Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 Oct;81(4):2122–2134. doi: 10.1016/S0006-3495(01)75860-2

Cholesterol organization in membranes at low concentrations: effects of curvature stress and membrane thickness.

R Rukmini 1, S S Rawat 1, S C Biswas 1, A Chattopadhyay 1
PMCID: PMC1301684  PMID: 11566783

Abstract

Cholesterol is often found distributed nonrandomly in domains in biological and model membranes and has been reported to be distributed heterogeneously among various intracellular membranes. Although a large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol. Using a fluorescent cholesterol analog (25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol, or NBD-cholesterol), we have previously shown that cholesterol may exhibit local organization even at very low concentrations in membranes, which could possibly be attributable to transbilayer tail-to-tail dimers. This is supported by similar observations reported by other groups using cholesterol or dehydroergosterol, a naturally occurring fluorescent cholesterol analog which closely mimics cholesterol. In this paper, we have tested the basic features of cholesterol organization in membranes at low concentrations using spectral features of dehydroergosterol. More importantly, we have investigated the role of membrane surface curvature and thickness on transbilayer dimer arrangement of cholesterol using NBD-cholesterol. We find that dimerization is not favored in membranes with high curvature. However, cholesterol dimers are observed again if the curvature stress is relieved. Further, we have monitored the effect of membrane thickness on the dimerization process. Our results show that the dimerization process is stringently controlled by a narrow window of membrane thickness. Interestingly, this type of local organization of NBD-cholesterol at low concentrations is also observed in sphingomyelin-containing membranes. These results could be significant in membranes that have very low cholesterol content, such as the endoplasmic reticulum and the inner mitochondrial membrane, and in trafficking and sorting of cellular cholesterol.

Full Text

The Full Text of this article is available as a PDF (150.8 KB).

Selected References

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

  1. Aridor M., Balch W. E. Principles of selective transport: coat complexes hold the key. Trends Cell Biol. 1996 Aug;6(8):315–320. doi: 10.1016/0962-8924(96)10027-1. [DOI] [PubMed] [Google Scholar]
  2. Bretscher M. S., Munro S. Cholesterol and the Golgi apparatus. Science. 1993 Sep 3;261(5126):1280–1281. doi: 10.1126/science.8362242. [DOI] [PubMed] [Google Scholar]
  3. Brown R. E. Sphingolipid organization in biomembranes: what physical studies of model membranes reveal. J Cell Sci. 1998 Jan;111(Pt 1):1–9. doi: 10.1242/jcs.111.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chattopadhyay A. Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes. Chem Phys Lipids. 1990 Mar;53(1):1–15. doi: 10.1016/0009-3084(90)90128-e. [DOI] [PubMed] [Google Scholar]
  5. Chattopadhyay A., Komath S. S., Raman B. Aggregation of lasalocid A in membranes: a fluorescence study. Biochim Biophys Acta. 1992 Feb 17;1104(1):147–150. doi: 10.1016/0005-2736(92)90143-a. [DOI] [PubMed] [Google Scholar]
  6. Chattopadhyay A., London E. Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry. 1987 Jan 13;26(1):39–45. doi: 10.1021/bi00375a006. [DOI] [PubMed] [Google Scholar]
  7. Chattopadhyay A., London E. Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids in model membranes. Biochim Biophys Acta. 1988 Feb 8;938(1):24–34. doi: 10.1016/0005-2736(88)90118-6. [DOI] [PubMed] [Google Scholar]
  8. Chattopadhyay A., Mukherjee S. Fluorophore environments in membrane-bound probes: a red edge excitation shift study. Biochemistry. 1993 Apr 13;32(14):3804–3811. doi: 10.1021/bi00065a037. [DOI] [PubMed] [Google Scholar]
  9. Craven B. M. Crystal structure of cholesterol monohydrate. Nature. 1976 Apr 22;260(5553):727–729. doi: 10.1038/260727a0. [DOI] [PubMed] [Google Scholar]
  10. DITTMER J. C., LESTER R. L. A SIMPLE, SPECIFIC SPRAY FOR THE DETECTION OF PHOSPHOLIPIDS ON THIN-LAYER CHROMATOGRAMS. J Lipid Res. 1964 Jan;5:126–127. [PubMed] [Google Scholar]
  11. Fielding C. J., Fielding P. E. Intracellular cholesterol transport. J Lipid Res. 1997 Aug;38(8):1503–1521. [PubMed] [Google Scholar]
  12. Gimpl G., Burger K., Fahrenholz F. Cholesterol as modulator of receptor function. Biochemistry. 1997 Sep 9;36(36):10959–10974. doi: 10.1021/bi963138w. [DOI] [PubMed] [Google Scholar]
  13. Grechishnikova I. V., Bergström F., Johansson L. B., Brown R. E., Molotkovsky J. G. New fluorescent cholesterol analogs as membrane probes. Biochim Biophys Acta. 1999 Aug 20;1420(1-2):189–202. doi: 10.1016/s0005-2736(99)00088-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grinvald A., Steinberg I. Z. On the analysis of fluorescence decay kinetics by the method of least-squares. Anal Biochem. 1974 Jun;59(2):583–598. doi: 10.1016/0003-2697(74)90312-1. [DOI] [PubMed] [Google Scholar]
  15. Harris J. S., Epps D. E., Davio S. R., Kézdy F. J. Evidence for transbilayer, tail-to-tail cholesterol dimers in dipalmitoylglycerophosphocholine liposomes. Biochemistry. 1995 Mar 21;34(11):3851–3857. doi: 10.1021/bi00011a043. [DOI] [PubMed] [Google Scholar]
  16. Huang C., Mason J. T. Geometric packing constraints in egg phosphatidylcholine vesicles. Proc Natl Acad Sci U S A. 1978 Jan;75(1):308–310. doi: 10.1073/pnas.75.1.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Incardona J. P., Eaton S. Cholesterol in signal transduction. Curr Opin Cell Biol. 2000 Apr;12(2):193–203. doi: 10.1016/s0955-0674(99)00076-9. [DOI] [PubMed] [Google Scholar]
  18. Kobayashi T., Pagano R. E. ATP-dependent fusion of liposomes with the Golgi apparatus of perforated cells. Cell. 1988 Dec 2;55(5):797–805. doi: 10.1016/0092-8674(88)90135-3. [DOI] [PubMed] [Google Scholar]
  19. Koval M., Pagano R. E. Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts. J Cell Biol. 1990 Aug;111(2):429–442. doi: 10.1083/jcb.111.2.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Koynova R., Caffrey M. Phases and phase transitions of the phosphatidylcholines. Biochim Biophys Acta. 1998 Jun 29;1376(1):91–145. doi: 10.1016/s0304-4157(98)00006-9. [DOI] [PubMed] [Google Scholar]
  21. Lange Y. Disposition of intracellular cholesterol in human fibroblasts. J Lipid Res. 1991 Feb;32(2):329–339. [PubMed] [Google Scholar]
  22. Lange Y., Swaisgood M. H., Ramos B. V., Steck T. L. Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts. J Biol Chem. 1989 Mar 5;264(7):3786–3793. [PubMed] [Google Scholar]
  23. Lange Y., Ye J., Rigney M., Steck T. L. Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol. J Lipid Res. 1999 Dec;40(12):2264–2270. [PubMed] [Google Scholar]
  24. Lewis B. A., Engelman D. M. Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles. J Mol Biol. 1983 May 15;166(2):211–217. doi: 10.1016/s0022-2836(83)80007-2. [DOI] [PubMed] [Google Scholar]
  25. Liao S., Lin J., Do H., Johnson A. E. Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration. Cell. 1997 Jul 11;90(1):31–41. doi: 10.1016/s0092-8674(00)80311-6. [DOI] [PubMed] [Google Scholar]
  26. Lin S., Struve W. S. Time-resolved fluorescence of nitrobenzoxadiazole-aminohexanoic acid: effect of intermolecular hydrogen-bonding on non-radiative decay. Photochem Photobiol. 1991 Sep;54(3):361–365. doi: 10.1111/j.1751-1097.1991.tb02028.x. [DOI] [PubMed] [Google Scholar]
  27. Liscum L., Munn N. J. Intracellular cholesterol transport. Biochim Biophys Acta. 1999 Apr 19;1438(1):19–37. doi: 10.1016/s1388-1981(99)00043-8. [DOI] [PubMed] [Google Scholar]
  28. Liscum L., Underwood K. W. Intracellular cholesterol transport and compartmentation. J Biol Chem. 1995 Jun 30;270(26):15443–15446. doi: 10.1074/jbc.270.26.15443. [DOI] [PubMed] [Google Scholar]
  29. Loura L. M., Prieto M. Dehydroergosterol structural organization in aqueous medium and in a model system of membranes. Biophys J. 1997 May;72(5):2226–2236. doi: 10.1016/S0006-3495(97)78866-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. MacDonald R. C., MacDonald R. I., Menco B. P., Takeshita K., Subbarao N. K., Hu L. R. Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta. 1991 Jan 30;1061(2):297–303. doi: 10.1016/0005-2736(91)90295-j. [DOI] [PubMed] [Google Scholar]
  31. Maccarrone M., Bellincampi L., Melino G., Finazzi Agrò A. Cholesterol, but not its esters, triggers programmed cell death in human erythroleukemia K562 cells. Eur J Biochem. 1998 Apr 1;253(1):107–113. doi: 10.1046/j.1432-1327.1998.2530107.x. [DOI] [PubMed] [Google Scholar]
  32. Mazères S., Schram V., Tocanne J. F., Lopez A. 7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled phospholipids in lipid membranes: differences in fluorescence behavior. Biophys J. 1996 Jul;71(1):327–335. doi: 10.1016/S0006-3495(96)79228-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McClare C. W. An accurate and convenient organic phosphorus assay. Anal Biochem. 1971 Feb;39(2):527–530. doi: 10.1016/0003-2697(71)90443-x. [DOI] [PubMed] [Google Scholar]
  34. Mitra B., Hammes G. G. Membrane-protein structural mapping of chloroplast coupling factor in asolectin vesicles. Biochemistry. 1990 Oct 23;29(42):9879–9884. doi: 10.1021/bi00494a018. [DOI] [PubMed] [Google Scholar]
  35. Mukherjee S., Chattopadhyay A. Membrane organization at low cholesterol concentrations: a study using 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled cholesterol. Biochemistry. 1996 Jan 30;35(4):1311–1322. doi: 10.1021/bi951953q. [DOI] [PubMed] [Google Scholar]
  36. Mukherjee S., Ghosh R. N., Maxfield F. R. Endocytosis. Physiol Rev. 1997 Jul;77(3):759–803. doi: 10.1152/physrev.1997.77.3.759. [DOI] [PubMed] [Google Scholar]
  37. Mukherjee S., Maxfield F. R. Cholesterol: stuck in traffic. Nat Cell Biol. 1999 Jun;1(2):E37–E38. doi: 10.1038/10030. [DOI] [PubMed] [Google Scholar]
  38. Mukherjee S., Soe T. T., Maxfield F. R. Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. J Cell Biol. 1999 Mar 22;144(6):1271–1284. doi: 10.1083/jcb.144.6.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nezil F. A., Bloom M. Combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer thickness in model membranes. Biophys J. 1992 May;61(5):1176–1183. doi: 10.1016/S0006-3495(92)81926-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nichols J. W., Pagano R. E. Kinetics of soluble lipid monomer diffusion between vesicles. Biochemistry. 1981 May 12;20(10):2783–2789. doi: 10.1021/bi00513a012. [DOI] [PubMed] [Google Scholar]
  41. Pelham H. R., Munro S. Sorting of membrane proteins in the secretory pathway. Cell. 1993 Nov 19;75(4):603–605. doi: 10.1016/0092-8674(93)90479-A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Porter J. A., Young K. E., Beachy P. A. Cholesterol modification of hedgehog signaling proteins in animal development. Science. 1996 Oct 11;274(5285):255–259. doi: 10.1126/science.274.5285.255. [DOI] [PubMed] [Google Scholar]
  43. Raffy S., Teissié J. Control of lipid membrane stability by cholesterol content. Biophys J. 1999 Apr;76(4):2072–2080. doi: 10.1016/S0006-3495(99)77363-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schroeder F. Fluorescent sterols: probe molecules of membrane structure and function. Prog Lipid Res. 1984;23(2):97–113. doi: 10.1016/0163-7827(84)90009-2. [DOI] [PubMed] [Google Scholar]
  45. Schroeder F., Frolov A. A., Murphy E. J., Atshaves B. P., Jefferson J. R., Pu L., Wood W. G., Foxworth W. B., Kier A. B. Recent advances in membrane cholesterol domain dynamics and intracellular cholesterol trafficking. Proc Soc Exp Biol Med. 1996 Nov;213(2):150–177. doi: 10.3181/00379727-213-44047. [DOI] [PubMed] [Google Scholar]
  46. Schroeder F., Jefferson J. R., Kier A. B., Knittel J., Scallen T. J., Wood W. G., Hapala I. Membrane cholesterol dynamics: cholesterol domains and kinetic pools. Proc Soc Exp Biol Med. 1991 Mar;196(3):235–252. doi: 10.3181/00379727-196-43185. [DOI] [PubMed] [Google Scholar]
  47. Schroeder F., Woodford J. K., Kavecansky J., Wood W. G., Joiner C. Cholesterol domains in biological membranes. Mol Membr Biol. 1995 Jan-Mar;12(1):113–119. doi: 10.3109/09687689509038505. [DOI] [PubMed] [Google Scholar]
  48. Simons K., Ikonen E. Functional rafts in cell membranes. Nature. 1997 Jun 5;387(6633):569–572. doi: 10.1038/42408. [DOI] [PubMed] [Google Scholar]
  49. Simons K., Ikonen E. How cells handle cholesterol. Science. 2000 Dec 1;290(5497):1721–1726. doi: 10.1126/science.290.5497.1721. [DOI] [PubMed] [Google Scholar]
  50. Smutzer G., Crawford B. F., Yeagle P. L. Physical properties of the fluorescent sterol probe dehydroergosterol. Biochim Biophys Acta. 1986 Nov 17;862(2):361–371. doi: 10.1016/0005-2736(86)90239-7. [DOI] [PubMed] [Google Scholar]
  51. Sparrow C. P., Patel S., Baffic J., Chao Y. S., Hernandez M., Lam M. H., Montenegro J., Wright S. D., Detmers P. A. A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro. J Lipid Res. 1999 Oct;40(10):1747–1757. [PubMed] [Google Scholar]
  52. Tulenko T. N., Chen M., Mason P. E., Mason R. P. Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis. J Lipid Res. 1998 May;39(5):947–956. [PubMed] [Google Scholar]
  53. Xu X., London E. The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid domain formation. Biochemistry. 2000 Feb 8;39(5):843–849. doi: 10.1021/bi992543v. [DOI] [PubMed] [Google Scholar]
  54. Yeagle P. L. Cholesterol and the cell membrane. Biochim Biophys Acta. 1985 Dec 9;822(3-4):267–287. doi: 10.1016/0304-4157(85)90011-5. [DOI] [PubMed] [Google Scholar]
  55. Zimmerberg J. Are the curves in all the right places? Traffic. 2000 Apr;1(4):366–368. doi: 10.1034/j.1600-0854.2000.010409.x. [DOI] [PubMed] [Google Scholar]
  56. van Meer G., Stelzer E. H., Wijnaendts-van-Resandt R. W., Simons K. Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J Cell Biol. 1987 Oct;105(4):1623–1635. doi: 10.1083/jcb.105.4.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES