Skip to main content
SpringerLink
Log in

Biophysical Analysis of Lipid Domains by Fluorescence Microscopy

  • Protocol
  • First Online:
Lipid Rafts

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2187))

Abstract

The study of the structure and dynamics of membrane domains in vivo is a challenging task. However, major advances could be achieved through the application of microscopic and spectroscopic techniques coupled with the use of model membranes, where the relations between lipid composition and the type, amount and properties of the domains present can be quantitatively studied.

This chapter provides protocols to study membrane organization and visualize membrane domains by fluorescence microscopy both in artificial membrane and living cell models of Gaucher Disease (GD ). We describe a bottom-up multiprobe methodology, which enables understanding how the specific lipid interactions established by glucosylceramide, the lipid that accumulates in GD , affect the biophysical properties of model and cell membranes, focusing on its ability to influence the formation, properties and organization of lipid raft domains. In this context, we address the preparation of (1) raft-mimicking giant unilamellar vesicles labeled with a combination of fluorophores that allow for the visualization and comprehensive characterization of those membrane domains and (2) human fibroblasts exhibiting GD phenotype to assess the biophysical properties of biological membrane in living cells using fluorescence microscopy.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
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. Saxton MJ, Jacobson K (1997) Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct 26:373–399. https://doi.org/10.1146/annurev.biophys.26.1.373

    Article  CAS  PubMed  Google Scholar 

  2. Kusumi A, Sako Y, Yamamoto M (1993) Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys J 65:2021–2040. https://doi.org/10.1016/S0006-3495(93)81253-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kusumi A, Nakada C, Ritchie K et al (2005) Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. Annu Rev Biophys Biomol Struct 34:351–378. https://doi.org/10.1146/annurev.biophys.34.040204.144637

    Article  CAS  PubMed  Google Scholar 

  4. Sezgin E, Levental I, Mayor S, Eggeling C (2017) The mystery of membrane organization: composition, regulation and roles of lipid rafts. Nat Rev Mol Cell Biol 18:361–374. https://doi.org/10.1038/nrm.2017.16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572. https://doi.org/10.1038/42408

    Article  CAS  PubMed  Google Scholar 

  6. Bieberich E (2018) Sphingolipids and lipid rafts: novel concepts and methods of analysis. Chem Phys Lipids 216:114–131. https://doi.org/10.1016/J.CHEMPHYSLIP.2018.08.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. de Almeida RFMM, Fedorov A, Prieto M (2003) Sphingomyelin/phosphatidylcholine/Cholesterol phase diagram: boundaries and composition of lipid rafts. Biophys J 85(4):2406–2416

    Article  Google Scholar 

  8. Castro BM, de Almeida RFM, Silva LC et al (2007) Formation of ceramide/sphingomyelin gel domains in the presence of an unsaturated phospholipid: a quantitative multiprobe approach. Biophys J 93:1639–1650. https://doi.org/10.1529/biophysj.107.107714

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Silva LC, de Almeida RFM, Castro BM et al (2007) Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid. Biophys J 92:502–516. https://doi.org/10.1529/biophysj.106.091876

    Article  CAS  PubMed  Google Scholar 

  10. Castro BM, Prieto M, Silva LC (2014) Ceramide: a simple sphingolipid with unique biophysical properties. Prog Lipid Res 54:53–67. https://doi.org/10.1016/j.plipres.2014.01.004

    Article  CAS  PubMed  Google Scholar 

  11. Zhang Y, Li X, Becker KA, Gulbins E (2009) Ceramide-enriched membrane domains—structure and function. Biochim Biophys Acta Biomembr 1788:178–183. https://doi.org/10.1016/j.bbamem.2008.07.030

    Article  CAS  Google Scholar 

  12. Ventura AE, Mestre B, Silva LC (2019) Ceramide domains in health and disease: a biophysical perspective. Adv Exp Med Biol 1159:79–108. https://doi.org/10.1007/978-3-030-21162-2_6

    Article  CAS  PubMed  Google Scholar 

  13. Pinto SN, Fernandes F, Fedorov A et al (2013) A combined fluorescence spectroscopy, confocal and 2-photon microscopy approach to re-evaluate the properties of sphingolipid domains. Biochim Biophys Acta 1828:2099–2110. https://doi.org/10.1016/j.bbamem.2013.05.011

    Article  CAS  PubMed  Google Scholar 

  14. Varela ARP, Ventura AE, Carreira AC et al (2016) Pathological levels of glucosylceramide change the biophysical properties of artificial and cell membranes. Phys Chem Chem Phys 19:340–346. https://doi.org/10.1039/c6cp07227e

    Article  PubMed  Google Scholar 

  15. Sezgin E (2017) Super-resolution optical microscopy for studying membrane structure and dynamics. J Phys Condens Matter 29:273001. https://doi.org/10.1088/1361-648X/aa7185

    Article  PubMed  PubMed Central  Google Scholar 

  16. Burgert A, Schlegel J, Becam J et al (2017) Characterization of plasma membrane ceramides by super-resolution microscopy. Angew Chem Int Ed Engl 56:6131–6135. https://doi.org/10.1002/anie.201700570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Fernandes F, Coutinho A, Prieto M, Loura LMS (2015) Electrostatically driven lipid-protein interaction: answers from FRET. Biochim Biophys Acta 1848:1837–1848. https://doi.org/10.1016/j.bbamem.2015.02.023

    Article  CAS  PubMed  Google Scholar 

  18. Bastos AEP, Scolari S, Stockl M, de Almeida RFM (2012) Applications of fluorescence lifetime spectroscopy and imaging to lipid domains in vivo. Methods Enzymol 504:57–81. https://doi.org/10.1016/B978-0-12-391857-4.00003-3

    Article  CAS  PubMed  Google Scholar 

  19. Cebecauer M, Amaro M, Jurkiewicz P et al (2018) Membrane lipid nanodomains. Chem Rev 118:11259–11297. https://doi.org/10.1021/acs.chemrev.8b00322

    Article  CAS  PubMed  Google Scholar 

  20. Koynova R, Caffrey M (1998) Phases and phase transitions of the phosphatidylcholines. Biochim Biophys Acta 1376:91–145. https://doi.org/10.1016/S0304-4157(98)00006-9

    Article  CAS  PubMed  Google Scholar 

  21. Ulrich AS, Sami M, Watts A (1994) Hydration of DOPC bilayers by differential scanning calorimetry. Biochim Biophys Acta Biomembr 1191:225–230. https://doi.org/10.1016/0005-2736(94)90253-4

    Article  CAS  Google Scholar 

  22. Biltonen RL, Lichtenberg D (1993) The use of differential scanning calorimetry as a tool to characterize liposome preparations. Chem Phys Lipids 64:129–142. https://doi.org/10.1016/0009-3084(93)90062-8

    Article  CAS  Google Scholar 

  23. Koynova R, Caffrey M (1995) Phases and phase transitions of the sphingolipids. Biochim Biophys Acta 1255:213–236. https://doi.org/10.1016/0005-2760(94)00202-A

    Article  PubMed  Google Scholar 

  24. Goñi FM, Alonso A, Bagatolli LA et al (2008) Phase diagrams of lipid mixtures relevant to the study of membrane rafts. Biochim Biophys Acta Mol Cell Biol Lipids 1781:665–684. https://doi.org/10.1016/J.BBALIP.2008.09.002

    Article  Google Scholar 

  25. de Almeida RFM, Loura LMS, Fedorov A, Prieto M (2005) Lipid rafts have different sizes depending on membrane composition: a time-resolved fluorescence resonance energy transfer study. J Mol Biol 346:1109–1120. https://doi.org/10.1016/j.jmb.2004.12.026

    Article  CAS  PubMed  Google Scholar 

  26. de Almeida RFM, Borst J, Fedorov A et al (2007) Complexity of lipid domains and rafts in giant unilamellar vesicles revealed by combining imaging and microscopic and macroscopic time-resolved fluorescence. Biophys J 93:539–553. https://doi.org/10.1529/biophysj.106.098822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Castro BM, Torreno-Pina JA, van Zanten TS, Gracia-Parajo MF (2013) Biochemical and imaging methods to study receptor membrane organization and association with lipid rafts. Methods Cell Biol 117:105–122. https://doi.org/10.1016/B978-0-12-408143-7.00006-2

    Article  CAS  PubMed  Google Scholar 

  28. Castro BM, de Almeida RFM, Fedorov A, Prieto M (2012) The photophysics of a Rhodamine head labeled phospholipid in the identification and characterization of membrane lipid phases. Chem Phys Lipids 165:311–319. https://doi.org/10.1016/j.chemphyslip.2012.02.007

    Article  CAS  PubMed  Google Scholar 

  29. Amaro M, Reina F, Hof M et al (2017) Laurdan and Di-4-ANEPPDHQ probe different properties of the membrane. J Phys D Appl Phys 50:134004. https://doi.org/10.1088/1361-6463/aa5dbc

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mazeres S, Fereidouni F, Joly E (2017) Using spectral decomposition of the signals from laurdan-derived probes to evaluate the physical state of membranes in live cells. F1000Res 6:763. https://doi.org/10.12688/f1000research.11577.2

    Article  PubMed  PubMed Central  Google Scholar 

  31. Owen DM, Rentero C, Magenau A et al (2011) Quantitative imaging of membrane lipid order in cells and organisms. Nat Protoc 7:24–35. https://doi.org/10.1038/nprot.2011.419

    Article  CAS  PubMed  Google Scholar 

  32. Varela ARPRP, Couto ASAS, Fedorov A et al (2016) Glucosylceramide reorganizes cholesterol-containing domains in a fluid phospholipid membrane. Biophys J 110:612–622. https://doi.org/10.1016/j.bpj.2015.12.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Khmelinskaia A, Ibarguren M, de Almeida RFM et al (2014) Changes in membrane organization upon spontaneous insertion of 2-hydroxylated unsaturated fatty acids in the lipid bilayer. Langmuir 30:2117–2128. https://doi.org/10.1021/la403977f

    Article  CAS  PubMed  Google Scholar 

  34. Varela ARP, Goncalves da Silva AMPS, Fedorov A et al (2013) Effect of glucosylceramide on the biophysical properties of fluid membranes. Biochim Biophys Acta 1828:1122–1130. https://doi.org/10.1016/j.bbamem.2012.11.018

    Article  CAS  PubMed  Google Scholar 

  35. Castro BM, Fedorov A, Hornillos V et al (2013) Edelfosine and miltefosine effects on lipid raft properties: membrane biophysics in cell death by antitumor lipids. J Phys Chem B 117:7929–7940. https://doi.org/10.1021/jp401407d

    Article  CAS  PubMed  Google Scholar 

  36. Rascol E, Devoisselle J-M, Chopineau J (2016) The relevance of membrane models to understand nanoparticles–cell membrane interactions. Nanoscale 8:4780–4798. https://doi.org/10.1039/C5NR07954C

    Article  CAS  PubMed  Google Scholar 

  37. Peetla C, Stine A, Labhasetwar V (2009) Biophysical interactions with model lipid membranes: applications in drug discovery and drug delivery. Mol Pharm 6:1264–1276. https://doi.org/10.1021/mp9000662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Stirnemann J, Belmatoug N, Camou F et al (2017) A review of gaucher disease pathophysiology, clinical presentation and treatments. Int J Mol Sci 18:E441. https://doi.org/10.3390/ijms18020441

    Article  CAS  PubMed  Google Scholar 

  39. Brady RO, Kanfer JN, Shapiro D (1965) Metabolism of glucocerebrosides. II. Evidence of an enzymatic deficiency in Gaucher’s disease. Biochem Biophys Res Commun 18:221–225. https://doi.org/10.1016/0006-291x(65)90743-6

    Article  CAS  PubMed  Google Scholar 

  40. Farfel-Becker T, Vitner EB, Kelly SL et al (2014) Neuronal accumulation of glucosylceramide in a mouse model of neuronopathic Gaucher disease leads to neurodegeneration. Hum Mol Genet 23:843–854. https://doi.org/10.1093/hmg/ddt468

    Article  CAS  PubMed  Google Scholar 

  41. Pandey MK, Burrow TA, Rani R et al (2017) Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease. Nature 543:108–112. https://doi.org/10.1038/nature21368

    Article  CAS  PubMed  Google Scholar 

  42. Haugland R (1996) The molecular probes handbook, 6th edn. CRC Press, Boca Raton

    Google Scholar 

  43. McClare CW (1971) An accurate and convenient organic phosphorus assay. Anal Biochem 39:527–530

    Article  CAS  Google Scholar 

  44. Silva L, de Almeida RFM, Fedorov A et al (2006) Ceramide-platform formation and -induced biophysical changes in a fluid phospholipid membrane. Mol Membr Biol 23:137–148. https://doi.org/10.1080/09687860500439474

    Article  CAS  PubMed  Google Scholar 

  45. Nikolaus J (2011) Hemifusion and lateral lipid domain partition in lipid membranes of different complexity

    Google Scholar 

  46. Pinto SN, Silva LC, Futerman AH, Prieto M (2011) Effect of ceramide structure on membrane biophysical properties: the role of acyl chain length and unsaturation. Biochim Biophys Acta Biomembr 1808:2753–2760. https://doi.org/10.1016/j.bbamem.2011.07.023

    Article  CAS  Google Scholar 

  47. Varela ARP, Gonçalves da Silva AMPS, Fedorov A et al (2014) Influence of intracellular membrane pH on Sphingolipid Organization and Membrane Biophysical Properties. Langmuir 30:4094–4104. https://doi.org/10.1021/la5003397

    Article  CAS  PubMed  Google Scholar 

  48. (2014) Characterization of ceramide-induced changes on the biophysical properties of cellular membranes. PT

    Google Scholar 

  49. Pinto SN, Silva LC, de Almeida RFM, Prieto M (2008) Membrane domain formation, interdigitation, and morphological alterations induced by the very long chain asymmetric C24:1 ceramide. Biophys J 95:2867–2879. https://doi.org/10.1529/biophysj.108.129858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Saxena K, Duclos RI, Zimmermann P et al (1999) Structure and properties of totally synthetic galacto- and gluco-cerebrosides. J Lipid Res 40:839–849

    CAS  PubMed  Google Scholar 

  51. Shah J, Atienza JM, Duclos RIJ et al (1995) Structural and thermotropic properties of synthetic C16:0 (palmitoyl) ceramide: effect of hydration. J Lipid Res 36:1936–1944

    CAS  PubMed  Google Scholar 

  52. Garidel P (2006) Structural organisation and phase behaviour of a stratum corneum lipid analogue: ceramide 3A. Phys Chem Chem Phys 8:2265–2275. https://doi.org/10.1039/b517540b

    Article  CAS  PubMed  Google Scholar 

  53. Marques JT, Cordeiro AM, Viana AS et al (2015) Formation and properties of membrane-ordered domains by phytoceramide: role of sphingoid base hydroxylation. Langmuir 31:9410–9421. https://doi.org/10.1021/acs.langmuir.5b02550

    Article  CAS  PubMed  Google Scholar 

  54. Sarmento MJ, Prieto M, Fernandes F (2012) Reorganization of lipid domain distribution in giant unilamellar vesicles upon immobilization with different membrane tethers. Biochim Biophys Acta 1818:2605–2615. https://doi.org/10.1016/j.bbamem.2012.05.028

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors acknowledge Fundação para a Ciência e Tecnologia, FCT Portugal, for SFRH/BD/110093/2015 to A.E.V.; SFRH/BD/102933/2014 to T.C.B.S.; PTDC/BBB-BQB/3710/2014, PTDC/BBB-BQB/6071/2014, PTDC/BIA-BFS/29448/2017, UIDB/00100/2020, and Investigador FCT to L.C. Silva (IF/00437/2014).

Author information

Author notes

  1. Ana E. Ventura and Tânia C. B. Santos contributed equally to this work.

Authors and Affiliations

  1. Research Institute for medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal

    Ana E. Ventura, Tânia C. B. Santos & Liana C. Silva

  2. CQFM-IN and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Ana E. Ventura & Tânia C. B. Santos

  3. Centro de Química e Bioquímica, Centro de Química Estrutural, DQB, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal

    Joaquim T. Marquês & Rodrigo F. M. de Almeida

Authors
  1. Ana E. Ventura
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tânia C. B. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joaquim T. Marquês
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rodrigo F. M. de Almeida
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Liana C. Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Liana C. Silva .

Editor information

Editors and Affiliations

  1. Department of Physiology, University of Kentucky Medical School, Lexington, KY, USA

    Erhard Bieberich

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ventura, A.E., Santos, T.C.B., Marquês, J.T., de Almeida, R.F.M., Silva, L.C. (2021). Biophysical Analysis of Lipid Domains by Fluorescence Microscopy. In: Bieberich, E. (eds) Lipid Rafts. Methods in Molecular Biology, vol 2187. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0814-2_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0814-2_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0813-5

  • Online ISBN: 978-1-0716-0814-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation