(08)61005-0. [DOI] [PubMed] [Google Scholar]
  17. Kreis T. E., Geiger B., Schmid E., Jorcano J. L., Franke W. W. De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin. Cell. 1983 Apr;32(4):1125–1137. doi: 10.1016/0092-8674(83)90296-9. [DOI] [PubMed] [Google Scholar]
  18. Kreis T. E., Winterhalter K. H., Birchmeier W. In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3814–3818. doi: 10.1073/pnas.76.8.3814. [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. Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature. 1980 Jan 17;283(5744):249–256. doi: 10.1038/283249a0. [DOI] [PubMed] [Google Scholar]
  21. Lehto V. P., Virtanen I. Immunolocalization of a novel, cytoskeleton-associated polypeptide of Mr 230,000 daltons (p230). J Cell Biol. 1983 Mar;96(3):703–716. doi: 10.1083/jcb.96.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lieska N., Chen J., Maisel H., Romero-Herrera A. E. Subunit characterization of lens intermediate filaments. Biochim Biophys Acta. 1980 Nov 20;626(1):136–153. doi: 10.1016/0005-2795(80)90205-6. [DOI] [PubMed] [Google Scholar]
  23. Ngai J., Capetanaki Y. G., Lazarides E. Differentiation of murine erythroleukemia cells results in the rapid repression of vimentin gene expression. J Cell Biol. 1984 Jul;99(1 Pt 1):306–314. doi: 10.1083/jcb.99.1.306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paulin-Levasseur M., Brown D. L. Vimentin dynamics during the mitogenic stimulation of mouse splenic lymphocytes. Cell Motil Cytoskeleton. 1987;8(3):227–237. doi: 10.1002/cm.970080304. [DOI] [PubMed] [Google Scholar]
  25. Quax W., van den Broek L., Egberts W. V., Ramaekers F., Bloemendal H. Characterization of the hamster desmin gene: expression and formation of desmin filaments in nonmuscle cells after gene transfer. Cell. 1985 Nov;43(1):327–338. doi: 10.1016/0092-8674(85)90038-8. [DOI] [PubMed] [Google Scholar]
  26. Sammak P. J., Gorbsky G. J., Borisy G. G. Microtubule dynamics in vivo: a test of mechanisms of turnover. J Cell Biol. 1987 Mar;104(3):395–405. doi: 10.1083/jcb.104.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Saxton W. M., Stemple D. L., Leslie R. J., Salmon E. D., Zavortink M., McIntosh J. R. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schulze E., Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. doi: 10.1083/jcb.102.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Soellner P., Quinlan R. A., Franke W. W. Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7929–7933. doi: 10.1073/pnas.82.23.7929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Soltys B. J., Borisy G. G. Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes. J Cell Biol. 1985 May;100(5):1682–1689. doi: 10.1083/jcb.100.5.1682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Starger J. M., Brown W. E., Goldman A. E., Goldman R. D. Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells. J Cell Biol. 1978 Jul;78(1):93–109. doi: 10.1083/jcb.78.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Steinert P. M., Steven A. C., Roop D. R. The molecular biology of intermediate filaments. Cell. 1985 Sep;42(2):411–420. doi: 10.1016/0092-8674(85)90098-4. [DOI] [PubMed] [Google Scholar]
  33. Taylor D. L., Wang Y. L. Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells. Proc Natl Acad Sci U S A. 1978 Feb;75(2):857–861. doi: 10.1073/pnas.75.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tölle H. G., Weber K., Osborn M. Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments. Eur J Cell Biol. 1985 Sep;38(2):234–244. [PubMed] [Google Scholar]
  36. Yang H. Y., Lieska N., Goldman A. E., Goldman R. D. A 300,000-mol-wt intermediate filament-associated protein in baby hamster kidney (BHK-21) cells. J Cell Biol. 1985 Feb;100(2):620–631. doi: 10.1083/jcb.100.2.620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zackroff R. V., Goldman R. D. In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6226–6230. doi: 10.1073/pnas.76.12.6226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. van den Heuvel R. M., van Eys G. J., Ramaekers F. C., Quax W. J., Vree Egberts W. T., Schaart G., Cuypers H. T., Bloemendal H. Intermediate filament formation after transfection with modified hamster vimentin and desmin genes. J Cell Sci. 1987 Nov;88(Pt 4):475–482. doi: 10.1242/jcs.88.4.475. [DOI] [PubMed] [Google Scholar]

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