Home LiteratureArticle Details
PMID: 1709167 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Keratin incorporation into intermediate filament networks is a rapid process.

The Journal of cell biology ·Vol. 113 ·No. 4 ·1991-05-00 ·Pages 843-55

Miller RK, Vikstrom K, Goldman RD

Abstract

The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements.

MeSH Terms
Animals Biotin Cell Line Cricetinae Dipodomys Fluorescent Antibody Technique In Vitro Techniques Intermediate Filaments/metabolism,ultrastructure Keratinocytes/metabolism,ultrastructure Keratins/chemistry,metabolism Mice Microinjections Time Factors
Chemicals
Keratins Biotin
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Miller R K
Northwestern University Medical School, Department of Cell, Molecular, and Structural Biology, Chicago, Illinois 60611.
Vikstrom K
Goldman R D
References (75)
75 references, click to expand
  1. Dynamic aspects of the supramolecular organization of intermediate filament networks in cultured epidermal cells.
    Cell Motil. 1982;2(3):197-213 PMID: 6756644
  2. The structure and function of spot desmosomes.
    Int J Dermatol. 1981 Jun;20(5):330-9 PMID: 7016773
  3. Organization and spatial arrangement of fluorescein-labeled native actin microinjected into normal locomoting and experimentally influenced Amoeba proteus.
    Cell Tissue Res. 1980;206(2):181-91 PMID: 6893014
  4. Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery.
    Cell. 1982 Jul;29(3):835-45 PMID: 6891291
  5. The use of the avidin-biotin complex as a tool in molecular biology.
    Methods Biochem Anal. 1980;26:1-45 PMID: 7392958
  6. 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):4350-4 PMID: 388439
  7. The intermediate-sized filaments in rat kangaroo PtK2 cells. I. Morphology in situ.
    Cytobiologie. 1978 Aug;17(2):365-91 PMID: 357224
  8. Immunofluorescent staining of keratin fibers in cultured cells.
    Cell. 1978 Jul;14(3):469-76 PMID: 357009
  9. Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells.
    Proc Natl Acad Sci U S A. 1978 Feb;75(2):857-61 PMID: 345279
  10. The organizational fate of intermediate filament networks in two epithelial cell types during mitosis.
    J Cell Biol. 1985 Jan;100(1):93-102 PMID: 2578129
  11. Molecular and cellular biology of intermediate filaments.
    Annu Rev Biochem. 1988;57:593-625 PMID: 3052284
  12. Expression of mutant keratin cDNAs in epithelial cells reveals possible mechanisms for initiation and assembly of intermediate filaments.
    J Cell Biol. 1989 Apr;108(4):1477-93 PMID: 2466849
  13. Fractionation of desmosomes and comparison of the polypeptide composition of desmosomes prepared from two bovine epithelial tissues.
    J Cell Biochem. 1988 Mar;36(3):223-36 PMID: 2454237
  14. 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 PMID: 2442174
  15. Turnover of cytokeratin polypeptides in mouse hepatocytes.
    Exp Cell Res. 1987 Nov;173(1):137-43 PMID: 2445591
  16. The transfection of epidermal keratin genes into fibroblasts and simple epithelial cells: evidence for inducing a type I keratin by a type II gene.
    Cell. 1987 Feb 13;48(3):453-63 PMID: 2433047
  17. The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments.
    J Cell Biol. 1986 May;102(5):1767-77 PMID: 2422179
  18. Organization of cytokeratin bundles by desmosomes in rat mammary cells.
    J Cell Biol. 1986 Feb;102(2):560-7 PMID: 2418033
  19. Structural and functional implications of amino acid sequences of keratin intermediate filament subunits.
    Ann N Y Acad Sci. 1985;455:451-61 PMID: 2417526
  20. Relation of the intermediate filament distribution center to keratin filament dynamics in vivo: cells at the edge of an experimental wound.
    Ann N Y Acad Sci. 1985;455:343-53 PMID: 2417520
  21. Amino acid sequences of mouse and human epidermal type II keratins of Mr 67,000 provide a systematic basis for the structural and functional diversity of the end domains of keratin intermediate filament subunits.
    J Biol Chem. 1985 Jun 10;260(11):7142-9 PMID: 2581964
  22. The coiled-coil molecules of intermediate filaments consist of two parallel chains in exact axial register.
    Biochem Biophys Res Commun. 1985 Mar 29;127(3):1012-8 PMID: 2580517
  23. Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides.
    J Cell Biol. 1985 Nov;101(5 Pt 1):1826-41 PMID: 2414304
  24. Expression of rat neurofilament proteins NF-L and NF-M in transfected non-neuronal cells.
    Eur J Cell Biol. 1989 Dec;50(2):475-90 PMID: 2516804
  25. Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study.
    J Cell Biol. 1989 Oct;109(4 Pt 1):1581-95 PMID: 2677022
  26. Visualization of intermediate filaments in living cells using fluorescently labeled desmin.
    Cell Motil Cytoskeleton. 1989;12(3):127-38 PMID: 2653644
  27. Phosphorylation and disassembly of intermediate filaments in mitotic cells.
    Proc Natl Acad Sci U S A. 1989 Mar;86(6):1885-9 PMID: 2648386
  28. Intermediate filaments: known structure, unknown function.
    Biochim Biophys Acta. 1989 Apr 12;1007(3):245-53 PMID: 2649151
  29. Dynamic aspects of intermediate filament networks in BHK-21 cells.
    Proc Natl Acad Sci U S A. 1989 Jan;86(2):549-53 PMID: 2643116
  30. Progressive and spatially differentiated stability of microtubules in developing neuronal cells.
    J Cell Biol. 1989 Jul;109(1):253-63 PMID: 2745551
  31. Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures.
    J Cell Biol. 1989 Apr;108(4):1495-506 PMID: 2925792
  32. Biotin stores in rodent lungs: localization to Clara and type II alveolar cells.
    Exp Lung Res. 1988;14(4):527-36 PMID: 3208717
  33. Microtubule dynamics in vivo: a test of mechanisms of turnover.
    J Cell Biol. 1987 Mar;104(3):395-405 PMID: 3546333
  34. Microtubule dynamics in interphase cells.
    J Cell Biol. 1986 Mar;102(3):1020-31 PMID: 3512576
  35. Sites of microtubule assembly and disassembly in the mitotic spindle.
    Cell. 1986 May 23;45(4):515-27 PMID: 3708686
  36. Probing the mechanism of incorporation of fluorescently labeled actin into stress fibers.
    J Cell Biol. 1986 Mar;102(3):1074-84 PMID: 3949874
  37. Intermediate filaments: possible functions as cytoskeletal connecting links between the nucleus and the cell surface.
    Ann N Y Acad Sci. 1985;455:1-17 PMID: 3909878
  38. Intermediate filaments and the initiation of desmosome assembly.
    J Cell Biol. 1985 Aug;101(2):506-17 PMID: 3894376
  39. In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells.
    Proc Natl Acad Sci U S A. 1979 Dec;76(12):6226-30 PMID: 293716
  40. Self-assembly of bovine epidermal keratin filaments in vitro.
    J Mol Biol. 1976 Dec 15;108(3):547-67 PMID: 1018318
  41. The polypeptide composition of bovine epidermal alpha-keratin.
    Biochem J. 1975 Dec;151(3):603-14 PMID: 56175
  42. Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro.
    Exp Cell Res. 1974 May;86(1):95-105 PMID: 4857507
  43. 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;72:248-54 PMID: 942051
  44. The extraction and characterization of bovine epidermal alpha-keratin.
    Biochem J. 1975 Jul;149(1):39-48 PMID: 1191265
  45. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  46. Analyzing dynamic properties of intermediate filaments.
    Methods Enzymol. 1991;196:506-25 PMID: 1709715
  47. Dynamic properties of intermediate filaments: disassembly and reassembly during mitosis in baby hamster kidney cells.
    Cell Motil Cytoskeleton. 1990;17(3):150-66 PMID: 2268873
  48. Coumarin-phalloidin: a new actin probe permitting triple immunofluorescence microscopy of the cytoskeleton.
    J Cell Sci. 1988 Jan;89 ( Pt 1):21-4 PMID: 2458368
  49. Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments.
    J Cell Biol. 1987 Jul;105(1):105-15 PMID: 3038923
  50. Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes.
    J Cell Biol. 1985 May;100(5):1682-9 PMID: 3886672
  51. A 300,000-mol-wt intermediate filament-associated protein in baby hamster kidney (BHK-21) cells.
    J Cell Biol. 1985 Feb;100(2):620-31 PMID: 3881459
  52. Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling.
    J Cell Biol. 1985 Aug;101(2):597-602 PMID: 4040521
  53. 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 PMID: 2188731
  54. The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression.
    J Cell Biol. 1990 Apr;110(4):1199-210 PMID: 1691189
  55. 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):553-65 PMID: 1696263
  56. Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: specific domain functions in keratin stabilization and filament formation.
    Cell. 1990 Aug 24;62(4):681-96 PMID: 1696851
  57. Elucidating the early stages of keratin filament assembly.
    J Cell Biol. 1990 Jul;111(1):153-69 PMID: 1694855
  58. The two-chain coiled-coil molecule of native epidermal keratin intermediate filaments is a type I-type II heterodimer.
    J Biol Chem. 1990 May 25;265(15):8766-74 PMID: 1692836
  59. Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin.
    Cell. 1990 Sep 21;62(6):1063-71 PMID: 2169348
  60. Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly.
    Cell. 1990 Feb 9;60(3):415-27 PMID: 2406021
  61. Disruption of the keratin filament network during epithelial cell division.
    EMBO J. 1982;1(11):1365-72 PMID: 6202508
  62. Fluorescently labelled molecules as probes of the structure and function of living cells.
    Nature. 1980 Apr 3;284(5755):405-10 PMID: 6987537
  63. 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):813-25 PMID: 6200235
  64. Reorganization of arrays of prekeratin filaments during mitosis. Immunofluorescence microscopy with multiclonal and monoclonal prekeratin antibodies.
    Exp Cell Res. 1981 Aug;134(2):281-90 PMID: 6168474
  65. Heterogeneity of intermediate filaments assembled in vitro.
    Cold Spring Harb Symp Quant Biol. 1982;46 Pt 1:465-74 PMID: 6179700
  66. Two distinct classes of keratin genes and their evolutionary significance.
    Cell. 1981 Nov;27(1 Pt 2):75-84 PMID: 6173133
  67. Intermediate filaments of the vimentin-type and the cytokeratin-type are distributed differently during mitosis.
    Exp Cell Res. 1980 Sep;129(1):149-65 PMID: 6159217
  68. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.
    Cell. 1982 Nov;31(1):11-24 PMID: 6186379
  69. Type I and type II keratins have evolved from lower eukaryotes to form the epidermal intermediate filaments in mammalian skin.
    Proc Natl Acad Sci U S A. 1983 Oct;80(19):5857-61 PMID: 6193525
  70. De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin.
    Cell. 1983 Apr;32(4):1125-37 PMID: 6188536
  71. Microinjection of fluorescent tubulin into dividing sea urchin cells.
    J Cell Biol. 1983 Oct;97(4):1249-54 PMID: 6684663
  72. Pattern and time course of rhodamine-actin incorporation in cardiac myocytes.
    J Cell Biol. 1983 Apr;96(4):1164-7 PMID: 6833396
  73. Intermediate filament proteins in nonfilamentous structures: transient disintegration and inclusion of subunit proteins in granular aggregates.
    Cell. 1982 Aug;30(1):103-13 PMID: 6751555
  74. Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression.
    J Cell Biol. 1984 Apr;98(4):1388-96 PMID: 6201491
  75. Synthetic peptides corresponding to keratin subunits elicit highly specific antibodies.
    J Biol Chem. 1984 Jul 10;259(13):8037-40 PMID: 6203901
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.113.4.843
Published
1991-05-00
Pages
843-55
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2288995
Subset
IM
Grants
PHS HHS · 36806-06 · United States
External Links
PubMed source DOI: 10.1083/jcb.113.4.843 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2