title: Single-molecule imaging of receptor-receptor interactions.
abstract: Single-molecule imaging is a powerful tool for the study of dynamic molecular interactions in living cell plasma membranes. Herein, we describe a single-molecule imaging microscopy technique that can be used to measure lifetimes and densities of receptor dimers and oligomers. This method can be performed using a total internal reflection fluorescent microscope equipped with one or two high-sensitivity cameras. For dual-color observation, two images obtained synchronously in different colors are spatially corrected and then overlaid. Receptors must be expressed at low density in cell plasma membranes because high-density expression (>2 molecules/μm(2)) creates difficulty for tracking individual fluorescent spots. In addition, the receptors should be labeled with highly photostable fluorophores at high efficiency because short photobleaching lifetimes and low labeling efficiency of receptors reduce the probability of detecting dimers and oligomers. In this chapter, we describe methods for observing and detecting colocalization of the individual fluorescent spots of receptors labeled with fluorophores via small tags and the estimation of true dimer and oligomer lifetimes after correction with photobleaching lifetimes of fluorophores.
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan; National Centre for Biological Sciences (NCBS)/Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, India.
authors.forename: Kenichi G N
authors.initials: KG
authors.lastname: Suzuki
authors.forename: Rinshi S
authors.initials: RS
authors.lastname: Kasai
authors.forename: Takahiro K
authors.initials: TK
authors.lastname: Fujiwara
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kusumi
mesh_terms: D000213:Acyl Carrier Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D018957:CD59 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002630:Chemotactic Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003412:Cricetulus; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000213:Acyl Carrier Protein; D018957:CD59 Antigens; D002630:Chemotactic Factors; C498127:F2L peptide, human; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; C051590:CD59 protein, human
keywords: ACP-tag; Colocalization; Dimer; Dual-color observation; G protein-coupled receptor; GPI-anchored protein; Halo-tag; Oligomer; Rafts; Single-molecule imaging