Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Sep 1;107(3):1037–1048. doi: 10.1083/jcb.107.3.1037

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study

PMCID: PMC2115304  PMID: 3417773

Abstract

In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brush-border cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the 110-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrootlet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actin-activated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and alpha-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the alpha-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and alpha- actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the 110-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.

Full Text

The Full Text of this article is available as a PDF (4.4 MB).

Selected References

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

  1. Bonder E. M., Mooseker M. S. Direct electron microscopic visualization of barbed end capping and filament cutting by intestinal microvillar 95-kdalton protein (villin): a new actin assembly assay using the Limulus acrosomal process. J Cell Biol. 1983 Apr;96(4):1097–1107. doi: 10.1083/jcb.96.4.1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bretscher A. Fimbrin is a cytoskeletal protein that crosslinks F-actin in vitro. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6849–6853. doi: 10.1073/pnas.78.11.6849. [DOI] [PMC free article] [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. Burgess D. R., Broschat K. O., Hayden J. M. Tropomyosin distinguishes between the two actin-binding sites of villin and affects actin-binding properties of other brush border proteins. J Cell Biol. 1987 Jan;104(1):29–40. doi: 10.1083/jcb.104.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burgess D. R., Prum B. E. Reevaluation of brush border motility: calcium induces core filament solution and microvillar vesiculation. J Cell Biol. 1982 Jul;94(1):97–107. doi: 10.1083/jcb.94.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burgess D. R. Reactivation of intestinal epithelial cell brush border motility: ATP-dependent contraction via a terminal web contractile ring. J Cell Biol. 1982 Dec;95(3):853–863. doi: 10.1083/jcb.95.3.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  11. Collins J. H., Borysenko C. W. The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase. J Biol Chem. 1984 Nov 25;259(22):14128–14135. [PubMed] [Google Scholar]
  12. Coluccio L. M., Bretscher A. Calcium-regulated cooperative binding of the microvillar 110K-calmodulin complex to F-actin: formation of decorated filaments. J Cell Biol. 1987 Jul;105(1):325–333. doi: 10.1083/jcb.105.1.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Conzelman K. A., Mooseker M. S. The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase. J Cell Biol. 1987 Jul;105(1):313–324. doi: 10.1083/jcb.105.1.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Coudrier E., Reggio H., Louvard D. The cytoskeleton of intestinal microvilli contains two polypeptides immunologically related to proteins of striated muscle. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):881–892. doi: 10.1101/sqb.1982.046.01.082. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Craig S. W., Powell L. D. Regulation of actin polymerization by villin, a 95,000 dalton cytoskeletal component of intestinal brush borders. Cell. 1980 Dec;22(3):739–746. doi: 10.1016/0092-8674(80)90550-4. [DOI] [PubMed] [Google Scholar]
  17. Dermietzel R., Leibstein A., Frixen U., Janssen-Timmen U., Traub O., Willecke K. Gap junctions in several tissues share antigenic determinants with liver gap junctions. EMBO J. 1984 Oct;3(10):2261–2270. doi: 10.1002/j.1460-2075.1984.tb02124.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Drenckhahn D., Bennett V. Polarized distribution of Mr 210,000 and 190,000 analogs of erythrocyte ankyrin along the plasma membrane of transporting epithelia, neurons and photoreceptors. Eur J Cell Biol. 1987 Jun;43(3):479–486. [PubMed] [Google Scholar]
  19. Drenckhahn D., Franz H. Identification of actin-, alpha-actinin-, and vinculin-containing plaques at the lateral membrane of epithelial cells. J Cell Biol. 1986 May;102(5):1843–1852. doi: 10.1083/jcb.102.5.1843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Drenckhahn D., Hofmann H. D., Mannherz H. G. Evidence for the association of villin with core filaments and rootlets of intestinal epithelial microvilli. Cell Tissue Res. 1983;228(2):409–414. doi: 10.1007/BF00204889. [DOI] [PubMed] [Google Scholar]
  22. Drenckhahn D., Merte C. Restriction of the human kidney band 3-like anion exchanger to specialized subdomains of the basolateral plasma membrane of intercalated cells. Eur J Cell Biol. 1987 Dec;45(1):107–115. [PubMed] [Google Scholar]
  23. Dudouet B., Robine S., Huet C., Sahuquillo-Merino C., Blair L., Coudrier E., Louvard D. Changes in villin synthesis and subcellular distribution during intestinal differentiation of HT29-18 clones. J Cell Biol. 1987 Jul;105(1):359–369. doi: 10.1083/jcb.105.1.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Geiger B., Dutton A. H., Tokuyasu K. T., Singer S. J. Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J Cell Biol. 1981 Dec;91(3 Pt 1):614–628. doi: 10.1083/jcb.91.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Glenney J. R., Jr, Glenney P. The microvillus 110K cytoskeletal protein is an integral membrane protein. Cell. 1984 Jul;37(3):743–751. doi: 10.1016/0092-8674(84)90410-0. [DOI] [PubMed] [Google Scholar]
  27. Glenney J. R., Jr, Glenney P., Weber K. Erythroid spectrin, brain fodrin, and intestinal brush border proteins (TW-260/240) are related molecules containing a common calmodulin-binding subunit bound to a variant cell type-specific subunit. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4002–4005. doi: 10.1073/pnas.79.13.4002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Glenney J. R., Jr, Glenney P., Weber K. The spectrin-related molecule, TW-260/240, cross-links the actin bundles of the microvillus rootlets in the brush borders of intestinal epithelial cells. J Cell Biol. 1983 May;96(5):1491–1496. doi: 10.1083/jcb.96.5.1491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Glenney J. R., Jr, Osborn M., Weber K. The intracellular localization of the microvillus 110K protein, a component considered to be involved in side-on membrane attachment of F-actin. Exp Cell Res. 1982 Mar;138(1):199–205. doi: 10.1016/0014-4827(82)90106-9. [DOI] [PubMed] [Google Scholar]
  30. Hagen S. J., Allan C. H., Trier J. S. Demonstration of microtubules in the terminal web of mature absorptive cells from the small intestine of the rat. Cell Tissue Res. 1987 Jun;248(3):709–711. doi: 10.1007/BF00216503. [DOI] [PubMed] [Google Scholar]
  31. Hirokawa N., Cheney R. E., Willard M. Location of a protein of the fodrin-spectrin-TW260/240 family in the mouse intestinal brush border. Cell. 1983 Mar;32(3):953–965. doi: 10.1016/0092-8674(83)90080-6. [DOI] [PubMed] [Google Scholar]
  32. Hirokawa N., Heuser J. E. Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J Cell Biol. 1981 Nov;91(2 Pt 1):399–409. doi: 10.1083/jcb.91.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hirokawa N., Keller T. C., 3rd, Chasan R., Mooseker M. S. Mechanism of brush border contractility studied by the quick-freeze, deep-etch method. J Cell Biol. 1983 May;96(5):1325–1336. doi: 10.1083/jcb.96.5.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hirokawa N., Tilney L. G., Fujiwara K., Heuser J. E. Organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells. J Cell Biol. 1982 Aug;94(2):425–443. doi: 10.1083/jcb.94.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Howe C. L., Mooseker M. S. Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells. J Cell Biol. 1983 Oct;97(4):974–985. doi: 10.1083/jcb.97.4.974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Howe C. L., Sacramone L. M., Mooseker M. S., Morrow J. S. Mechanisms of cytoskeletal regulation: modulation of membrane affinity in avian brush border and erythrocyte spectrins. J Cell Biol. 1985 Oct;101(4):1379–1385. doi: 10.1083/jcb.101.4.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Keller T. C., 3rd, Conzelman K. A., Chasan R., Mooseker M. S. Role of myosin in terminal web contraction in isolated intestinal epithelial brush borders. J Cell Biol. 1985 May;100(5):1647–1655. doi: 10.1083/jcb.100.5.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Koob R., Zimmermann M., Schoner W., Drenckhahn D. Colocalization and coprecipitation of ankyrin and Na+,K+-ATPase in kidney epithelial cells. Eur J Cell Biol. 1988 Feb;45(2):230–237. [PubMed] [Google Scholar]
  40. Langanger G., Moeremans M., Daneels G., Sobieszek A., De Brabander M., De Mey J. The molecular organization of myosin in stress fibers of cultured cells. J Cell Biol. 1986 Jan;102(1):200–209. doi: 10.1083/jcb.102.1.200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lecount T. S., Grey R. D. Transient shortening of microvilli induced by cycloheximide in the duodenal epithelium of the chicken. J Cell Biol. 1972 May;53(2):601–605. doi: 10.1083/jcb.53.2.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Madara J. L., Barenberg D., Carlson S. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol. 1986 Jun;102(6):2125–2136. doi: 10.1083/jcb.102.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Madara J. L. Intestinal absorptive cell tight junctions are linked to cytoskeleton. Am J Physiol. 1987 Jul;253(1 Pt 1):C171–C175. doi: 10.1152/ajpcell.1987.253.1.C171. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Matsudaira P. T., Burgess D. R. Organization of the cross-filaments in intestinal microvilli. J Cell Biol. 1982 Mar;92(3):657–664. doi: 10.1083/jcb.92.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Matsudaira P. T., Burgess D. R. Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca++-dependent, actin-bundling/depolymerizing protein. J Cell Biol. 1982 Mar;92(3):648–656. doi: 10.1083/jcb.92.3.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Matsudaira P., Mandelkow E., Renner W., Hesterberg L. K., Weber K. Role of fimbrin and villin in determining the interfilament distances of actin bundles. Nature. 1983 Jan 20;301(5897):209–214. doi: 10.1038/301209a0. [DOI] [PubMed] [Google Scholar]
  48. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  49. Mooseker M. S., Bonder E. M., Grimwade B. G., Howe C. L., Keller T. C., 3rd, Wasserman R. H., Wharton K. A. Regulation of contractility, cytoskeletal structure, and filament assembly in the brush border of intestinal epithelial cells. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):855–870. doi: 10.1101/sqb.1982.046.01.080. [DOI] [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., Graves T. A., Wharton K. A., Falco N., Howe C. L. Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells. J Cell Biol. 1980 Dec;87(3 Pt 1):809–822. doi: 10.1083/jcb.87.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Mooseker M. S. Organization, chemistry, and assembly of the cytoskeletal apparatus of the intestinal brush border. Annu Rev Cell Biol. 1985;1:209–241. doi: 10.1146/annurev.cb.01.110185.001233. [DOI] [PubMed] [Google Scholar]
  53. Mooseker M. S., Pollard T. D., Wharton K. A. Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border. J Cell Biol. 1982 Oct;95(1):223–233. doi: 10.1083/jcb.95.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Murphy D. B. Assembly-disassembly purification and characterization of microtubule protein without glycerol. Methods Cell Biol. 1982;24:31–49. doi: 10.1016/s0091-679x(08)60646-9. [DOI] [PubMed] [Google Scholar]
  56. Pollard T. D., Mooseker M. S. Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores. J Cell Biol. 1981 Mar;88(3):654–659. doi: 10.1083/jcb.88.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. Sandoz D., Lainé M. C., Nicolas G. Distribution of microtubules within the intestinal terminal web as revealed by quick-freezing and cryosubstitution. Eur J Cell Biol. 1986 Jan;39(2):481–484. [PubMed] [Google Scholar]
  59. Shibayama T., Carboni J. M., Mooseker M. S. Assembly of the intestinal brush border: appearance and redistribution of microvillar core proteins in developing chick enterocytes. J Cell Biol. 1987 Jul;105(1):335–344. doi: 10.1083/jcb.105.1.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Stidwill R. P., Burgess D. R. Regulation of intestinal brush border microvillus length during development by the G- to F-actin ratio. Dev Biol. 1986 Apr;114(2):381–388. doi: 10.1016/0012-1606(86)90202-2. [DOI] [PubMed] [Google Scholar]
  61. Talian J. C., Olmsted J. B., Goldman R. D. A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture. J Cell Biol. 1983 Oct;97(4):1277–1282. doi: 10.1083/jcb.97.4.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  63. Verner K., Bretscher A. Microvillus 110K-calmodulin: effects of nucleotides on isolated cytoskeletons and the interaction of the purified complex with F-actin. J Cell Biol. 1985 May;100(5):1455–1465. doi: 10.1083/jcb.100.5.1455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Volberg T., Geiger B., Kartenbeck J., Franke W. W. Changes in membrane-microfilament interaction in intercellular adherens junctions upon removal of extracellular Ca2+ ions. J Cell Biol. 1986 May;102(5):1832–1842. doi: 10.1083/jcb.102.5.1832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Walsh T. P., Weber A., Higgins J., Bonder E. M., Mooseker M. S. Effect of villin on the kinetics of actin polymerization. Biochemistry. 1984 Jun 5;23(12):2613–2621. doi: 10.1021/bi00307a012. [DOI] [PubMed] [Google Scholar]

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

RESOURCES