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. 1982 Oct 1;95(1):223–233. doi: 10.1083/jcb.95.1.223

Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border

PMCID: PMC2112343  PMID: 6890554

Abstract

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G- actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G- actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.

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Selected References

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  1. Altmann G. G. Influence of starvation and refeeding on mucosal size and epithelial renewal in the rat small intestine. Am J Anat. 1972 Apr;133(4):391–400. doi: 10.1002/aja.1001330403. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bergen L. G., Borisy G. G. Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly. J Cell Biol. 1980 Jan;84(1):141–150. doi: 10.1083/jcb.84.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brenner S. L., Korn E. D. Substoichiometric concentrations of cytochalasin D inhibit actin polymerization. Additional evidence for an F-actin treadmill. J Biol Chem. 1979 Oct 25;254(20):9982–9985. [PubMed] [Google Scholar]
  5. Brenner S. L., Korn E. D. The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization. J Biol Chem. 1980 Feb 10;255(3):841–844. [PubMed] [Google Scholar]
  6. Brown S. S., Spudich J. A. Cytochalasin inhibits the rate of elongation of actin filament fragments. J Cell Biol. 1979 Dec;83(3):657–662. doi: 10.1083/jcb.83.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Flanagan M. D., Lin S. Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin. J Biol Chem. 1980 Feb 10;255(3):835–838. [PubMed] [Google Scholar]
  9. Hayashi T., Ip W. Polymerization polarity of actin. J Mechanochem Cell Motil. 1976 Mar;3(3):163–169. [PubMed] [Google Scholar]
  10. Hitchcock-DeGregori S. E. Actin assembly. Nature. 1980 Dec 4;288(5790):437–438. doi: 10.1038/288437a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Isenberg G., Aebi U., Pollard T. D. An actin-binding protein from Acanthamoeba regulates actin filament polymerization and interactions. Nature. 1980 Dec 4;288(5790):455–459. doi: 10.1038/288455a0. [DOI] [PubMed] [Google Scholar]
  13. Kirschner M. W. Implications of treadmilling for the stability and polarity of actin and tubulin polymers in vivo. J Cell Biol. 1980 Jul;86(1):330–334. doi: 10.1083/jcb.86.1.330. [DOI] [PMC free article] [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. 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]
  16. Lin D. C., Tobin K. D., Grumet M., Lin S. Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation. J Cell Biol. 1980 Feb;84(2):455–460. doi: 10.1083/jcb.84.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Mooseker M. S., Howe C. L. The brush border of intestinal epithelium: a model system for analysis of cell-surface architecture and motility. Methods Cell Biol. 1982;25(Pt B):143–174. doi: 10.1016/s0091-679x(08)61424-7. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Tilney L. G., Kallenbach N. Polymerization of actin. VI. The polarity of the actin filaments in the acrosomal process and how it might be determined. J Cell Biol. 1979 Jun;81(3):608–623. doi: 10.1083/jcb.81.3.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]

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