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. 1989 Dec 1;109(6):2833–2840. doi: 10.1083/jcb.109.6.2833

Evidence for a direct, nucleotide-sensitive interaction between actin and liver cell membranes

PMCID: PMC2115935  PMID: 2592407

Abstract

We have investigated the association of actin with membranes isolated from rat liver. A plasma membrane-enriched fraction prepared by homogenization in a low salt/CaCl2 buffer was found to contain a substantial amount of residual actin which could be removed by treatment with 1 M Na2CO3/NaHCO3, pH 10.5. Using a sedimentation binding assay that uses gelsolin to shorten actin filaments and render membrane binding saturable (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102:2067-2075), we found that membranes stripped of endogenous actin bound 125I-actin in a specific and saturable manner. Scatchard plots of binding data were linear, indicating a single class of binding sites with a Kd of 1.6 microns; 66 micrograms actin bound/mg membrane protein at saturation. Binding of actin to liver cell membranes was negligible with unstripped membranes, was competed by excess unlabeled actin, and was greatly reduced by preheating or proteolytic digestion of the membranes. Kinetic measurements showed that binding had an initial lag phase and was strongly temperature dependent. The binding of actin to liver cell membranes was also found to be competitively inhibited by ATP and other nucleotides, including the nonhydrolyzable analogue AMP-PNP. We conclude that we have reconstituted an interaction between actin and integral membrane proteins from the rat liver. This interaction exhibits a number of distinctive features which have not been observed in other actin- membrane systems.

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Selected References

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  1. Bennett V. The spectrin-actin junction of erythrocyte membrane skeletons. Biochim Biophys Acta. 1989 Jan 18;988(1):107–121. doi: 10.1016/0304-4157(89)90006-3. [DOI] [PubMed] [Google Scholar]
  2. Brown S. S., Malinoff H. L., Wicha M. S. Connectin: cell surface protein that binds both laminin and actin. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5927–5930. doi: 10.1073/pnas.80.19.5927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carey D. J., Rafferty C. M., Schramm M. M. Association of heparan sulfate proteoglycan and laminin with the cytoskeleton in rat liver. J Biol Chem. 1987 Mar 5;262(7):3376–3381. [PubMed] [Google Scholar]
  4. Carey D. J., Todd M. S. A cytoskeleton-associated plasma membrane heparan sulfate proteoglycan in Schwann cells. J Biol Chem. 1986 Jun 5;261(16):7518–7525. [PubMed] [Google Scholar]
  5. Carraway C. A., Jung G., Carraway K. L. Isolation of actin-containing transmembrane complexes from ascites adenocarcinoma sublines having mobile and immobile receptors. Proc Natl Acad Sci U S A. 1983 Jan;80(2):430–434. doi: 10.1073/pnas.80.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cooper J. A., Bryan J., Schwab B., 3rd, Frieden C., Loftus D. J., Elson E. L. Microinjection of gelsolin into living cells. J Cell Biol. 1987 Mar;104(3):491–501. doi: 10.1083/jcb.104.3.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dorling P. R., Le Page R. N. A rapid high yield method for the preparation of rat liver cell plasma membranes. Biochim Biophys Acta. 1973 Aug 9;318(1):33–40. doi: 10.1016/0005-2736(73)90333-7. [DOI] [PubMed] [Google Scholar]
  8. French S. W., Davies P. L. Ultrastructural localization of actin-like filaments in rat hepatocytes. Gastroenterology. 1975 Apr;68(4 Pt 1):765–774. [PubMed] [Google Scholar]
  9. Gabbiani G., Montesano R., Tuchweber B., Salas M., Orci L. Phalloidin-induced hyperplasia of actin filaments in rat hepatocytes. Lab Invest. 1975 Nov;33(5):562–569. [PubMed] [Google Scholar]
  10. Geiger B. Membrane-cytoskeleton interaction. Biochim Biophys Acta. 1983 Aug 11;737(3-4):305–341. doi: 10.1016/0304-4157(83)90005-9. [DOI] [PubMed] [Google Scholar]
  11. Govindan V. M., Wieland T. Isolation and identification of an actin from rat liver. FEBS Lett. 1975 Nov 1;59(1):117–119. doi: 10.1016/0014-5793(75)80354-1. [DOI] [PubMed] [Google Scholar]
  12. Hubbard A. L., Ma A. Isolation of rat hepatocyte plasma membranes. II. Identification of membrane-associated cytoskeletal proteins. J Cell Biol. 1983 Jan;96(1):230–239. doi: 10.1083/jcb.96.1.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ishii M., Miyazaki Y., Ohta S., Otsuki M., Goto Y. Three-dimensional structure of cytoskeletal system in human hepatocytes viewed by polyethylene-glycol-embedding method. Tohoku J Exp Med. 1985 Sep;147(1):47–63. doi: 10.1620/tjem.147.47. [DOI] [PubMed] [Google Scholar]
  14. Ishikawa H. Plasmalemmal undercoat: the cytoskeleton supporting the plasmalemma. Arch Histol Cytol. 1988 May;51(2):127–145. doi: 10.1679/aohc.51.127. [DOI] [PubMed] [Google Scholar]
  15. Jacobson B. S. Interaction of the plasma membrane with the cytoskeleton: an overview. Tissue Cell. 1983;15(6):829–852. doi: 10.1016/0040-8166(83)90053-8. [DOI] [PubMed] [Google Scholar]
  16. Korn E. D. Biochemistry of actomyosin-dependent cell motility (a review). Proc Natl Acad Sci U S A. 1978 Feb;75(2):588–599. doi: 10.1073/pnas.75.2.588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Marchesi V. T. Stabilizing infrastructure of cell membranes. Annu Rev Cell Biol. 1985;1:531–561. doi: 10.1146/annurev.cb.01.110185.002531. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Oda M., Price V. M., Fisher M. M., Phillips M. J. Ultrastructure of bile canaliculi, with special reference to the surface coat and the pericanalicular web. Lab Invest. 1974 Oct;31(4):314–323. [PubMed] [Google Scholar]
  23. Oshio C., Phillips M. J. Contractility of bile canaliculi: implications for liver function. Science. 1981 May 29;212(4498):1041–1042. doi: 10.1126/science.7015506. [DOI] [PubMed] [Google Scholar]
  24. Oyamada M., Mori M. Immunohistochemical demonstration of tubulin and actin in rat hepatocytes in situ using a perfusion extraction-fixation procedure. J Histochem Cytochem. 1985 Dec;33(12):1197–1204. doi: 10.1177/33.12.2415574. [DOI] [PubMed] [Google Scholar]
  25. Phillips M. J., Oda M., Mak E., Fisher M. M., Jeejeebhoy K. N. Microfilament dysfunction as a possible cause of intrahepatic cholestasis. Gastroenterology. 1975 Jul;69(1):48–58. [PubMed] [Google Scholar]
  26. Phillips M. J., Oshio C., Miyairi M., Watanabe S., Smith C. R. What is actin doing in the liver cell? Hepatology. 1983 May-Jun;3(3):433–436. doi: 10.1002/hep.1840030325. [DOI] [PubMed] [Google Scholar]
  27. Schwartz M. A., Luna E. J. Binding and assembly of actin filaments by plasma membranes from Dictyostelium discoideum. J Cell Biol. 1986 Jun;102(6):2067–2075. doi: 10.1083/jcb.102.6.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schwartz M. A., Luna E. J. How actin binds and assembles onto plasma membranes from Dictyostelium discoideum. J Cell Biol. 1988 Jul;107(1):201–209. doi: 10.1083/jcb.107.1.201. [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. 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]
  31. Wuestehube L. J., Luna E. J. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes. J Cell Biol. 1987 Oct;105(4):1741–1751. doi: 10.1083/jcb.105.4.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yousef I. M., Murray R. K. Studies on the preparation of rat liver plasma membrane fractions and on their polypeptide patterns. Can J Biochem. 1978 Jul;56(7):713–721. doi: 10.1139/o78-107. [DOI] [PubMed] [Google Scholar]

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