Advertisement

Exploring Membrane Lipid and Protein Diffusion by FRAP

  • Parijat Sarkar
  • Amitabha ChattopadhyayEmail author
Protocol
  • 27 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Knowledge of membrane dynamics is crucial since it allows us to understand membrane function. Fluorescence recovery after photobleaching (FRAP) is a widely used technique to monitor diffusion of lipids and proteins in biological membranes. We outline here general aspects of FRAP, followed by a step-by-step guide to carry out FRAP measurements for exploring diffusion of fluorescently labeled lipids and proteins in membranes of attached cells and membranes of Candida albicans. In this process, we have provided detailed hands-on tips, judicious use of which would ensure reliability and quality of acquired FRAP data and associated analysis.

Keywords

FRAP Confocal microscopy Lateral diffusion Mobile fraction Diffusion coefficient DiI GFP 

Notes

Acknowledgments

A.C. gratefully acknowledges support from SERB Distinguished Fellowship (Department of Science and Technology, Govt. of India). P.S. thanks the Council of Scientific and Industrial Research for the award of a Shyama Prasad Mukherjee Fellowship. A.C. is a Distinguished Visiting Professor at the Indian Institute of Technology Bombay (Mumbai), Adjunct Professor at the RMIT University (Melbourne, Australia), Tata Institute of Fundamental Research (Mumbai), and Indian Institute of Science Education and Research (Kolkata), and an Honorary Professor at the Jawaharlal Nehru Centre for Advanced Scientific Research (Bengaluru). We thank members of the Chattopadhyay laboratory for their comments and discussions.

Conflicts of Interest

There are no conflicts of interest to declare.

References

  1. 1.
    Edidin M (1994) Fluorescence photobleaching and recovery, FPR, in the analysis of membrane structure and dynamics. In: Damjanovich S, Edidin M, Szöllõsi J, Trón L (eds) Mobility and proximity in biological membranes. CRC, Boca Raton, pp 109–135Google Scholar
  2. 2.
    Lippincott-Schwartz J, Snapp E, Kenworthy A (2001) Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2:444–456PubMedGoogle Scholar
  3. 3.
    Kenworthy AK (2007) Fluorescence recovery after photobleaching studies of lipid rafts. In: McIntosh TJ (ed) Lipid Rafts, Methods in molecular biology, vol 398. Humana, Totowa, pp 179–192Google Scholar
  4. 4.
    Carisey A, Stroud M, Tsang R, Ballestrem C (2011) Fluorescence recovery after photobleaching. In: Wells CM, Parsons M (eds) Cell migration: developmental methods and protocols, Methods in molecular biology, vol 769. Humana, New York, pp 387–402Google Scholar
  5. 5.
    Jafurulla M, Chattopadhyay A (2015) Novel insights in membrane biology utilizing fluorescence recovery after photobleaching. Adv Exp Med Biol 842:27–40PubMedGoogle Scholar
  6. 6.
    Sarkar P, Chattopadhyay A (2019) Exploring membrane organization at varying spatiotemporal resolutions utilizing fluorescence-based approaches: implications in membrane biology. Phys Chem Chem Phys 21:11554–11563PubMedGoogle Scholar
  7. 7.
    Lippincott-Schwartz J, Snapp EL, Phair RD (2018) The development and enhancement of FRAP as a key tool for investigating protein dynamics. Biophys J 115:1146–1155PubMedPubMedCentralGoogle Scholar
  8. 8.
    Poo M, Cone RA (1974) Lateral diffusion of rhodopsin in the photoreceptor membrane. Nature 247:438–441PubMedGoogle Scholar
  9. 9.
    Liebman PA, Entine G (1974) Lateral diffusion of visual pigment in photoreceptor disk membranes. Science 185:457–459PubMedGoogle Scholar
  10. 10.
    Peters R, Peters J, Tews KH, Bähr W (1974) A microfluorimetric study of translational diffusion in erythrocyte membranes. Biochim Biophys Acta 367:282–294PubMedGoogle Scholar
  11. 11.
    Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16:1055–1069PubMedPubMedCentralGoogle Scholar
  12. 12.
    Jacobson K, Derzko Z, Wu E-S, Hou Y, Poste G (1976) Measurement of the lateral mobility of cell surface components in single, living cells by fluorescence recovery after photobleaching. J Supramol Struct 5:565(417)–576(428)Google Scholar
  13. 13.
    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805PubMedGoogle Scholar
  14. 14.
    Reits EAJ, Neefjes JJ (2001) From fixed to FRAP: measuring protein mobility and activity in living cells. Nat Cell Biol 3:E145–E147PubMedGoogle Scholar
  15. 15.
    Lagerholm BC, Weinreb GE, Jacobson K, Thompson NL (2005) Detecting microdomains in intact cell membranes. Annu Rev Phys Chem 56:309–336PubMedGoogle Scholar
  16. 16.
    Valdez-Taubas J, Pelham HRB (2003) Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling. Curr Biol 13:1636–1640PubMedGoogle Scholar
  17. 17.
    Ganguly S, Pucadyil TJ, Chattopadhyay A (2008) Actin cytoskeleton-dependent dynamics of the human serotonin1A receptor correlates with receptor signaling. Biophys J 95:451–463PubMedPubMedCentralGoogle Scholar
  18. 18.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544PubMedGoogle Scholar
  19. 19.
    White J, Stelzer E (1999) Photobleaching GFP reveals protein dynamics inside live cells. Trends Cell Biol 9:61–65PubMedGoogle Scholar
  20. 20.
    Lippincott-Schwartz J, Patterson GH (2003) Development and use of fluorescent protein markers in living cells. Science 300:87–91PubMedGoogle Scholar
  21. 21.
    Haldar S, Chattopadhyay A (2009) Green fluorescent protein: the molecular lantern that illuminates the cellular interior. J Biosci 34:169–172PubMedGoogle Scholar
  22. 22.
    Sarkar P, Chattopadhyay A (2018) GFP fluorescence: a few lesser-known nuggets that make it work. J Biosci 43:421–430PubMedGoogle Scholar
  23. 23.
    Fucile S, Palma E, Martínez-Torres A, Miledi R, Eusebi F (2002) The single-channel properties of human acetylcholine α7 receptors are altered by fusing α7 to the green fluorescent protein. Proc Natl Acad Sci USA 99:3956–3961PubMedGoogle Scholar
  24. 24.
    Saffman PG, Delbrück M (1975) Brownian motion in biological membranes. Proc Natl Acad Sci USA 72:3111–3113PubMedGoogle Scholar
  25. 25.
    Maekawa M, Fairn GD (2014) Molecular probes to visualize the location, organization and dynamics of lipids. J Cell Sci 127:4801–4812PubMedGoogle Scholar
  26. 26.
    Klymchenko AS, Kreder R (2014) Fluorescent probes for lipid rafts: from model membranes to living cells. Chem Biol 21:97–113PubMedGoogle Scholar
  27. 27.
    Klausner RD, Wolf DE (1980) Selectivity of fluorescent lipid analogues for lipid domains. Biochemistry 19:6199–6203PubMedGoogle Scholar
  28. 28.
    Spink CH, Yeager MD, Feigenson GW (1990) Partitioning behavior of indocarbocyanine probes between coexisting gel and fluid phases in model membranes. Biochim Biophys Acta 1023:25–33PubMedGoogle Scholar
  29. 29.
    Kalipatnapu S, Chattopadhyay A (2004) A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin1A receptor. FEBS Lett 576:455–460PubMedGoogle Scholar
  30. 30.
    Baumgart T, Hunt G, Farkas ER, Webb WW, Feigenson GW (2007) Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. Biochim Biophys Acta 1768:2182–2194PubMedPubMedCentralGoogle Scholar
  31. 31.
    Mukherjee S, Soe TT, Maxfield FR (1999) Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. J Cell Biol 144:1271–1284PubMedPubMedCentralGoogle Scholar
  32. 32.
    Pucadyil TJ, Chattopadhyay A (2006) Effect of cholesterol on lateral diffusion of fluorescent lipid probes in native hippocampal membranes. Chem Phys Lipids 143:11–21PubMedGoogle Scholar
  33. 33.
    Mukhopadhyay K, Prasad T, Saini P, Pucadyil TJ, Chattopadhyay A, Prasad R (2004) Membrane sphingolipid-ergosterol interactions are important determinants of multidrug resistance in Candida albicans. Antimicrob Agents Chemother 48:1778–1787PubMedPubMedCentralGoogle Scholar
  34. 34.
    Soumpasis DM (1983) Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys J 41:95–97PubMedPubMedCentralGoogle Scholar
  35. 35.
    Lippincott-Schwartz J, Presley JF, Zaal KJM, Hirschberg K, Miller CD, Ellenberg J (1999) Monitoring the dynamics and mobility of membrane proteins tagged with green fluorescent protein. Methods Cell Biol 58:261–281PubMedGoogle Scholar
  36. 36.
    Yang F, Moss LG, Phillips GN Jr (1996) The molecular structure of green fluorescent protein. Nat Biotechnol 14:1246–1251PubMedGoogle Scholar
  37. 37.
    Prendergast FG (1999) Biophysics of the green fluorescent protein. Methods Cell Biol 58:1–18PubMedGoogle Scholar
  38. 38.
    Haldar S, Chattopadhyay A (2007) Dipolar relaxation within the protein matrix of the green fluorescent protein: a red edge excitation shift study. J Phys Chem B 111:14436–14439PubMedGoogle Scholar
  39. 39.
    Weiss M (2004) Challenges and artifacts in quantitative photobleaching experiments. Traffic 5:662–671PubMedGoogle Scholar
  40. 40.
    Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388:355–358PubMedGoogle Scholar
  41. 41.
    McAnaney TB, Zeng W, Doe CFE, Bhanji N, Wakelin S, Pearson DS, Abbyad P, Shi X, Boxer SG, Bagshaw CR (2005) Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism. Biochemistry 44:5510–5524PubMedGoogle Scholar
  42. 42.
    Lippincott-Schwartz J, Altan-Bonnet N, Patterson GH (2003) Photobleaching and photoactivation: following protein dynamics in living cells. Nat Cell Biol 5:S7–S14Google Scholar
  43. 43.
    Snapp EL, Altan N, Lippincott-Schwartz J (2003) Measuring protein mobility by photobleaching GFP chimeras in living cells. Curr Prot Cell Biol 21(1):1–21.1.24Google Scholar
  44. 44.
    Khandelwal NK, Sarkar P, Gaur NA, Chattopadhyay A, Prasad R (2018) Phosphatidylserine decarboxylase governs plasma membrane fluidity and impacts drug susceptibilities of Candida albicans cells. Biochim Biophys Acta 1860:2308–2319Google Scholar
  45. 45.
    Pucadyil TJ, Chattopadhyay A (2006) Confocal fluorescence recovery after photobleaching of green fluorescent protein in solution. J Fluoresc 16:87–94PubMedGoogle Scholar
  46. 46.
    Axelrod D (1977) Cell surface heating during fluorescence photobleaching recovery experiments. Biophys J 18:129–131PubMedPubMedCentralGoogle Scholar
  47. 47.
    De Los SC, Chang C-W, Mycek M-A, Cardullo RA (2015) FRAP, FLIM, and FRET: detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy. Mol Reprod Dev 82:587–604Google Scholar
  48. 48.
    Koppel DE, Sheetz MP, Schindler M (1980) Lateral diffusion in biological membranes. A normal-mode analysis of diffusion on a spherical surface. Biophys J 30:187–192PubMedPubMedCentralGoogle Scholar
  49. 49.
    Pucadyil TJ, Chattopadhyay A (2007) Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells. Biochim Biophys Acta 1768:655–668PubMedGoogle Scholar
  50. 50.
    Ganguly S, Chattopadhyay A (2010) Cholesterol depletion mimics the effect of cytoskeletal destabilization on membrane dynamics of the serotonin1A receptor: a zFCS study. Biophys J 99:1397–1407PubMedPubMedCentralGoogle Scholar
  51. 51.
    Kreutzberger AJB, Ji M, Aaron J, Mihaljević L, Urban S (2019) Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion. Science 363:eaao0076PubMedPubMedCentralGoogle Scholar
  52. 52.
    Feder TJ, Brust-Mascher I, Slattery JP, Baird B, Webb WW (1996) Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys J 70:2767–2773PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.CSIR-Centre for Cellular and Molecular BiologyHyderabadIndia

Personalised recommendations