Advertisement

Novel Insights in Membrane Biology Utilizing Fluorescence Recovery After Photobleaching

  • Amitabha ChattopadhyayEmail author
  • Md. Jafurulla
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 842)

Abstract

Understanding cellular signaling by membrane receptors in terms of their lateral dynamics represents a challenging area in contemporary biology. Fluorescence recovery after photobleaching (FRAP) offers a convenient approach to measure lateral diffusion and is extensively used for measuring lateral diffusion of lipids and proteins in membranes. In this review, we have provided an overview of the type of questions that could be addressed in membrane and receptor biology utilizing FRAP, with representative examples chosen from work carried out in our group. A major focus is on exploring new horizons in the organization and dynamics of G protein-coupled receptors (GPCRs) utilizing FRAP. We discuss how lateral dynamics of membrane receptors could serve as crucial determinants of their signaling. We envision that FRAP, along with confocal microscopy, could provide novel insight into dynamics of intracellular organelles.

Abbreviations

25-NBD-cholesterol

25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)methyl]amino]-27-norcholesterol

5-HT1A receptor

5-Hydroxytryptamine-1A receptor

5-HT1AR-EYFP

5-Hydroxytryptamine-1A receptor tagged to enhanced yellow fluorescent protein

DiIC18(3)

1,1′-Dioctadecyl-3,3,3′,3′,-tetramethylindocarbocyanine perchlorate

EYFP

Enhanced yellow fluorescent protein

FAST DiI

1,1′-Dilinoleyl-3,3,3′,3′,-tetramethylindocarbocyanine 4-chlorobenzenesulfonate

FRAP

Fluorescence recovery after photobleaching

GFP

Green fluorescent protein

GPCR

G protein-coupled receptor

NBD-PE

1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)

p-MPPI

4-(2′-Methoxy)phenyl-1-[2′-(N-2″-pyridinyl)-p-iodobenzamido]ethylpiperazine

Notes

Acknowledgments

We dedicate this paper to Prof. Michael Edidin (The Johns Hopkins University, Baltimore, MD) who pioneered the application of FRAP in biological membranes and in whose laboratory one of us (A.C.) learnt the nuts and bolts of FRAP measurements during a visit as a CSIR-Raman Fellow. Work in A.C.’s laboratory was supported by the Council of Scientific and Industrial Research, Govt. of India. A.C. is an Adjunct Professor at the Special Centre for Molecular Medicine of Jawaharlal Nehru University (New Delhi, India) and Indian Institute of Science Education and Research (Mohali, India), and Honorary Professor of the Jawaharlal Nehru Centre for Advanced Scientific Research (Bangalore, India). A.C. gratefully acknowledges J.C. Bose Fellowship (Dept. of Science and Technology, Govt. of India). Some of the work described in this article was carried out by former members of A.C.’s group whose contributions are gratefully acknowledged. We thank members of our laboratory for critically reading the manuscript.

References

  1. Aguila B, Simaan M, Laporte SA (2011) Study of G protein-coupled receptor/β-arrestin interactions within endosomes using FRAP. Methods Mol Biol 756:371–380CrossRefGoogle Scholar
  2. Baker A, Saulière A, Dumas F, Millot C, Mazères S, Lopez A, Salomé L (2007a) Functional membrane diffusion of G-protein coupled receptors. Eur Biophys J 36:849–860CrossRefGoogle Scholar
  3. Baker A-M, Saulière A, Gaibelet G, Lagane B, Mazères S, Fourage M, Bachelerie F, Salomé L, Lopez A, Dumas F (2007b) CD4 interacts constitutively with multiple CCR5 at the plasma membrane of living cells. A fluorescence recovery after photobleaching at variable radii approach. J Biol Chem 282:35163–35168CrossRefGoogle Scholar
  4. Calvert PD, Govardovskii VI, Krasnoperova N, Anderson RE, Lem J, Makino CL (2001) Membrane protein diffusion sets the speed of rod phototransduction. Nature 411:90–94CrossRefGoogle Scholar
  5. Celada P, Bortolozzi A, Artigas F (2013) Serotonin 5-HT1A receptors as targets for agents to treat psychiatric disorders: rationale and current status of research. CNS Drugs 27:703–716CrossRefGoogle Scholar
  6. Cézanne L, Lecat S, Lagane B, Millot C, Vollmer J-Y, Matthes H, Galzi J-L, Lopez A (2004) Dynamic confinement of NK2 receptors in the plasma membrane. Improved FRAP analysis and biological relevance. J Biol Chem 279:45057–45067CrossRefGoogle Scholar
  7. Dundr M, Misteli T (2003) Measuring dynamics of nuclear proteins by photobleaching. Curr Protoc Cell Biol 18:13.5.1–13.5.18CrossRefGoogle Scholar
  8. Edidin M (1992) Patches, posts and fences: proteins and plasma membrane domains. Trends Cell Biol 2:376–380Google Scholar
  9. 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, FL pp 109–135Google Scholar
  10. Edidin M, Stroynowski I (1991) Differences between the lateral organization of conventional and inositol phospholipid-anchored membrane proteins. A further definition of micrometer scale membrane domains. J Cell Biol 112:1143–1150CrossRefGoogle Scholar
  11. Ganguly S, Pucadyil TJ, Chattopadhyay A (2008) Actin cytoskeleton-dependent dynamics of the human serotonin1A receptor correlates with receptor signaling. Biophys J 95:451–463CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hagen GM, Roess DA, de León GC, Barisas BG (2005) High probe intensity photobleaching measurement of lateral diffusion in cell membranes. J Fluoresc 15:873–882CrossRefGoogle Scholar
  13. Haldar S, Chattopadhyay A (2009) Green fluorescent protein: a molecular lantern that illuminates the cellular interior. J Biosci 34:169–172CrossRefGoogle Scholar
  14. Haldar S, Chattopadhyay A (2013) Application of NBD-labeled lipids in membrane and cell biology. In: Mely Y, Duportail G (eds) Springer series on fluorescence, vol 13. Springer, Heidelberg, pp 37–50Google Scholar
  15. Heilker R, Wolff M, Tautermann CS, Bieler M (2009) G-protein-coupled receptor-focused drug discovery using a target class platform approach. Drug Discov Today 14:231–240CrossRefPubMedPubMedCentralGoogle Scholar
  16. Kahn CR (1976) Membrane receptors for hormones and neurotransmitters. J Cell Biol 70:261–286CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kalipatnapu S, Chattopadhyay A (2004) A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin1A receptor. FEBS Lett 576:455–460CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kalipatnapu S, Chattopadhyay A (2007) Membrane organization and function of the serotonin1A receptor. Cell Mol Neurobiol 27:1097–1116CrossRefPubMedPubMedCentralGoogle Scholar
  19. Klausner RD, Wolf DE (1980) Selectivity of fluorescent lipid analogues for lipid domains. Biochemistry 19:6199–6203CrossRefPubMedPubMedCentralGoogle Scholar
  20. Klonis N, Rug M, Harper I, Wickham M, Cowman A, Tilley L (2002) Fluorescence photobleaching analysis for the study of cellular dynamics. Eur Biophys J 31:36–51CrossRefGoogle Scholar
  21. Lippincott-Schwartz J, Snapp E, Kenworthy A (2001) Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2:444–456CrossRefGoogle Scholar
  22. Marguet D, Lenne P-F, Rigneault H, He H-T (2006) Dynamics in the plasma membrane: how to combine fluidity and order. EMBO J 25:3446–3457CrossRefPubMedPubMedCentralGoogle Scholar
  23. Mariappan I, Parnaik VK (2005) Sequestration of pRb by cyclin D3 causes intranuclear reorganization of lamin A/C during muscle cell differentiation. Mol Biol Cell 16:1948–1960CrossRefPubMedPubMedCentralGoogle Scholar
  24. 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 48:1778–1787CrossRefPubMedPubMedCentralGoogle Scholar
  25. Müller CP, Carey RJ, Huston JP, De Souza Silva MA (2007) Serotonin and psychostimulant addiction: focus on 5-HT1A-receptors. Prog Neurobiol 81:133–178CrossRefGoogle Scholar
  26. Nygaard R, Zou Y, Dror RO, Mildorf TJ, Arlow DH, Manglik A, Pan AC, Liu CW, Fung JJ, Bokoch MP, Thian FS, Kobilka TS, Shaw DE, Mueller L, Prosser RS, Kobilka BK (2013) The dynamic process of β2-adrenergic receptor activation. Cell 152:532–542CrossRefPubMedPubMedCentralGoogle Scholar
  27. Parker PD, Tilley L, Klonis N (2004) Plasmodium falciparum induces reorganization of host membrane proteins during intraerythrocytic growth. Blood 103:2404–2406CrossRefGoogle Scholar
  28. Peters R (1988) Lateral mobility of proteins and lipids in the red cell membrane and the activation of adenylate cyclase by β-adrenergic receptors. FEBS Lett 234:1–7CrossRefGoogle Scholar
  29. Pierce KL, Premont RT, Lefkowitz RJ (2002) Seven-transmembrane receptors. Nat Rev Mol Cell Biol 3:639–650CrossRefPubMedPubMedCentralGoogle Scholar
  30. Pucadyil TJ, Chattopadhyay A (2006) Effect of cholesterol on lateral diffusion of fluorescent lipid probes in native hippocampal membranes. Chem Phys Lipids 143:11–21CrossRefGoogle Scholar
  31. Pucadyil TJ, Chattopadhyay A (2007a) The human serotonin1A receptor exhibits G-protein-dependent cell surface dynamics. Glycoconj J 24:25–31CrossRefGoogle Scholar
  32. Pucadyil TJ, Chattopadhyay A (2007b) Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells. Biochim Biophys Acta 1768:655–668CrossRefPubMedPubMedCentralGoogle Scholar
  33. Pucadyil TJ, Kalipatnapu S, Harikumar KG, Rangaraj N, Karnik SS, Chattopadhyay A (2004) G-protein-dependent cell surface dynamics of the human serotonin1A receptor tagged to yellow fluorescent protein. Biochemistry 43:15852–15862CrossRefGoogle Scholar
  34. Pucadyil TJ, Kalipatnapu S, Chattopadhyay A (2005) The serotonin1A receptor: a representative member of the serotonin receptor family. Cell Mol Neurobiol 25:553–580CrossRefGoogle Scholar
  35. Pucadyil TJ, Mukherjee S, Chattopadhyay A (2007) Organization and dynamics of NBD-labeled lipids in membranes analyzed by fluorescence recovery after photobleaching. J Phys Chem B 111:1975–1983CrossRefGoogle Scholar
  36. Rawat SS, Zimmerman C, Johnson BT, Cho E, Lockett SJ, Blumenthal R, Puri A (2008) Restricted lateral mobility of plasma membrane CD4 impairs HIV-1 envelope glycoprotein mediated fusion. Mol Membr Biol 25:83–94CrossRefPubMedPubMedCentralGoogle Scholar
  37. Rosenbaum DM, Rasmussen SGF, Kobilka BK (2009) The structure and function of G-protein-coupled receptors. Nature 459:356–363CrossRefPubMedPubMedCentralGoogle Scholar
  38. Salomé L, Cazeils JL, Lopez A, Tocanne JF (1998) Characterization of membrane domains by frap experiments at variable observation areas. Eur Biophys J 27:391–402CrossRefGoogle Scholar
  39. Saulière-Nzeh Ndong A, Millot C, Corbani M, Mazères S, Lopez A, Salomé L (2010) Agonist-selective dynamic compartmentalization of human mu opioid receptor as revealed by resolutive FRAP analysis. J Biol Chem 285:14514–14520CrossRefPubMedPubMedCentralGoogle Scholar
  40. Schlyer S, Horuk R (2006) I want a new drug: G-protein-coupled receptors in drug development. Drug Discov Today 11:481–493CrossRefGoogle Scholar
  41. Schmidt P, Thomas L, Müller P, Scheidt HA, Huster D (2014) The G-protein-coupled neuropeptide Y receptor type 2 is highly dynamic in lipid membranes as revealed by solid-state NMR spectroscopy. Chemistry 20:4986–4992CrossRefGoogle Scholar
  42. Spink CH, Yeager MD, Feigenson GW (1990) Partitioning behavior of indocarbocyanine probes between coexisting gel and fluid phases in model membranes. Biochim Biophys Acta 1023:25–33CrossRefGoogle Scholar
  43. Staras K, Mikulincer D, Gitler D (2013) Monitoring and quantifying dynamic physiological processes in live neurons using fluorescence recovery after photobleaching. J Neurochem 126:213–222CrossRefGoogle Scholar
  44. Yechiel E, Edidin M (1987) Micrometer-scale domains in fibroblast plasma membranes. J Cell Biol 105:755–760CrossRefGoogle Scholar
  45. Zhang Y, DeVries ME, Skolnick J (2006) Structure modeling of all identified G protein-coupled receptors in the human genome. PLoS Comput Biol 2:e13CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

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

Personalised recommendations