Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 May;74(5):2171–2183. doi: 10.1016/S0006-3495(98)77926-3

Structure of the erythrocyte membrane skeleton as observed by atomic force microscopy.

M Takeuchi 1, H Miyamoto 1, Y Sako 1, H Komizu 1, A Kusumi 1
PMCID: PMC1299560  PMID: 9591644

Abstract

The structure of the membrane skeleton on the cytoplasmic surface of the erythrocyte plasma membrane was observed in dried human erythrocyte ghosts by atomic force microscopy (AFM), taking advantage of its high sensitivity to small height variations in surfaces. The majority of the membrane skeleton can be imaged, even on the extracellular surface of the membrane. Various fixation and drying methods were examined for preparation of ghost membrane samples for AFM observation, and it was found that freeze-drying (freezing by rapid immersion in a cryogen) of unfixed specimens was a fast and simple way to obtain consistently good results for observation without removing the membrane or extending the membrane skeleton. Observation of the membrane skeleton at the external surface of the cell was possible mainly because the bilayer portion of the membrane sank into the cell during the drying process. The average mesh size of the spectrin network observed at the extracellular and cytoplasmic surfaces of the plasma membrane was 4800 and 3000 nm2, respectively, which indicates that spectrin forms a three-dimensionally folded meshwork, and that 80% of spectrin can be observed at the extracellular surface of the plasma membrane.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akahori H., Ishii H., Nonaka I., Yoshida H. A simple freeze-drying device using t-butyl alcohol for SEM specimens. J Electron Microsc (Tokyo) 1988;37(6):351–352. [PubMed] [Google Scholar]
  2. Almqvist N., Backman L., Fredriksson S. Imaging human erythrocyte spectrin with atomic force microscopy. Micron. 1994;25(3):227–232. doi: 10.1016/0968-4328(94)90027-2. [DOI] [PubMed] [Google Scholar]
  3. Beaven G. H., Jean-Baptiste L., Ungewickell E., Baines A. J., Shahbakhti F., Pinder J. C., Lux S. E., Gratzer W. B. An examination of the soluble oligomeric complexes extracted from the red cell membrane and their relation to the membrane cytoskeleton. Eur J Cell Biol. 1985 Mar;36(2):299–306. [PubMed] [Google Scholar]
  4. Bennett V., Gilligan D. M. The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane. Annu Rev Cell Biol. 1993;9:27–66. doi: 10.1146/annurev.cb.09.110193.000331. [DOI] [PubMed] [Google Scholar]
  5. Bennett V. Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiol Rev. 1990 Oct;70(4):1029–1065. doi: 10.1152/physrev.1990.70.4.1029. [DOI] [PubMed] [Google Scholar]
  6. Boal D. H., Boey S. K. Barrier-free paths of directed protein motion in the erythrocyte plasma membrane. Biophys J. 1995 Aug;69(2):372–379. doi: 10.1016/S0006-3495(95)79909-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boal D. H. Computer simulation of a model network for the erythrocyte cytoskeleton. Biophys J. 1994 Aug;67(2):521–529. doi: 10.1016/S0006-3495(94)80511-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brandow S. L., Turner D. C., Ratna B. R., Gaber B. P. Modification of supported lipid membranes by atomic force microscopy. Biophys J. 1993 Mar;64(3):898–902. doi: 10.1016/S0006-3495(93)81450-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Butt H. J., Wolff E. K., Gould S. A., Dixon Northern B., Peterson C. M., Hansma P. K. Imaging cells with the atomic force microscope. J Struct Biol. 1990 Oct-Dec;105(1-3):54–61. doi: 10.1016/1047-8477(90)90098-w. [DOI] [PubMed] [Google Scholar]
  10. Byers T. J., Branton D. Visualization of the protein associations in the erythrocyte membrane skeleton. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6153–6157. doi: 10.1073/pnas.82.18.6153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chang L., Kious T., Yorgancioglu M., Keller D., Pfeiffer J. Cytoskeleton of living, unstained cells imaged by scanning force microscopy. Biophys J. 1993 Apr;64(4):1282–1286. doi: 10.1016/S0006-3495(93)81493-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clarke M., Schatten G., Mazia D., Spudich J. A. Visualization of actin fibers associated with the cell membrane in amoebae of Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1975 May;72(5):1758–1762. doi: 10.1073/pnas.72.5.1758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Coleman T. R., Fishkind D. J., Mooseker M. S., Morrow J. S. Functional diversity among spectrin isoforms. Cell Motil Cytoskeleton. 1989;12(4):225–247. doi: 10.1002/cm.970120405. [DOI] [PubMed] [Google Scholar]
  14. Costello M. J. Ultra-rapid freezing of thin biological samples. Scan Electron Microsc. 1980;(Pt 2):361–370. [PubMed] [Google Scholar]
  15. Elder H. Y., Gray C. C., Jardine A. G., Chapman J. N., Biddlecombe W. H. Optimum conditions for cryoquenching of small tissue blocks in liquid coolants. J Microsc. 1982 Apr;126(Pt 1):45–61. doi: 10.1111/j.1365-2818.1982.tb00356.x. [DOI] [PubMed] [Google Scholar]
  16. Evans E. A. Structure and deformation properties of red blood cells: concepts and quantitative methods. Methods Enzymol. 1989;173:3–35. doi: 10.1016/s0076-6879(89)73003-2. [DOI] [PubMed] [Google Scholar]
  17. Gilligan D. M., Bennett V. The junctional complex of the membrane skeleton. Semin Hematol. 1993 Jan;30(1):74–83. [PubMed] [Google Scholar]
  18. Hainfeld J. F., Steck T. L. The sub-membrane reticulum of the human erythrocyte: a scanning electron microscope study. J Supramol Struct. 1977;6(3):301–311. doi: 10.1002/jss.400060303. [DOI] [PubMed] [Google Scholar]
  19. Hammerton R. W., Krzeminski K. A., Mays R. W., Ryan T. A., Wollner D. A., Nelson W. J. Mechanism for regulating cell surface distribution of Na+,K(+)-ATPase in polarized epithelial cells. Science. 1991 Nov 8;254(5033):847–850. doi: 10.1126/science.1658934. [DOI] [PubMed] [Google Scholar]
  20. Han W., Mou J., Sheng J., Yang J., Shao Z. Cryo atomic force microscopy: a new approach for biological imaging at high resolution. Biochemistry. 1995 Jul 4;34(26):8215–8220. doi: 10.1021/bi00026a001. [DOI] [PubMed] [Google Scholar]
  21. Henderson E., Haydon P. G., Sakaguchi D. S. Actin filament dynamics in living glial cells imaged by atomic force microscopy. Science. 1992 Sep 25;257(5078):1944–1946. doi: 10.1126/science.1411511. [DOI] [PubMed] [Google Scholar]
  22. Hitt A. L., Luna E. J. Membrane interactions with the actin cytoskeleton. Curr Opin Cell Biol. 1994 Feb;6(1):120–130. doi: 10.1016/0955-0674(94)90125-2. [DOI] [PubMed] [Google Scholar]
  23. Hörber J. K., Mosbacher J., Häberle W., Ruppersberg J. P., Sakmann B. A look at membrane patches with a scanning force microscope. Biophys J. 1995 May;68(5):1687–1693. doi: 10.1016/S0006-3495(95)80346-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Inoué T., Osatake H. A new drying method of biological specimens for scanning electron microscopy: the t-butyl alcohol freeze-drying method. Arch Histol Cytol. 1988 Mar;51(1):53–59. doi: 10.1679/aohc.51.53. [DOI] [PubMed] [Google Scholar]
  25. Jacobson K., Sheets E. D., Simson R. Revisiting the fluid mosaic model of membranes. Science. 1995 Jun 9;268(5216):1441–1442. doi: 10.1126/science.7770769. [DOI] [PubMed] [Google Scholar]
  26. Kusumi A., Sako Y. Cell surface organization by the membrane skeleton. Curr Opin Cell Biol. 1996 Aug;8(4):566–574. doi: 10.1016/s0955-0674(96)80036-6. [DOI] [PubMed] [Google Scholar]
  27. Lal R., Drake B., Blumberg D., Saner D. R., Hansma P. K., Feinstein S. C. Imaging real-time neurite outgrowth and cytoskeletal reorganization with an atomic force microscope. Am J Physiol. 1995 Jul;269(1 Pt 1):C275–C285. doi: 10.1152/ajpcell.1995.269.1.C275. [DOI] [PubMed] [Google Scholar]
  28. Liu S. C., Derick L. H., Palek J. Visualization of the hexagonal lattice in the erythrocyte membrane skeleton. J Cell Biol. 1987 Mar;104(3):527–536. doi: 10.1083/jcb.104.3.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu S. C., Windisch P., Kim S., Palek J. Oligomeric states of spectrin in normal erythrocyte membranes: biochemical and electron microscopic studies. Cell. 1984 Jun;37(2):587–594. doi: 10.1016/0092-8674(84)90389-1. [DOI] [PubMed] [Google Scholar]
  30. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. McGough A. M., Josephs R. On the structure of erythrocyte spectrin in partially expanded membrane skeletons. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5208–5212. doi: 10.1073/pnas.87.13.5208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mohandas N., Chasis J. A. Red blood cell deformability, membrane material properties and shape: regulation by transmembrane, skeletal and cytosolic proteins and lipids. Semin Hematol. 1993 Jul;30(3):171–192. [PubMed] [Google Scholar]
  35. Nermut M. V. Visualization of the "membrane skeleton" in human erythrocytes by freeze-etching. Eur J Cell Biol. 1981 Oct;25(2):265–271. [PubMed] [Google Scholar]
  36. Ohanian V., Wolfe L. C., John K. M., Pinder J. C., Lux S. E., Gratzer W. B. Analysis of the ternary interaction of the red cell membrane skeletal proteins spectrin, actin, and 4.1. Biochemistry. 1984 Sep 11;23(19):4416–4420. doi: 10.1021/bi00314a027. [DOI] [PubMed] [Google Scholar]
  37. Pietrasanta L. I., Schaper A., Jovin T. M. Imaging subcellular structures of rat mammary carcinoma cells by scanning force microscopy. J Cell Sci. 1994 Sep;107(Pt 9):2427–2437. doi: 10.1242/jcs.107.9.2427. [DOI] [PubMed] [Google Scholar]
  38. Sako Y., Kusumi A. Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether. J Cell Biol. 1995 Jun;129(6):1559–1574. doi: 10.1083/jcb.129.6.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sako Y., Kusumi A. Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis. J Cell Biol. 1994 Jun;125(6):1251–1264. doi: 10.1083/jcb.125.6.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sheets E. D., Simson R., Jacobson K. New insights into membrane dynamics from the analysis of cell surface interactions by physical methods. Curr Opin Cell Biol. 1995 Oct;7(5):707–714. doi: 10.1016/0955-0674(95)80113-8. [DOI] [PubMed] [Google Scholar]
  41. Sheetz M. P., Sawyer D. Triton shells of intact erythrocytes. J Supramol Struct. 1978;8(4):399–412. doi: 10.1002/jss.400080403. [DOI] [PubMed] [Google Scholar]
  42. Sheetz M. P., Schindler M., Koppel D. E. Lateral mobility of integral membrane proteins is increased in spherocytic erythrocytes. Nature. 1980 Jun 12;285(5765):510–511. doi: 10.1038/285510a0. [DOI] [PubMed] [Google Scholar]
  43. Shen B. W., Josephs R., Steck T. L. Ultrastructure of the intact skeleton of the human erythrocyte membrane. J Cell Biol. 1986 Mar;102(3):997–1006. doi: 10.1083/jcb.102.3.997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Timme A. H. The ultrastructure of the erythrocyte cytoskeleton at neutral and reduced pH. J Ultrastruct Res. 1981 Nov;77(2):199–209. doi: 10.1016/s0022-5320(81)80041-x. [DOI] [PubMed] [Google Scholar]
  46. Tsuji A., Kawasaki K., Ohnishi S., Merkle H., Kusumi A. Regulation of band 3 mobilities in erythrocyte ghost membranes by protein association and cytoskeletal meshwork. Biochemistry. 1988 Sep 20;27(19):7447–7452. doi: 10.1021/bi00419a041. [DOI] [PubMed] [Google Scholar]
  47. Tsukita S., Tsukita S., Ishikawa H. Cytoskeletal network underlying the human erythrocyte membrane. Thin-section electron microscopy. J Cell Biol. 1980 Jun;85(3):567–576. doi: 10.1083/jcb.85.3.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tsukita S., Tsukita S., Nagafuchi A., Yonemura S. Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions. Curr Opin Cell Biol. 1992 Oct;4(5):834–839. doi: 10.1016/0955-0674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  49. Ursitti J. A., Pumplin D. W., Wade J. B., Bloch R. J. Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane. Cell Motil Cytoskeleton. 1991;19(4):227–243. doi: 10.1002/cm.970190402. [DOI] [PubMed] [Google Scholar]
  50. Ursitti J. A., Wade J. B. Ultrastructure and immunocytochemistry of the isolated human erythrocyte membrane skeleton. Cell Motil Cytoskeleton. 1993;25(1):30–42. doi: 10.1002/cm.970250105. [DOI] [PubMed] [Google Scholar]
  51. Vertessy B. G., Steck T. L. Elasticity of the human red cell membrane skeleton. Effects of temperature and denaturants. Biophys J. 1989 Feb;55(2):255–262. doi: 10.1016/S0006-3495(89)82800-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Weinstein R. S., Tazelaar H. D., Loew J. M. Red cell comets: ultrastructure of axial elongation of the membrane skeleton. Blood Cells. 1986;11(3):343–366. [PubMed] [Google Scholar]
  53. Zhang Y., Sheng S., Shao Z. Imaging biological structures with the cryo atomic force microscope. Biophys J. 1996 Oct;71(4):2168–2176. doi: 10.1016/S0006-3495(96)79418-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES