title: Analyzing receptor assemblies in the cell membrane using fluorescence anisotropy imaging with TIRF microscopy.
abstract: Signaling within and between animal cells is controlled by the many receptor proteins in their membrane. They variously operate as trans-membrane monomers and homo- or hetero-dimers, and may assemble with ion-channels: analyses thereof are needed in studies of receptor actions in tissue physiology and pathology. Interactions between membrane proteins are detectable when pre-labeled with fluorophores, but a much fuller analysis is achievable via advanced optical techniques on living cells. In this context, the measurement of polarization anisotropy in the emitted fluorescence has been the least exploited. Here we demonstrate its methodology and particular advantages in the study of receptor protein assembly. Through excitation in both TIRF and EPI fluorescence illumination modes we are able to quantify and suppress contributions to the signal from extraneous intra-cellular fluorescence, and we show that the loss of fluorescence-polarization measured in membrane proteins reports on receptor protein assembly in real time. Receptor monomers and homo-dimers in the cell membrane can be analyzed quantitatively and for homo-dimers only a single fluorescent marker is needed, thus suppressing ambiguities that arise in alternative assays, which require multiple label moieties and which are thus subject to stoichiometric uncertainty.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom; Analytical Science Division, National Physical Laboratory, Teddington, Middlesex, United Kingdom; Department of Optics and Quantum Electronics, University of Szeged, Szeged, Hungary.
authors.forename: Miklos
authors.initials: M
authors.lastname: Erdelyi
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Joseph
authors.initials: J
authors.lastname: Simon
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Barnard
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D005454:Fluorescence Polarization; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D058912:Purinergic P2Y Receptor Agonists / Q000494:pharmacology; D058921:Purinergic P2Y Receptor Antagonists / Q000494:pharmacology; D058472:Receptors, Purinergic P2Y1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
chemical_list: C495475:2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate; C485182:Cyan Fluorescent Protein; D003838:Deoxyadenine Nucleotides; D058912:Purinergic P2Y Receptor Agonists; D058921:Purinergic P2Y Receptor Antagonists; D058472:Receptors, Purinergic P2Y1; D013873:Thionucleotides; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; C008501:2-methylthio-ATP