Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 Jul 1;90(1):116–127. doi: 10.1083/jcb.90.1.116

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border

PMCID: PMC2111844  PMID: 6166624

Abstract

Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-order fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1.5 M KCl) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of Mr 55,000, 48,000, and 40,000 was noted. On two- dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (Mr 48,000, and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the Mr 55,000 protein which focused at pH values higher than 6.4. The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins. Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell. The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue- specific patterns of their protein subunits.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Baden H. P., Lee L. D. Fibrous protein of human epidermis. J Invest Dermatol. 1978 Aug;71(2):148–151. doi: 10.1111/1523-1747.ep12546905. [DOI] [PubMed] [Google Scholar]
  2. Billington T., Nayudu P. R. Studies on the brush border membrane of the mouse duodenum. I. Membrane isolation and analysis of protein components. J Membr Biol. 1975 Apr 23;21(1-2):49–64. doi: 10.1007/BF01941061. [DOI] [PubMed] [Google Scholar]
  3. Bretscher A., Weber K. Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures. J Cell Biol. 1980 Jul;86(1):335–340. doi: 10.1083/jcb.86.1.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bretscher A., Weber K. Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy. J Cell Biol. 1978 Dec;79(3):839–845. doi: 10.1083/jcb.79.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bretscher A., Weber K. Purification of microvilli and an analysis of the protein components of the microfilament core bundle. Exp Cell Res. 1978 Oct 15;116(2):397–407. doi: 10.1016/0014-4827(78)90463-9. [DOI] [PubMed] [Google Scholar]
  6. Bretscher A., Weber K. Villin: the major microfilament-associated protein of the intestinal microvillus. Proc Natl Acad Sci U S A. 1979 May;76(5):2321–2325. doi: 10.1073/pnas.76.5.2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bruder G., Bretscher A., Franke W. W., Jarasch E. D. Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420. Biochim Biophys Acta. 1980 Aug 14;600(3):739–755. doi: 10.1016/0005-2736(80)90477-0. [DOI] [PubMed] [Google Scholar]
  8. Brunser O., Luft H. J. Fine structure of the apex of absorptive cell from rat small intestine. J Ultrastruct Res. 1970 May;31(3):291–311. doi: 10.1016/s0022-5320(70)90133-4. [DOI] [PubMed] [Google Scholar]
  9. Brysk M. M., Gray R. H., Bernstein I. A. Tonofilament protein from newborn rat epidermis. Isolation, localization, and biosynthesis of marker of epidermal differentiation. J Biol Chem. 1977 Mar 25;252(6):2127–2133. [PubMed] [Google Scholar]
  10. Craig S. W., Lancashire C. L. Comparison of intestinal brush-border 95-Kdalton polypeptide and alpha-actinins. J Cell Biol. 1980 Mar;84(3):655–667. doi: 10.1083/jcb.84.3.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Craig S. W., Pardo J. V. alpha-Actinin localization in the junctional complex of intestinal epithelial cells. J Cell Biol. 1979 Jan;80(1):203–210. doi: 10.1083/jcb.80.1.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Robertis E. M., Partington G. A., Longthorne R. F., Gurdon J. B. Somatic nuclei in amphibian oocytes: evidence for selective gene expression. J Embryol Exp Morphol. 1977 Aug;40:199–214. [PubMed] [Google Scholar]
  13. Drenckhahn D., Gröschel-Stewart U. Localization of myosin, actin, and tropomyosin in rat intestinal epithelium: immunohistochemical studies at the light and electron microscope levels. J Cell Biol. 1980 Aug;86(2):475–482. doi: 10.1083/jcb.86.2.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Drochmans P., Freudenstein C., Wanson J. C., Laurent L., Keenan T. W., Stadler J., Leloup R., Franke W. W. Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis. J Cell Biol. 1978 Nov;79(2 Pt 1):427–443. doi: 10.1083/jcb.79.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Evans E. M., Wrigglesworth J. M., Burdett K., Pover W. F. Studies on epithelial cells isolated from guinea pig small intestine. J Cell Biol. 1971 Nov;51(21):452–464. doi: 10.1083/jcb.51.2.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. FARQUHAR M. G., PALADE G. E. Junctional complexes in various epithelia. J Cell Biol. 1963 May;17:375–412. doi: 10.1083/jcb.17.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Forstner G. G., Sabesin S. M., Isselbacher K. J. Rat intestinal microvillus membranes. Purification and biochemical characterization. Biochem J. 1968 Jan;106(2):381–390. doi: 10.1042/bj1060381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Franke W. W., Appelhans B., Schmid E., Freudenstein C., Osborn M., Weber K. Identification and characterization of epithelial cells in mammalian tissues by immunofluorescence microscopy using antibodies to prekeratin. Differentiation. 1979;15(1):7–25. doi: 10.1111/j.1432-0436.1979.tb01030.x. [DOI] [PubMed] [Google Scholar]
  19. Franke W. W., Appelhans B., Schmid E., Freudenstein C., Osborn M., Weber K. The organization of cytokeratin filaments in the intestinal epithelium. Eur J Cell Biol. 1979 Aug;19(3):255–268. [PubMed] [Google Scholar]
  20. Franke W. W., Denk H., Kalt R., Schmid E. Biochemical and immunological identification of cytokeratin proteins present in hepatocytes of mammalian liver tissue. Exp Cell Res. 1981 Feb;131(2):299–318. doi: 10.1016/0014-4827(81)90234-2. [DOI] [PubMed] [Google Scholar]
  21. Franke W. W., Schmid E., Breitkreutz D., Lüder M., Boukamp P., Fusenig N. E., Osborn M., Weber K. Simultaneous expression of two different types of intermediate sized filaments in mouse keratinocytes proliferating in vitro. Differentiation. 1979;14(1-2):35–50. doi: 10.1111/j.1432-0436.1979.tb01010.x. [DOI] [PubMed] [Google Scholar]
  22. Franke W. W., Schmid E., Freudenstein C., Appelhans B., Osborn M., Weber K., Keenan T. W. Intermediate-sized filaments of the prekeratin type in myoepithelial cells. J Cell Biol. 1980 Mar;84(3):633–654. doi: 10.1083/jcb.84.3.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Franke W. W., Schmid E., Osborn M., Weber K. Different intermediate-sized filaments distinguished by immunofluorescence microscopy. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5034–5038. doi: 10.1073/pnas.75.10.5034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Franke W. W., Schmid E., Osborn M., Weber K. Intermediate-sized filaments of human endothelial cells. J Cell Biol. 1979 Jun;81(3):570–580. doi: 10.1083/jcb.81.3.570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Franke W. W., Schmid E., Osborn M., Weber K. The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments. Cytobiologie. 1978 Aug;17(2):392–411. [PubMed] [Google Scholar]
  26. Franke W. W., Schmid E., Vandekerckhove J., Weber K. Permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type. J Cell Biol. 1980 Dec;87(3 Pt 1):594–600. doi: 10.1083/jcb.87.3.594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Franke W. W., Schmid E., Weber K., Osborn M. HeLa cells contain intermediate-sized filaments of the prekeratin type. Exp Cell Res. 1979 Jan;118(1):95–109. doi: 10.1016/0014-4827(79)90587-1. [DOI] [PubMed] [Google Scholar]
  28. Franke W. W., Schmid E., Winter S., Osborn M., Weber K. Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates. Exp Cell Res. 1979 Oct 1;123(1):25–46. doi: 10.1016/0014-4827(79)90418-x. [DOI] [PubMed] [Google Scholar]
  29. Franke W. W., Weber K., Osborn M., Schmid E., Freudenstein C. Antibody to prekeratin. Decoration of tonofilament like arrays in various cells of epithelial character. Exp Cell Res. 1978 Oct 15;116(2):429–445. doi: 10.1016/0014-4827(78)90466-4. [DOI] [PubMed] [Google Scholar]
  30. Freudenstein C., Franke W. W., Osborn M., Weber K. Reaction of tonofilament-like intermediate-sized filaments with antibodies raised against isolated defined polypeptides of bovine hoof prekeratin. Cell Biol Int Rep. 1978 Nov;2(6):591–600. doi: 10.1016/0309-1651(78)90068-1. [DOI] [PubMed] [Google Scholar]
  31. Fuchs E., Green H. Changes in keratin gene expression during terminal differentiation of the keratinocyte. Cell. 1980 Apr;19(4):1033–1042. doi: 10.1016/0092-8674(80)90094-x. [DOI] [PubMed] [Google Scholar]
  32. Fuchs E., Green H. Multiple keratins of cultured human epidermal cells are translated from different mRNA molecules. Cell. 1979 Jul;17(3):573–582. doi: 10.1016/0092-8674(79)90265-4. [DOI] [PubMed] [Google Scholar]
  33. Fuchs E., Green H. The expression of keratin genes in epidermis and cultured epidermal cells. Cell. 1978 Nov;15(3):887–897. doi: 10.1016/0092-8674(78)90273-8. [DOI] [PubMed] [Google Scholar]
  34. Gard D. L., Bell P. B., Lazarides E. Coexistence of desmin and the fibroblastic intermediate filament subunit in muscle and nonmuscle cells: identification and comparative peptide analysis. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3894–3898. doi: 10.1073/pnas.76.8.3894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Geiger B., Tokuyasu K. T., Dutton A. H., Singer S. J. Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4127–4131. doi: 10.1073/pnas.77.7.4127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Geiger B., Tokuyasu K. T., Singer S. J. Immunocytochemical localization of alpha-actinin in intestinal epithelial cells. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2833–2837. doi: 10.1073/pnas.76.6.2833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Gipson I. K., Anderson R. A. Comparison of 10 nm filaments from three bovine tissues. Exp Cell Res. 1980 Aug;128(2):395–406. doi: 10.1016/0014-4827(80)90075-0. [DOI] [PubMed] [Google Scholar]
  38. Howe C. L., Mooseker M. S., Graves T. A. Brush-border calmodulin. A major component of the isolated microvillus core. J Cell Biol. 1980 Jun;85(3):916–923. doi: 10.1083/jcb.85.3.916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Hubbard B. D., Lazarides E. Copurification of actin and desmin from chicken smooth muscle and their copolymerization in vitro to intermediate filaments. J Cell Biol. 1979 Jan;80(1):166–182. doi: 10.1083/jcb.80.1.166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Hull B. E., Staehelin L. A. The terminal web. A reevaluation of its structure and function. J Cell Biol. 1979 Apr;81(1):67–82. doi: 10.1083/jcb.81.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Jackson B. W., Grund C., Schmid E., Bürki K., Franke W. W., Illmensee K. Formation of cytoskeletal elements during mouse embryogenesis. Intermediate filaments of the cytokeratin type and desmosomes in preimplantation embryos. Differentiation. 1980;17(3):161–179. doi: 10.1111/j.1432-0436.1980.tb01093.x. [DOI] [PubMed] [Google Scholar]
  42. Kelly P. T., Cotman C. W. Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide. J Cell Biol. 1978 Oct;79(1):173–183. doi: 10.1083/jcb.79.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Lazarides E., Weber K. Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2268–2272. doi: 10.1073/pnas.71.6.2268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Lee L. D., Baden H. P. Organisation of the polypeptide chains in mammalian keratin. Nature. 1976 Nov 25;264(5584):377–379. doi: 10.1038/264377a0. [DOI] [PubMed] [Google Scholar]
  47. Lee L. D., Kubilus J., Baden H. P. Intraspecies heterogeneity of epidermal keratins isolated from bovine hoof and snout. Biochem J. 1979 Jan 1;177(1):187–196. doi: 10.1042/bj1770187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Matoltsy A. G. Desmosomes, filaments, and keratohyaline granules: their role in the stabilization and keratinization of the epidermis. J Invest Dermatol. 1975 Jul;65(1):127–142. doi: 10.1111/1523-1747.ep12598093. [DOI] [PubMed] [Google Scholar]
  49. Matsudaira P. T., Burgess D. R. Identification and organization of the components in the isolated microvillus cytoskeleton. J Cell Biol. 1979 Dec;83(3):667–673. doi: 10.1083/jcb.83.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Mooseker M. S. Brush border motility. Microvillar contraction in triton-treated brush borders isolated from intestinal epithelium. J Cell Biol. 1976 Nov;71(2):417–433. doi: 10.1083/jcb.71.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Mooseker M. S., Pollard T. D., Fujiwara K. Characterization and localization of myosin in the brush border of intestinal epithelial cells. J Cell Biol. 1978 Nov;79(2 Pt 1):444–453. doi: 10.1083/jcb.79.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Mooseker M. S., Stephens R. E. Brush-border alpha-actinin? Comparison of two proteins of the microvillus core with alpha-actinin by two-dimensional peptide mapping. J Cell Biol. 1980 Aug;86(2):466–474. doi: 10.1083/jcb.86.2.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Renart J., Reiser J., Stark G. R. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3116–3120. doi: 10.1073/pnas.76.7.3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Rodewald R., Newman S. B., Karnovsky M. J. Contraction of isolated brush borders from the intestinal epithelium. J Cell Biol. 1976 Sep;70(3):541–554. doi: 10.1083/jcb.70.3.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Skerrow C. J., Matoltsy A. G. Chemical characterization of isolated epidermal desmosomes. J Cell Biol. 1974 Nov;63(2 Pt 1):524–530. doi: 10.1083/jcb.63.2.524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Skerrow C. J., Matoltsy A. G. Isolation of epidermal desmosomes. J Cell Biol. 1974 Nov;63(2 Pt 1):515–523. doi: 10.1083/jcb.63.2.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Skerrow D., Matoltsy A. G., Matoltsy M. N. Isolation and characterization of the helical regions of epidermal prekeratin. J Biol Chem. 1973 Jul 10;248(13):4820–4826. [PubMed] [Google Scholar]
  60. Staehelin L. A. Structure and function of intercellular junctions. Int Rev Cytol. 1974;39:191–283. doi: 10.1016/s0074-7696(08)60940-7. [DOI] [PubMed] [Google Scholar]
  61. Steinert P. M., Idler W. W., Goldman R. D. Intermediate filaments of baby hamster kidney (BHK-21) cells and bovine epidermal keratinocytes have similar ultrastructures and subunit domain structures. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4534–4538. doi: 10.1073/pnas.77.8.4534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Steinert P. M., Idler W. W., Zimmerman S. B. Self-assembly of bovine epidermal keratin filaments in vitro. J Mol Biol. 1976 Dec 15;108(3):547–567. doi: 10.1016/s0022-2836(76)80136-2. [DOI] [PubMed] [Google Scholar]
  63. Steinert P. M. Structure of the three-chain unit of the bovine epidermal keratin filament. J Mol Biol. 1978 Jul 25;123(1):49–70. doi: 10.1016/0022-2836(78)90376-5. [DOI] [PubMed] [Google Scholar]
  64. Sun T. T., Green H. Immunofluorescent staining of keratin fibers in cultured cells. Cell. 1978 Jul;14(3):469–476. doi: 10.1016/0092-8674(78)90233-7. [DOI] [PubMed] [Google Scholar]
  65. Sun T. T., Green H. Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation. J Biol Chem. 1978 Mar 25;253(6):2053–2060. [PubMed] [Google Scholar]
  66. Sun T. T., Shih C., Green H. Keratin cytoskeletons in epithelial cells of internal organs. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2813–2817. doi: 10.1073/pnas.76.6.2813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. 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]
  68. Weber K., Bibring T., Osborn M. Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence. Cytoplasmic microtubules, vinblastine-induced paracrystals, and mitotic figures. Exp Cell Res. 1975 Oct 1;95(1):111–120. doi: 10.1016/0014-4827(75)90615-1. [DOI] [PubMed] [Google Scholar]
  69. Weber K., Osborn M., Franke W. W. Antibodies against merokeratin from sheep wool decorate cytokeratin filaments in non-keratinizing epithelial cells. Eur J Cell Biol. 1980 Dec;23(1):110–114. [PubMed] [Google Scholar]

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

RESOURCES