Skip to main content
SpringerLink
Log in

Multiphoton Laser-Scanning Microscopy and Spatial Analysis of Dehydroergosterol Distributions on Plasma Membrane of Living Cells

  • Protocol
Lipid Rafts

Part of the book series: Methods in Molecular Biology ((MIMB,volume 398))

Abstract

Multiphoton laser-scanning microscopy (MPLSM) imaging in combination with advanced image analysis techniques provides unique opportunities to visualize the arrangement of cholesterol in the plasma membrane (PM) of living cells. MPLSM makes possible the use of a naturally occurring sterol, dehydroergosterol (DHE), for observing sterol-enriched areas of the PM. Pure DHE has properties similar to cholesterol as observed in model and cellular membranes but with a conjugated double-bond system that fluoresces at ultraviolet wavelengths. MPLSM enables the excitation of DHE at infrared wavelengths that many laser-scanning microscopy systems are able to transmit effectively and that are less harmful to the cell. Thus, with the incorporation of DHE into living cells and the advent of MPLSM, real-time images of the cellular distribution of DHE can be obtained. In juxtaposition, notably the application of newly advanced techniques in image analysis, aids not only the identification and segmentation of sterol-rich regions of the PM of cells, but also the elucidation of the statistical nature of the observed patterns. In studies involving murine L-cell (Larpt-+K−) fibroblasts, DHE is shown to exhibit strong cluster patterns within the PM.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Singer, S. J. and Nicolson, G. L. (1972) The fluid mosaic model of the structure of cell membranes. Science 175, 720–731.

    Article  CAS  PubMed  Google Scholar 

  2. Schroeder, F., Atshaves, B. P., Gallegos, A. M., et al. (2005) Lipid rafts and caveolae organization, in Advances in Molecular and Cell Biology, (Frank, P. G. and Lisanti, M. P., eds.), Elsevier, Amsterdam, pp. 3–36.

    Google Scholar 

  3. Schroeder, F., Frolov, A. A., Murphy, E. J., et al. (1996) Recent advances in membrane cholesterol domain dynamics and intracellular cholesterol trafficking. Proc. Soc. Exp. Biol. Med. 213, 150–177.

    CAS  PubMed  Google Scholar 

  4. Brown, D. A. and London, E. (1998) Structure and origin of ordered lipid domains in biological membranes. J. Membr. Biol. 164, 103–114.

    Article  CAS  PubMed  Google Scholar 

  5. Hooper, N. M. (1999) Detergent-insoluble glycosphingolipid/cholesterol rich membrane domains, lipid rafts, and caveolae. Mol. Membr. Biol. 16, 145–156.

    Article  CAS  PubMed  Google Scholar 

  6. Schroeder, F., Gallegos, A. M., Atshaves, B. P., et al. (2001) Recent advances in membrane microdomains: rafts, caveolae and intracellular cholesterol trafficking. Exp. Biol. Med. 226, 873–890.

    CAS  Google Scholar 

  7. Edidin, M. (2001) Shrinking patches and slippery rafts: scales of domains in the plasma membrane. Trends Cell Biol. 11, 492–496.

    Article  CAS  PubMed  Google Scholar 

  8. Anderson, R. G. W. and Jacobson, K. (2002) A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 296, 1821–1825.

    Article  CAS  PubMed  Google Scholar 

  9. Lin, S. L. and Tian, P. (2003) Detailed computational analysis of a comprehensive set of group A rotavirus NSP4 proteins. Virus Genes 26, 271–282.

    Article  CAS  PubMed  Google Scholar 

  10. Bretscher, M. S. and Munro, S. (1993) Cholesterol and the Golgi apparatus. Science 261, 1280–1281.

    Article  CAS  PubMed  Google Scholar 

  11. Brown, R. E. (1998) Sphingolipid organization in biomembranes: what physical studies of model membranes reveal. J. Cell Sci. 111, 1–9.

    CAS  PubMed  Google Scholar 

  12. Lavie, Y. and Liscovitch, M. (2000) Changes in lipid and protein constituents of rafts and caveolae in multidrug resistant cancer cells and their functional consequences. Glycoconjugate J. 17, 253–259.

    Article  CAS  Google Scholar 

  13. Everson, W. V. and Smart, E. J. (2005) Caveolae and the regulation of cellular cholesterol homeostasis, in Caveolae and Lipid Rafts: Roles in Signal Transduction and the Pathogenesis of Human Disease, (Lisanti, M. P. and Frank, P. G. eds.), Elsevier Academic Press, San Diego, pp. 37–55.

    Google Scholar 

  14. Smart, E. J. (2005) Caveolae and the regulation of cellular cholesterol homeostasis, in Advances in Molecular and Cell Biology, (Lisanti, M. P. and Frank, P. G., eds.), Elsevier B. V., Amsterdam, 35p.

    Google Scholar 

  15. Smart, E. J. and van der Westhuyzen, D. R. (1998) Scavenger receptors, caveolae, caveolin, and cholesterol trafficking, in Intracellular Cholesterol Trafficking, (Chang, T. Y. and Freeman, D. A., eds.), Kluwer Academic Publishers, Boston, pp. 253–272.

    Google Scholar 

  16. Huang, H., Schroeder, F., Zeng, C., Estes, M. K., Schoer, J., and Ball, J. A. (2001) Membrane interactions of a novel viral enterotoxin: rotavirus nonstructural glycoprotein NSP4. Biochemistry 40, 4169–4180.

    Article  CAS  PubMed  Google Scholar 

  17. Huang, H., Schroeder, F., Estes, M. K., McPherson, T., and Ball, J. M. (2004) The interactions of rotavirus NSP4 C-terminal peptides with model membranes. Biochem. J. 380, 723–733.

    Article  CAS  PubMed  Google Scholar 

  18. Swaggerty, C. L., Huang, H., Lim, W. S., Schroeder, F., and Ball, J. A. (2004) Comparison of SIVmac239(352–382) and SIVsmmPBjJ41(360–390) enterotoxic synthetic peptides. Virology 320, 243–257.

    Article  CAS  PubMed  Google Scholar 

  19. Abrami, L., Liu, S., Cosson, P., Leppla, S. H., and van der Groot, F. G. (2003) Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-independent process. J. Cell Biol. 160, 321–328.

    Article  CAS  PubMed  Google Scholar 

  20. Sandvig, K. and van Deurs, B. (1999) Endocytosis and intracellular transport of ricin: recent discoveries. FEBS Lett. 452, 67–70.

    Article  CAS  PubMed  Google Scholar 

  21. Shin, J.-S. and Abraham, S. N. (2002) Caveolae as portals of entry for microbes. Microbes Infect. 3, 755–761.

    Article  Google Scholar 

  22. Norkin, L. C. (2001) Caveolae in the uptake and targeting of infectious agents and secreted toxins. Adv. Drug Delivery Rev. 49, 301–315.

    Article  CAS  Google Scholar 

  23. Bavari, S., Bosio, C. M., Wiegand, E., et al. (2002) Lipid raft microdomains. A gateway for comportmentalized trafficking of Ebola and Marburg viruses. J. Exp. Med. 195, 593–602.

    Article  CAS  PubMed  Google Scholar 

  24. Empig, C. J. and Goldsmith, M. A. (2002) Association of the caveola vesicular system with cellular entry by filoviruses. J. Virol. 76, 5266–5270.

    Article  CAS  PubMed  Google Scholar 

  25. Marjomaki, V., Pietiainen, V., Upla, P., et al. (2002) Internalization of Echovirus 1 in caveolae. J. Virol. 76, 1856–1865.

    Article  CAS  PubMed  Google Scholar 

  26. Scheiffele, P., Roth, M. G., and Simons, K. (1997) Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain. EMBO J. 16, 5501–5508.

    Article  CAS  PubMed  Google Scholar 

  27. Marsh, M. and Pelchen-Matthews, A. (2000) Endocytosis in viral replication. Traffic 1, 525–532.

    Article  CAS  PubMed  Google Scholar 

  28. Sieczkarski, S. B. and Whittaker, G. R. (2002) Dissecting virus entry via endocytosis. J. Gen. Virol. 83, 1535–1545.

    CAS  PubMed  Google Scholar 

  29. McIntosh, A., Gallegos, A., Atshaves, B. P., Storey, S., Kannoju, D., and Schroeder, F. (2003) Fluorescence and multiphoton imaging resolve unique structural forms of sterol in membranes of living cells. J. Biol. Chem. 278, 6384–6403.

    Article  CAS  PubMed  Google Scholar 

  30. Zhang, W., McIntosh, A., Xu, H., et al. (2005) Structural analysis of sterol distribution in the plasma membrane of living cells. Biochemistry 44, 2864–2984.

    Article  CAS  PubMed  Google Scholar 

  31. Gallegos, A. M., McIntosh, A. L., Atshaves, B. P., and Schroeder, F. (2004) Structure and cholesterol domain dynamics of an enriched caveolae/raft isolate. Biochem. J. 382, 451–461.

    Article  CAS  PubMed  Google Scholar 

  32. Atshaves, B. P., Gallegos, A., McIntosh, A. L., Kier, A. B., and Schroeder, F. (2003) Sterol carrier protein-2 selectively alters lipid composition and cholesterol dynamics of caveolae/lipid raft vs non-raft domains in L-cell fibroblast plasma membranes. Biochemistry 42, 14,583–14,598.

    Article  CAS  PubMed  Google Scholar 

  33. Eckert, G. P., Igbavboa, U., Muller, W., and Wood, W. G. (2003) Lipid rafts of purified mouse brain synaptosomes prepared with or without detergent reveal different lipid and protein domains. Brain Res. 962, 144–150.

    Article  CAS  PubMed  Google Scholar 

  34. Pike, L. J., Han, X., Chung, K.-N., and Gross, R. W. (2002) Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis. Biochemistry 41, 2075–2088.

    Article  CAS  PubMed  Google Scholar 

  35. Gallegos, A. M., Atshaves, B. P., Storey, S. M., et al. (2001) Gene structure, intracellular localization, and functional roles of sterol carrier protein-2. Prog. Lipid Res. 40, 498–563.

    Article  CAS  PubMed  Google Scholar 

  36. Gimpl, G. and Fahrenholz, F. (2000) Human oxytocin receptors in cholesterol-rich vs cholesterol-poor microdomains of the plasma membrane. Eur. J. Biochem. 267, 2483–2497.

    Article  CAS  PubMed  Google Scholar 

  37. Burger, K., Gimpl, G., and Fahrenholz, F. (2000) Regulation of receptor function by cholesterol. Cell Mol. Life Sci. 57, 1577–1592.

    Article  CAS  PubMed  Google Scholar 

  38. Smart, E. J., Ying, Y., Conrad, P. A., and Anderson, R. G. W. (1994) Caveolin moves from caveolae to the golgi apparatus in response to cholesterol oxidation. J. Cell Biol. 127, 1185–1197.

    Article  CAS  PubMed  Google Scholar 

  39. Fischer, R. T., Stephenson, F. A., Shafiee, A., and Schroeder, F. (1985) Structure and dynamic properties of dehydroergosterol, delta 5,7,9(11),22-ergostatetraen-3 beta-ol. J. Biol. Phys. 13, 13–24.

    Article  CAS  Google Scholar 

  40. Sica, D., Boniforti, L., and DiGiacomo, G. (1982) Sterols of Candida tropicalis grown on n-alkanes. Phytochemistry 21, 234–236.

    Article  CAS  Google Scholar 

  41. Delseth, C., Kashman, Y., and Djerassi, C. (1979) Ergosta-5,7,9(11),22-tetraen-3beta-ol and its 24epsilon-ethyl homolog, two new marine sterols from the red sea sponge Biemna fortis. Helv. Chim. Acta 62, 2037–2045.

    Article  CAS  Google Scholar 

  42. Nemecz, G., Fontaine, R. N., and Schroeder, F. (1988) A fluorescence and radiolabel study of sterol exchange between membranes. Biochim. Biophys. Acta 943, 511–521.

    Article  CAS  PubMed  Google Scholar 

  43. Schroeder, F. (1984) Fluorescent sterols: probe molecules of membrane structure and function. Prog. Lipid Res. 23, 97–113.

    Article  CAS  PubMed  Google Scholar 

  44. Schroeder, F., Jefferson, J. R., Kier, A. B., et al. (1991) Membrane cholesterol dynamics: cholesterol domains and kinetic pools. Proc. Soc. Exp. Biol. Med. 196, 235–252.

    CAS  PubMed  Google Scholar 

  45. Gallegos, A. M., Atshaves, B. P., Storey, S. M., Schoer, J., Kier, A. B., and Schroeder, F. (2002) Molecular and fluorescent sterol approaches to probing lysosomal membrane lipid dynamics. Chem. Phys. Lipids 116, 19–38.

    Article  CAS  PubMed  Google Scholar 

  46. Schroeder, F., Goh, E. H., and Heimberg, M. (1979) Regulation of the surface physical properties of the very low density lipoprotein. J. Biol. Chem. 254, 2456–2463.

    CAS  PubMed  Google Scholar 

  47. Bergeron, R. J. and Scott, J. (1982) Cholestatriene and ergostatetraene as in vivo and in vitro membrane and lipoprotein probes. J. Lipid Res. 23, 391–404.

    CAS  PubMed  Google Scholar 

  48. Fischer, R. T., Cowlen, M. S., Dempsey, M. E., and Schroeder, F. (1985) Fluorescence of delta 5,7,9(11),22-ergostatetraen-3 beta-ol in micelles, sterol carrier protein complexes, and plasma membranes. Biochemistry 24, 3322–3331.

    Article  CAS  PubMed  Google Scholar 

  49. Schroeder, F., Frolov, A. Schoer, J., et al. (1998) Intracellular sterol binding proteins, cholesterol transport and membrane domains, in Intracellular Cholesterol Trafficking. (Chang, T. Y. and Freeman, D. A., eds.), Kluwer Academic Publishers, Boston, pp. 213–234.

    Google Scholar 

  50. Stolowich, N. J., Petrescu, A. D., Huang, H., Martin, G., Scott, A. I., and Schroeder, F. (2002) Sterol carrier protein-2: structure reveals function. Cell Mol. Life Sci. 59, 193–212.

    Article  CAS  PubMed  Google Scholar 

  51. Loura, L. M. S. and Prieto, M. (1997) Dehydroergosterol structural organization in aqueous medium and in a model system of membranes. Biophys. J. 72, 2226–2236.

    Article  CAS  PubMed  Google Scholar 

  52. John, K., Kubelt, J., Muller, P., Wustner, D., and Hermann, A. (2002) Rapid transbilayer movement of the fluorescent sterol dehydroergosterol in lipid membranes. Biophys. J. 83, 1525–1534.

    Article  CAS  PubMed  Google Scholar 

  53. Schroeder, F., Butko, P., Nemecz, G., and Scallen, T. J. (1990) Interaction of fluorescent delta 5,7,9(11),22-ergostatetraen-3β-ol with sterol carrier protein-2. J. Biol. Chem. 265, 151–157.

    CAS  PubMed  Google Scholar 

  54. Anderson, R. (1998) The caveolae membrane system. Annu. Rev. Biochem. 67, 199–225.

    Article  CAS  PubMed  Google Scholar 

  55. Everson, W. V. and Smart, E. J. (2001) Influence of caveolin, cholesterol, and lipoproteins on nitric oxide synthase. TCM 11, 246–250.

    CAS  PubMed  Google Scholar 

  56. Brown, D. A. and London, E. (2000) Structure and function of sphingolipid-and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17,221–17,224.

    Article  CAS  PubMed  Google Scholar 

  57. Kirsch, C., Eckert, G. P., and Mueller, W. E. (2002) Statins affect cholesterol micro-domains in brain plasma membranes. Biochem. Pharmacol. 65, 843–856.

    Article  Google Scholar 

  58. Ruyle, W. V., Jacob, T. A., Chemerda, J. M., et al. (1953) The preparation of delta7,9(11)-allo-steroids by the action of mercuric acetate on delta7-allo steroids. J. Am. Chem. Soc. 75, 2604–2609.

    Article  CAS  Google Scholar 

  59. Fischer, R. T., Stephenson, F. A., Shafiee, A., and Schroeder, F. (1984) Delta 5,7,9(11)-Cholestatrien-3 beta-ol: a fluorescent cholesterol analogue. Chem. Phys. Lipids 36, 1–14.

    Article  CAS  PubMed  Google Scholar 

  60. Gonzalez, R. C., and Woods, R. E. (2002) Digital Image Processing, 2nd ed., Prentice Hall, NJ, 222–275.

    Google Scholar 

  61. Mukundan, R. and Ramakrishnan, K. R. (1998) Moment Function in Image Analysis: Theory and Applications. World Scientific, River Edge, NJ.

    Google Scholar 

  62. Sluzek, A. (1995) Identification and Inspection of 2-D Objects Using New Moment-Based Shape Descriptors. Patt. Recog. Lett. 16, 687–697.

    Article  Google Scholar 

  63. Hu, M. (1962) Visual-Pattern Recognition by Moment Invariants. IRE Trans. Info. Theory 8, 179–187.

    Google Scholar 

  64. Neter, J., Kutner, M. H., Wasserman, W., and Nachtsheim, C. J. (1996) Applied Linear Statistical Models, 4th ed., McGraw-Hill/Irwin, Chicago, IL, 188–210.

    Google Scholar 

  65. Diggle, P. J. (2003) Statistical Analysis of Spatial Point Patterns, 2nd ed., A Hodder Arnold Publication, London, 6–7, 9–10, 30–100.

    Google Scholar 

  66. Barnard, G. A. (1963) Contribution to the discussion of Professor Bartlett’s paper. J. R. Stat. Soc. B25, 294.

    Google Scholar 

  67. Baddeley, A. and Gill, R. D. (1997) Kaplan-Meier estimators of distance distributions for spatial point processes. Ann. Stat. 25, 263–292.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dept. of Physiology and Pharmacology, Texas A&M University, College Station, TX, 77843, USA

    Avery L. McIntosh, Barbara P. Atshaves, Huan Huang & Friedhelm Schroeder

  2. Dept. of Physiology, Texas A&M University, College Station, TX, 77843, USA

    Adalberto M. Gallegos & Ann B. Kier

  3. Computer Science Department, Texas A&M University, College Station, TX, 77843, USA

    Hai Xu & Jyh-Charn Liu

  4. Department of Statistics, Texas A&M University, College Station, TX, 77843, USA

    Weimin Zhang & Suojin Wang

Authors
  1. Avery L. McIntosh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Barbara P. Atshaves
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Adalberto M. Gallegos
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ann B. Kier
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Friedhelm Schroeder
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hai Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Weimin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Suojin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jyh-Charn Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Cell Biology, Duke University Medical Center, Durham, NC

    Thomas J. McIntosh

Rights and permissions

Reprints and permissions

Copyright information

© 2007 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

McIntosh, A.L. et al. (2007). Multiphoton Laser-Scanning Microscopy and Spatial Analysis of Dehydroergosterol Distributions on Plasma Membrane of Living Cells. In: McIntosh, T.J. (eds) Lipid Rafts. Methods in Molecular Biology, vol 398. Humana Press. https://doi.org/10.1007/978-1-59745-513-8_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-513-8_8

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-729-7

  • Online ISBN: 978-1-59745-513-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation