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. 1985 May 1;100(5):1369–1378. doi: 10.1083/jcb.100.5.1369

Triton X-100 extraction of P815 tumor cells: evidence for a plasma membrane skeleton structure

PMCID: PMC2113856  PMID: 3921551

Abstract

It has been shown that a Triton X-100-insoluble protein matrix can be isolated from the plasma membranes of P815 tumor cells and murine lymphoid cells (Mescher, M. F., M. J. L. Jose and S. P. Balk, 1981, Nature (Lond.), 289:139-144). The properties of the matrix suggested that this set of proteins might form a membrane skeletal structure, stable in the absence of the lipid bilayer. Since purification of plasma membrane results in yields of only 20 to 40%, it was not clear whether the matrix was associated with the entire plasma membrane. To determine if a detergent-insoluble structure was present over the entire cell periphery and stable in the absence of the membrane bilayer or cytoskeletal components, we have examined extraction of whole cells with Triton X-100. Using the same conditions as those used for isolation of the matrix from membranes, we found that extraction of intact cells resulted in structures consisting of a continuous layer of protein at the periphery, a largely empty cytoplasmic space, and a nuclear remnant. Little or no lipid bilayer structure was evident in association with the peripheral layer, and no filamentous cytoskeletal structures could be seen in the cytoplasmic space by thin-section electron microscopy. Analysis of these Triton shells showed them to retain approximately 15% of the total cell protein, most of which was accounted for by low molecular weight nuclear proteins. 5'- Nucleotidase, a cell surface enzyme that remains associated with the plasma membrane matrix, was quantitatively recovered with the shells. Included among the polypeptides present in the shells was a set with mobilities identical to those of the set that makes up the plasma membrane matrix. The polypeptide composition of the shells further confirmed that cytoskeletal proteins were present to a very low extent, if at all, after the extraction. The results demonstrate that a detergent-insoluble protein matrix associated with the periphery of these cells forms a continuous, intact macrostructure whose stability is independent of the membrane bilayer or filamentous cytoskeletal elements, and thus has the properties of a membrane skeletal structure. Although not yet directly demonstrated, the results also strongly suggest that this peripheral layer is composed of the previously described set of plasma membrane matrix proteins. This article discusses possible roles for this proposed membrane skeletal structure in stabilizing the membrane bilayer and affecting the dynamics of other membrane proteins.

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Selected References

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  1. Avruch J., Wallach D. F. Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochim Biophys Acta. 1971 Apr 13;233(2):334–347. doi: 10.1016/0005-2736(71)90331-2. [DOI] [PubMed] [Google Scholar]
  2. Ben-Ze'ev A., Duerr A., Solomon F., Penman S. The outer boundary of the cytoskeleton: a lamina derived from plasma membrane proteins. Cell. 1979 Aug;17(4):859–865. doi: 10.1016/0092-8674(79)90326-x. [DOI] [PubMed] [Google Scholar]
  3. Bennett V. Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues. Nature. 1979 Oct 18;281(5732):597–599. doi: 10.1038/281597a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Bretscher M. S., Raff M. C. Mammalian plasma membranes. Nature. 1975 Nov 6;258(5530):43–49. doi: 10.1038/258043a0. [DOI] [PubMed] [Google Scholar]
  7. Brown S., Levinson W., Spudich J. A. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J Supramol Struct. 1976;5(2):119–130. doi: 10.1002/jss.400050203. [DOI] [PubMed] [Google Scholar]
  8. Cherry R. J. Rotational and lateral diffusion of membrane proteins. Biochim Biophys Acta. 1979 Dec 20;559(4):289–327. doi: 10.1016/0304-4157(79)90009-1. [DOI] [PubMed] [Google Scholar]
  9. Crumpton M. J., Snary D. Preparation and properties of lymphocyte plasma membrane. Contemp Top Mol Immunol. 1974;3:27–56. doi: 10.1007/978-1-4684-2838-4_2. [DOI] [PubMed] [Google Scholar]
  10. Davies A. A., Wigglesworth N. M., Allan D., Owens R. J., Crumpton M. J. Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue. Biochem J. 1984 Apr 1;219(1):301–308. doi: 10.1042/bj2190301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fleit H., Conklyn M., Stebbins R. D., Silber R. Function of 5'-nucleotidase in the uptake of adenosine from AMP by human lymphocytes. J Biol Chem. 1975 Dec 10;250(23):8889–8892. [PubMed] [Google Scholar]
  12. Fulton A. B., Prives J., Farmer S. R., Penman S. Developmental reorganization of the skeletal framework and its surface lamina in fusing muscle cells. J Cell Biol. 1981 Oct;91(1):103–112. doi: 10.1083/jcb.91.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haest C. W., Plasa G., Kamp D., Deuticke B. Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane. Biochim Biophys Acta. 1978 May 4;509(1):21–32. doi: 10.1016/0005-2736(78)90004-4. [DOI] [PubMed] [Google Scholar]
  14. Herrmann S. H., Mescher M. F. Secondary cytolytic T lymphocyte stimulation by purified H-2Kk in liposomes. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2488–2492. doi: 10.1073/pnas.78.4.2488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  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. Lange Y., Hadesman R. A., Steck T. L. Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane. J Cell Biol. 1982 Mar;92(3):714–721. doi: 10.1083/jcb.92.3.714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature. 1980 Jan 17;283(5744):249–256. doi: 10.1038/283249a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lehto V. P., Vartio T., Badley R. A., Virtanen I. Characterization of a detergent-resistant surface lamina in cultured human fibroblasts. Exp Cell Res. 1983 Feb;143(2):287–294. doi: 10.1016/0014-4827(83)90053-8. [DOI] [PubMed] [Google Scholar]
  21. Lehto V. P., Virtanen I., Kurki P. Intermediate filaments anchor the nuclei in nuclear monolayers of cultured human fibroblasts. Nature. 1978 Mar 9;272(5649):175–177. doi: 10.1038/272175a0. [DOI] [PubMed] [Google Scholar]
  22. Lemonnier F., Mescher T. M., sherman L., Burakoff S. The induction of cytolytic T lymphocytes with purified plasma membranes. J Immunol. 1978 Apr;120(4):1114–1120. [PubMed] [Google Scholar]
  23. Lubin B., Chiu D., Bastacky J., Roelofsen B., Van Deenen L. L. Abnormalities in membrane phospholipid organization in sickled erythrocytes. J Clin Invest. 1981 Jun;67(6):1643–1649. doi: 10.1172/JCI110200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lux S. E. Dissecting the red cell membrane skeleton. Nature. 1979 Oct 11;281(5731):426–429. doi: 10.1038/281426a0. [DOI] [PubMed] [Google Scholar]
  25. Marchesi V. T., Furthmayr H., Tomita M. The red cell membrane. Annu Rev Biochem. 1976;45:667–698. doi: 10.1146/annurev.bi.45.070176.003315. [DOI] [PubMed] [Google Scholar]
  26. Maupin P., Pollard T. D. Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation. J Cell Biol. 1983 Jan;96(1):51–62. doi: 10.1083/jcb.96.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mescher M. F., Jose M. J., Balk S. P. Actin-containing matrix associated with the plasma membrane of murine tumour and lymphoid cells. Nature. 1981 Jan 15;289(5794):139–144. doi: 10.1038/289139a0. [DOI] [PubMed] [Google Scholar]
  28. Mescher M. F., Pollock R. R. Murine cell surface immunoglobulin: two forms of delta-heavy chain. J Immunol. 1979 Sep;123(3):1155–1161. [PubMed] [Google Scholar]
  29. Mescher M. F., Stallcup K. C., Sullivan C. P., Turkewitz A. P., Herrmann S. H. Purification of murine MHC antigens by monoclonal antibody affinity chromatography. Methods Enzymol. 1983;92:86–109. doi: 10.1016/0076-6879(83)92011-6. [DOI] [PubMed] [Google Scholar]
  30. Moore P. B., Ownby C. L., Carraway K. L. Interactions of cytoskeletal elements with the plasma membrane of sarcoma180 ascites tumor cells. Exp Cell Res. 1978 Sep;115(2):331–342. doi: 10.1016/0014-4827(78)90287-2. [DOI] [PubMed] [Google Scholar]
  31. Morrison M. The determination of the exposed proteins on membranes by the use of lactoperoxidase. Methods Enzymol. 1974;32:103–109. doi: 10.1016/0076-6879(74)32013-7. [DOI] [PubMed] [Google Scholar]
  32. Nicolson G. L. Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over surface components. Biochim Biophys Acta. 1976 Apr 13;457(1):57–108. doi: 10.1016/0304-4157(76)90014-9. [DOI] [PubMed] [Google Scholar]
  33. Osborn M., Weber K. The detertent-resistant cytoskeleton of tissue culture cells includes the nucleus and the microfilament bundles. Exp Cell Res. 1977 May;106(2):339–349. doi: 10.1016/0014-4827(77)90179-3. [DOI] [PubMed] [Google Scholar]
  34. Owens R. J., Crumpton M. J. Isolation and characterization of a novel 68,000-Mr Ca2+-binding protein of lymphocyte plasma membrane. Biochem J. 1984 Apr 1;219(1):309–316. doi: 10.1042/bj2190309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Owens R. J., Gallagher C. J., Crumpton M. J. Cellular distribution of p68, a new calcium-binding protein from lymphocytes. EMBO J. 1984 May;3(5):945–952. doi: 10.1002/j.1460-2075.1984.tb01912.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Singer S. J., Nicolson G. L. The fluid mosaic model of the structure of cell membranes. Science. 1972 Feb 18;175(4023):720–731. doi: 10.1126/science.175.4023.720. [DOI] [PubMed] [Google Scholar]
  38. Streuli C. H., Patel B., Critchley D. R. The cholera toxin receptor ganglioside GM remains associated with triton X-100 cytoskeletons of BALB/c-3T3 cells. Exp Cell Res. 1981 Dec;136(2):247–254. doi: 10.1016/0014-4827(81)90002-1. [DOI] [PubMed] [Google Scholar]
  39. Williamson P., Bateman J., Kozarsky K., Mattocks K., Hermanowicz N., Choe H. R., Schlegel R. A. Involvement of spectrin in the maintenance of phase-state asymmetry in the erythrocyte membrane. Cell. 1982 Oct;30(3):725–733. doi: 10.1016/0092-8674(82)90277-x. [DOI] [PubMed] [Google Scholar]
  40. Yu J., Fischman D. A., Steck T. L. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J Supramol Struct. 1973;1(3):233–248. doi: 10.1002/jss.400010308. [DOI] [PubMed] [Google Scholar]

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