Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Jan 1;98(1):66–78. doi: 10.1083/jcb.98.1.66

Studies on the spectrin-like protein from the intestinal brush border, TW 260/240, and characterization of its interaction with the cytoskeleton and actin

PMCID: PMC2112984  PMID: 6538573

Abstract

The terminal web of the intestinal brush border contains a spectrin- like protein, TW 260/240 (Glenney, J. R., Jr., P. Glenney, M. Osborne, and K. Weber, 1982, Cell, 28:843-854.) that interconnects the "rootlet" ends of microvillar filament bundles in the terminal web (Hirokawa, N., R. E. Cheng, and M. Willard, 1983, Cell, 32:953-965; Glenney J. R., P. Glenney, and K. Weber, 1983, J. Cell Biol., 96:1491-1496). We have investigated further the structural properties of TW 260/240 and the interaction of this protein with actin. Salt extraction of TW 260/240 from isolated brush borders results in a loss of terminal web cross- linkers primarily from the apical zone directly beneath the plasma membrane. Morphological studies on purified TW 260/240 using the rotary shadowing technique confirm earlier results that this protein is spectrin-like and is in the tetrameric state in buffers of low ionic strength. However, examination of TW 260/240 tetramers by negative staining revealed a molecule much straighter and more uniform in diameter than rotary-shadowed molecules. At salt concentrations at (150 mM KCl) and above (300 mM KCl) the physiological range, we observed a partial dissociation of tetramers into dimers that occurred at both 0 degree and 37 degrees C. We also observed (in the presence of 75 mM KCl) a concentration-dependent self-association of TW 260/240 into sedimentable aggregates. We have studied the interaction of TW 260/240 with actin using techniques of co-sedimentation, viscometry, and both light and electron microscopy. We observed that TW 260/240 can bind and cross-link actin filaments and that this interaction is salt- and pH- dependent. Under optimum conditions (25-75 mM KCl, at pH 7.0) TW 260/240 cross-linked F-actin into long, large-diameter bundles. The filaments within these bundles were tightly packed but loosely ordered. At higher pH (7.5) such bundles were not observed, although binding and cross-linking were detectable by co-sedimentation and viscometry. At higher salt (greater than 150 mM KCl), the binding of TW 260/240 to actin was inhibited. The presence of skeletal muscle tropomyosin had no significant effect on the salt-dependent binding of TW 260/240 to F- actin.

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. Abrahamson D. R., Rodewald R. Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine. J Cell Biol. 1981 Oct;91(1):270–280. doi: 10.1083/jcb.91.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailey K. Tropomyosin: a new asymmetric protein component of the muscle fibril. Biochem J. 1948;43(2):271–279. doi: 10.1042/bj0430271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Begg D. A., Rodewald R., Rebhun L. I. The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments. J Cell Biol. 1978 Dec;79(3):846–852. doi: 10.1083/jcb.79.3.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bennett G. Migration of glycoprotein from golgi apparatus to cell coat in the columnar cells of the duodenal epithelium. J Cell Biol. 1970 Jun;45(3):668–673. doi: 10.1083/jcb.45.3.668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bennett V., Davis J., Fowler W. E. Brain spectrin, a membrane-associated protein related in structure and function to erythrocyte spectrin. Nature. 1982 Sep 9;299(5879):126–131. doi: 10.1038/299126a0. [DOI] [PubMed] [Google Scholar]
  6. Bennett V., Stenbuck P. J. Identification and partial purification of ankyrin, the high affinity membrane attachment site for human erythrocyte spectrin. J Biol Chem. 1979 Apr 10;254(7):2533–2541. [PubMed] [Google Scholar]
  7. Branton D., Cohen C. M., Tyler J. Interaction of cytoskeletal proteins on the human erythrocyte membrane. Cell. 1981 Apr;24(1):24–32. doi: 10.1016/0092-8674(81)90497-9. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Bretscher A., Weber K. Villin is a major protein of the microvillus cytoskeleton which binds both G and F actin in a calcium-dependent manner. Cell. 1980 Jul;20(3):839–847. doi: 10.1016/0092-8674(80)90330-x. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Burridge K., Kelly T., Mangeat P. Nonerythrocyte spectrins: actin-membrane attachment proteins occurring in many cell types. J Cell Biol. 1982 Nov;95(2 Pt 1):478–486. doi: 10.1083/jcb.95.2.478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DeRosier D. J., Tilney L. G. How actin filaments pack into bundles. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):525–540. doi: 10.1101/sqb.1982.046.01.049. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Eisenberg E., Kielley W. W. Troponin-tropomyosin complex. Column chromatographic separation and activity of the three, active troponin components with and without tropomyosin present. J Biol Chem. 1974 Aug 10;249(15):4742–4748. [PubMed] [Google Scholar]
  16. Glenney J. R., Jr, Glenney P., Osborn M., Weber K. An F-actin- and calmodulin-binding protein from isolated intestinal brush borders has a morphology related to spectrin. Cell. 1982 Apr;28(4):843–854. doi: 10.1016/0092-8674(82)90063-0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Glenney J. R., Jr, Glenney P., Weber K. F-actin-binding and cross-linking properties of porcine brain fodrin, a spectrin-related molecule. J Biol Chem. 1982 Aug 25;257(16):9781–9787. [PubMed] [Google Scholar]
  19. 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]
  20. Glenney J. R., Jr, Kaulfus P., Matsudaira P., Weber K. F-actin binding and bundling properties of fimbrin, a major cytoskeletal protein of microvillus core filaments. J Biol Chem. 1981 Sep 10;256(17):9283–9288. [PubMed] [Google Scholar]
  21. Glenney J. R., Jr, Weber K. Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells. J Biol Chem. 1980 Nov 25;255(22):10551–10554. [PubMed] [Google Scholar]
  22. Herman I. M., Pollard T. D. Electron microscopic localization of cytoplasmic myosin with ferritin-labeled antibodies. J Cell Biol. 1981 Feb;88(2):346–351. doi: 10.1083/jcb.88.2.346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. Howe C. L., Keller T. C., 3rd, Mooseker M. S., Wasserman R. H. Analysis of cytoskeletal proteins and Ca2+-dependent regulation of structure in intestinal brush borders from rachitic chicks. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1134–1138. doi: 10.1073/pnas.79.4.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Keller T. C., 3rd, Mooseker M. S. Ca++-calmodulin-dependent phosphorylation of myosin, and its role in brush border contraction in vitro. J Cell Biol. 1982 Dec;95(3):943–959. doi: 10.1083/jcb.95.3.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  31. 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]
  32. Lazarides E., Nelson W. J. Expression of spectrin in nonerythroid cells. Cell. 1982 Dec;31(3 Pt 2):505–508. doi: 10.1016/0092-8674(82)90306-3. [DOI] [PubMed] [Google Scholar]
  33. Levine J., Willard M. Fodrin: axonally transported polypeptides associated with the internal periphery of many cells. J Cell Biol. 1981 Sep;90(3):631–642. doi: 10.1083/jcb.90.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. MacLean-Fletcher S., Pollard T. D. Mechanism of action of cytochalasin B on actin. Cell. 1980 Jun;20(2):329–341. doi: 10.1016/0092-8674(80)90619-4. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Matsudaira P. T., Burgess D. R. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal Biochem. 1978 Jul 1;87(2):386–396. doi: 10.1016/0003-2697(78)90688-7. [DOI] [PubMed] [Google Scholar]
  37. Matsudaira P. T., Burgess D. R. Structure and function of the brush-border cytoskeleton. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):845–854. doi: 10.1101/sqb.1982.046.01.079. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. Mooseker M. S., Keller T. C., 3rd, Hirokawa N. Regulation of cytoskeletal structure and contractility in the brush border. Ciba Found Symp. 1983;95:195–215. doi: 10.1002/9780470720769.ch12. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Morrow J. S., Marchesi V. T. Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton. J Cell Biol. 1981 Feb;88(2):463–468. doi: 10.1083/jcb.88.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Palfrey H. C., Schiebler W., Greengard P. A major calmodulin-binding protein common to various vertebrate tissues. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3780–3784. doi: 10.1073/pnas.79.12.3780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Patterson D. S., Brush P. J., Foulkes J. A., Sweasey D. Copper metabolism and the composition of wool in Border disease. Vet Rec. 1974 Sep 7;95(10):214–215. doi: 10.1136/vr.95.10.214. [DOI] [PubMed] [Google Scholar]
  47. Shotton D. M., Burke B. E., Branton D. The molecular structure of human erythrocyte spectrin. Biophysical and electron microscopic studies. J Mol Biol. 1979 Jun 25;131(2):303–329. doi: 10.1016/0022-2836(79)90078-0. [DOI] [PubMed] [Google Scholar]
  48. Sobue K., Kanda K., Inui M., Morimoto K., Kakiuchi S. Actin polymerization induced by calspectin, a calmodulin-binding spectrin-like protein. FEBS Lett. 1982 Nov 8;148(2):221–225. doi: 10.1016/0014-5793(82)80811-9. [DOI] [PubMed] [Google Scholar]
  49. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  50. Tyler J. M., Branton D. Rotary shadowing of extended molecules dried from glycerol. J Ultrastruct Res. 1980 May;71(2):95–102. doi: 10.1016/s0022-5320(80)90098-2. [DOI] [PubMed] [Google Scholar]
  51. Ungewickell E., Gratzer W. Self-association of human spectrin. A thermodynamic and kinetic study. Eur J Biochem. 1978 Aug 1;88(2):379–385. doi: 10.1111/j.1432-1033.1978.tb12459.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES