title: Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis.
abstract: Measuring protein interactions is key to understanding cell signaling mechanisms, but quantitative analysis of these interactions in situ has remained a major challenge. Here, we present spatial intensity distribution analysis (SpIDA), an analysis technique for image data obtained using standard fluorescence microscopy. SpIDA directly measures fluorescent macromolecule densities and oligomerization states sampled within single images. The method is based on fitting intensity histograms calculated from images to obtain density maps of fluorescent molecules and their quantal brightness. Because spatial distributions are acquired by imaging, SpIDA can be applied to the analysis of images of chemically fixed tissue as well as live cells. However, the technique does not rely on spatial correlations, freeing it from biases caused by subcellular compartmentalization and heterogeneity within tissue samples. Analysis of computer-based simulations and immunocytochemically stained GABA(B) receptors in spinal cord samples shows that the approach yields accurate measurements over a broader range of densities than established procedures. SpIDA is applicable to sampling within small areas (6 μm(2)) and reveals the presence of monomers and dimers with single-dye labeling. Finally, using GFP-tagged receptor subunits, we show that SpIDA can resolve dynamic changes in receptor oligomerization in live cells. The advantages and greater versatility of SpIDA over current techniques open the door to quantificative studies of protein interactions in native tissue using standard fluorescence microscopy.
authors.affiliation: Department of Physics, Alan Edwards Center for Research of Pain, Chemistry, and Anatomy and Cell Biology, McGill University Montréal, Québec, Canada H3A 2T5.
authors.forename: Antoine G
authors.initials: AG
authors.lastname: Godin
authors.forename: Santiago
authors.initials: S
authors.lastname: Costantino
authors.forename: Louis-Etienne
authors.initials: LE
authors.lastname: Lorenzo
authors.forename: Jody L
authors.initials: JL
authors.lastname: Swift
authors.forename: Mikhail
authors.initials: M
authors.lastname: Sergeev
authors.forename: Alfredo
authors.initials: A
authors.lastname: Ribeiro-da-Silva
authors.forename: Yves
authors.initials: Y
authors.lastname: De Koninck
authors.forename: Paul W
authors.initials: PW
authors.lastname: Wiseman
mesh_terms: D000818:Animals; D016466:CHO Cells; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018080:Receptors, GABA-B / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism
chemical_list: D018080:Receptors, GABA-B; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins