CIUSSS Capitale nationale logo

Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis.

Publication Type:

Journal Article

Source:

Proc Natl Acad Sci U S A, Volume 108, Issue 17, p.7010-5 (2011)

Keywords:

Animals, CHO Cells, Computer Simulation, Cricetinae, Cricetulus, Green Fluorescent Proteins, Protein Multimerization, Rats, Rats, Sprague-Dawley, Receptors, GABA-B, Recombinant Fusion Proteins, Spinal Cord

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.

Funding / Support / Partners

logo FRQ-S logo ctrn logo fci logo cihr irsc logo nserc logo MESISentinelle nord