Chapter 22: Quantitation of protein-protein interactions: confocal FRET microscopy.

Förster resonance energy transfer (FRET) is an effective and high resolution method to monitor protein-protein interactions in live or fixed specimens. FRET can be used to estimate the distance between interacting protein molecules in vivo or in vitro using laser-scanning confocal FRET microscopy. The spectral overlap of donor and acceptor-essential for FRET-also generates a contamination of the FRET signal, which should be removed in order to carry out quantitative data analysis with confidence. Quantitative FRET data analysis addresses the wealth of information contained in the data set, once optimized FRET imaging has been completed. In this chapter, we describe step-by-step what the issues are in quantitative FRET data analysis, using membrane receptor trafficking and organization as an example. The assays described are applicable to many other biological applications.

[1]  Ammasi Periasamy,et al.  Protein localization in living cells and tissues using FRET and FLIM. , 2003, Differentiation; research in biological diversity.

[2]  S. Harrison,et al.  Crystal structure of the ectodomain of human transferrin receptor. , 1999, Science.

[3]  Y. Liu,et al.  Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. , 2001, Biophysical journal.

[4]  Ignacio A. Demarco,et al.  Dynamic Interactions between Pit-1 and C/EBPα in the Pituitary Cell Nucleus , 2006, Molecular and Cellular Biology.

[5]  B. Masters,et al.  Molecular Imaging, FRET Microscopy and Spectroscopy , 2006 .

[6]  Richard N. Day,et al.  Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. , 2001, Methods.

[7]  Cynthia F. Booker,et al.  Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein alpha in the living pituitary cell nucleus. , 2003, Molecular endocrinology.

[8]  D. Richardson.  Mysteries of the Transferrin-Transferrin Receptor 1 Interaction Uncovered , 2004, Cell.

[9]  Roger Y. Tsien,et al.  Seeing the Machinery of Live Cells , 1998, Science.

[10]  M. Matsuda,et al.  Analysis of the spatiotemporal activation of rho GTPases using Raichu probes. , 2006, Methods in enzymology.

[11]  T. Zal,et al.  Inhibition of T cell receptor-coreceptor interactions by antagonist ligands visualized by live FRET imaging of the T-hybridoma immunological synapse. , 2002, Immunity.

[12]  G. Bonamy,et al.  Receptor complexes cotransported via polarized endocytic pathways form clusters with distinct organizations. , 2007, Molecular biology of the cell.

[13]  M. Rao,et al.  Use of Forster's resonance energy transfer microscopy to study lipid rafts. , 2005, Biochimica et biophysica acta.

[14]  H. Wallrabe,et al.  5 – Confocal FRET Microscopy: Study of Clustered Distribution of Receptor–Ligand Complexes in Endocytic Membranes , 2005 .

[15]  T M Jovin,et al.  FRET microscopy demonstrates molecular association of non‐specific lipid transfer protein (nsL‐TP) with fatty acid oxidation enzymes in peroxisomes , 1998, The EMBO journal.

[16]  M. Edidin,et al.  Cutting Edge: Tapasin Is Retained in the Endoplasmic Reticulum by Dynamic Clustering and Exclusion from Endoplasmic Reticulum Exit Sites1 , 2002, The Journal of Immunology.

[17]  G. Bonamy,et al.  Cancer promoted by the oncoprotein v-ErbA may be due to subcellular mislocalization of nuclear receptors. , 2005, Molecular endocrinology.

[18]  Richard N. Day,et al.  Visualizing protein interactions in living cells using digitized GFP imaging and FRET microscopy. , 1999, Methods in cell biology.

[19]  Gaudenz Danuser,et al.  FRET or no FRET: a quantitative comparison. , 2003, Biophysical journal.

[20]  Horst Wallrabe,et al.  Imaging protein molecules using FRET and FLIM microscopy. , 2005, Current opinion in biotechnology.

[21]  S. Almo,et al.  Different cell surface oligomeric states of B7-1 and B7-2: Implications for signaling , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Horst Wallrabe,et al.  Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. , 2003, Methods.

[23]  T. Waldmann,et al.  IL-2 and IL-15 receptor alpha-subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Lakowicz,et al.  Anisotropy-based sensing with reference fluorophores. , 1999, Analytical biochemistry.

[25]  Ye Chen,et al.  Issues in confocal microscopy for quantitative FRET analysis , 2006, Microscopy research and technique.

[26]  A. Kenworthy,et al.  High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. , 2000, Molecular biology of the cell.

[27]  M. Edidin,et al.  Probing for membrane domains in the endoplasmic reticulum: retention and degradation of unassembled MHC class I molecules. , 2002, Molecular biology of the cell.

[28]  K. Hahn,et al.  Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR. , 2000, Methods in enzymology.

[29]  Benjamin Geiger,et al.  Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer , 2006, Journal of Cell Science.

[30]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[31]  Ammasi Periasamy,et al.  Characterization of two‐photon excitation fluorescence lifetime imaging microscopy for protein localization , 2004, Microscopy research and technique.

[32]  Da Xing,et al.  Mechanistic study of apoptosis induced by high-fluence low-power laser irradiation using fluorescence imaging techniques. , 2007, Journal of biomedical optics.

[33]  D. A. Lee,et al.  Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death. , 2003, American journal of physiology. Cell physiology.

[34]  R. Tsien,et al.  The Fluorescent Toolbox for Assessing Protein Location and Function , 2006, Science.

[35]  A. Kenworthy,et al.  In silico characterization of resonance energy transfer for disk-shaped membrane domains. , 2007, Biophysical journal.

[36]  A. Kenworthy,et al.  Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. , 2001, Methods.

[37]  Thomas Walz,et al.  Structure of the Human Transferrin Receptor-Transferrin Complex , 2004, Cell.

[38]  Joshua P. Mauldin,et al.  Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions , 2007, Journal of microscopy.

[39]  A. Kenworthy,et al.  Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer , 1998, The Journal of cell biology.

[40]  Horst Wallrabe,et al.  Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes. , 2003, Biophysical journal.

[41]  Ignacio A. Demarco,et al.  Monitoring dynamic protein interactions with photoquenching FRET , 2006, Nature Methods.

[42]  R. Clegg.  Fluorescence resonance energy transfer. , 2020, Current Opinion in Biotechnology.

[43]  M. Edidin,et al.  Clustering of Peptide-Loaded MHC Class I Molecules for Endoplasmic Reticulum Export Imaged by Fluorescence Resonance Energy Transfer1 , 2001, The Journal of Immunology.

[44]  Horst Wallrabe,et al.  One- and two-photon fluorescence resonance energy transfer microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes. , 2003, Journal of biomedical optics.

[45]  J. Swanson,et al.  Fluorescence resonance energy transfer-based stoichiometry in living cells. , 2002, Biophysical journal.

[46]  J. Mazurkiewicz,et al.  Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells. , 2004, Biochemistry.

[47]  Ammasi Periasamy,et al.  Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy. , 2003, Journal of biomedical optics.

[48]  D. Kelsell,et al.  Connexin interaction patterns in keratinocytes revealed morphologically and by FRET analysis , 2005, Journal of Cell Science.

[49]  B. Herman,et al.  Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. , 1998, Biophysical journal.

[50]  George H. Patterson,et al.  A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells , 2002, Science.

[51]  E. Sztul,et al.  Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome , 1994, The Journal of cell biology.

[52]  T M Jovin,et al.  Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I. , 1996, Proceedings of the National Academy of Sciences of the United States of America.