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. 1983 Apr 1;96(4):1097–1107. doi: 10.1083/jcb.96.4.1097

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

EM Bonder, MS Mooseker
PMCID: PMC2112331  PMID: 6682116

Abstract

We have re-examined the Ca(++)-dependent interaction of an intestinal microvillar 95- kdalton protein (MV-95K) and actin using the isolated acrosomal process bundles from limulus sperm. Making use of the processes as nuclei for assembling actin filaments, we quantitatively and qualitatively examined MV-95K’s effect on filament assembly and on F- actin, both in the presence and in the absence of Ca(++). The acrosomal processes are particularly advantageous for this approach because they nucleate large numbers of filaments, they are extremely stable, and their morphology can be used to determine the polarity of any nucleated filaments. When filament nucleation was initiated in the presence of MV-95K and the absence of Ca(++), there was biased filament assembly from the bundle ends. The calculated elongation rates from both the barbed and pointed filament ends were virtually indistinguishable from control preparations. In the presence of Ca(++), MV-95K completely inhibited filament assembly from the barbed filament end without affecting the initial rate of assembly from the pointed filament end. The inhibition of assembly results from MV-95K binding to and capping the barbed filament end, thereby preventing monomer addition. This indicates that, while MV-95K is a potent nucleator of actin assembly, it is also a potent inhibitor of actin filament elongation. To examine the effects of MV-95K on F-actin in the presence of Ca(++), we developed an assay where MV-95K is added to filaments previously assembled from acrosomal processes without causing filament breakage during mixing. These results clearly demonstrated that rapid filament shortening by MV-95K results through a mechanism of disrupting intrafilament monomer-monomer interactions. Finally, we show that tropomyosin-containing actin filaments are insensitive to cutting, but not to capping, by MV-95K in the presence of Ca(++).

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Selected References

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  1. 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]
  2. 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]
  3. Brown S. S., Spudich J. A. Mechanism of action of cytochalasin: evidence that it binds to actin filament ends. J Cell Biol. 1981 Mar;88(3):487–491. doi: 10.1083/jcb.88.3.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown S. S., Spudich J. A. Nucleation of polar actin filament assembly by a positively charged surface. J Cell Biol. 1979 Feb;80(2):499–504. doi: 10.1083/jcb.80.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown S. S., Yamamoto K., Spudich J. A. A 40,000-dalton protein from Dictyostelium discoideum affects assembly properties of actin in a Ca2+-dependent manner. J Cell Biol. 1982 Apr;93(1):205–210. doi: 10.1083/jcb.93.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Glenney J. R., Jr, Geisler N., Kaulfus P., Weber K. Demonstration of at least two different actin-binding sites in villin, a calcium-regulated modulator of F-actin organization. J Biol Chem. 1981 Aug 10;256(15):8156–8161. [PubMed] [Google Scholar]
  10. Glenney J. R., Jr, Kaulfus P., Weber K. F actin assembly modulated by villin: Ca++-dependent nucleation and capping of the barbed end. Cell. 1981 May;24(2):471–480. doi: 10.1016/0092-8674(81)90338-x. [DOI] [PubMed] [Google Scholar]
  11. Glenney J. R., Jr, Weber K. Calcium control of microfilaments: uncoupling of the F-actin-severing and -bundling activity of villin by limited proteolysis in vitro. Proc Natl Acad Sci U S A. 1981 May;78(5):2810–2814. doi: 10.1073/pnas.78.5.2810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hasegawa T., Takahashi S., Hayashi H., Hatano S. Fragmin: a calcium ion sensitive regulatory factor on the formation of actin filaments. Biochemistry. 1980 Jun 10;19(12):2677–2683. doi: 10.1021/bi00553a021. [DOI] [PubMed] [Google Scholar]
  13. Hayashi T., Ip W. Polymerization polarity of actin. J Mechanochem Cell Motil. 1976 Mar;3(3):163–169. [PubMed] [Google Scholar]
  14. Kondo H., Ishiwata S. Uni-directional growth of F-actin. J Biochem. 1976 Jan;79(1):159–171. doi: 10.1093/oxfordjournals.jbchem.a131043. [DOI] [PubMed] [Google Scholar]
  15. Korn E. D. Actin polymerization and its regulation by proteins from nonmuscle cells. Physiol Rev. 1982 Apr;62(2):672–737. doi: 10.1152/physrev.1982.62.2.672. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Lehrer S. S. Damage to actin filaments by glutaraldehyde: protection by tropomyosin. J Cell Biol. 1981 Aug;90(2):459–466. doi: 10.1083/jcb.90.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. MacLean-Fletcher S., Pollard T. D. Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association. Biochem Biophys Res Commun. 1980 Sep 16;96(1):18–27. doi: 10.1016/0006-291x(80)91175-4. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Nunnally M. H., Powell L. D., Craig S. W. Reconstitution and regulation of actin gel-sol transformation with purified filamin and villin. J Biol Chem. 1981 Mar 10;256(5):2083–2086. [PubMed] [Google Scholar]
  26. Pardee J. D., Simpson P. A., Stryer L., Spudich J. A. Actin filaments undergo limited subunit exchange in physiological salt conditions. J Cell Biol. 1982 Aug;94(2):316–324. doi: 10.1083/jcb.94.2.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Simpson P. A., Spudich J. A. ATP-driven steady-state exchange of monomeric and filamentous actin from Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4610–4613. doi: 10.1073/pnas.77.8.4610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Tilney L. G. Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments. J Cell Biol. 1975 Feb;64(2):289–310. doi: 10.1083/jcb.64.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tilney L. G., Bonder E. M., DeRosier D. J. Actin filaments elongate from their membrane-associated ends. J Cell Biol. 1981 Aug;90(2):485–494. doi: 10.1083/jcb.90.2.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tseng P. C., Pollard T. D. Mechanism of action of Acanthamoeba profilin: demonstration of actin species specificity and regulation by micromolar concentrations of MgCl2. J Cell Biol. 1982 Jul;94(1):213–218. doi: 10.1083/jcb.94.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang L. L., Bryan J. Isolation of calcium-dependent platelet proteins that interact with actin. Cell. 1981 Sep;25(3):637–649. doi: 10.1016/0092-8674(81)90171-9. [DOI] [PubMed] [Google Scholar]
  34. Wang Y. L., Taylor D. L. Probing the dynamic equilibrium of actin polymerization by fluorescence energy transfer. Cell. 1981 Dec;27(3 Pt 2):429–436. doi: 10.1016/0092-8674(81)90384-6. [DOI] [PubMed] [Google Scholar]
  35. Wegner A. Head to tail polymerization of actin. J Mol Biol. 1976 Nov;108(1):139–150. doi: 10.1016/s0022-2836(76)80100-3. [DOI] [PubMed] [Google Scholar]
  36. Wegner A., Neuhaus J. M. Requirement of divalent cations for fast exchange of actin monomers and actin filament subunits. J Mol Biol. 1981 Dec 15;153(3):681–693. doi: 10.1016/0022-2836(81)90413-7. [DOI] [PubMed] [Google Scholar]
  37. Woodrum D. T., Rich S. A., Pollard T. D. Evidence for biased bidirectional polymerization of actin filaments using heavy meromyosin prepared by an improved method. J Cell Biol. 1975 Oct;67(1):231–237. doi: 10.1083/jcb.67.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yin H. L., Zaner K. S., Stossel T. P. Ca2+ control of actin gelation. Interaction of gelsolin with actin filaments and regulation of actin gelation. J Biol Chem. 1980 Oct 10;255(19):9494–9500. [PubMed] [Google Scholar]

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