Protein-Protein-Protein Interactions in Membranes Measured by Triple Cross-Correlation of Confocal Images

Abstract

Protein-protein interactions in binary complexes have been measured successfully and quantitatively by fluorescence correlation spectroscopy in solution (Elson, BJ 101, 2855-2870 (2011)) and image correlation spectroscopy on cell surfaces (Kolin and Wiseman, Cell Biochem Biophys 49, 141-164 (2007)). These tools fail, however, to provide information about ternary complexes i.e. about protein-protein-protein interactions. It has been known for some time that higher order moments or correlations contain the relevant information (Palmer and Thompson, BJ 52, 257-270 (1987); Heinze, Jahnz, and Schwille, BJ 86, 506-516 (2004)) but it is only recently that triple correlation functions of ternary complexes have been measured in solution (Ridgeway, Millar, and Williamson, J. Phys Chem B 116, 1908-1919 (2012) and PNAS 109, 13614-13619 (2012)). Our present work shows how complete and quantitative information can be obtained for ternary complexes of membrane proteins from three confocal images from each of three distinctly labeled protein species by employing a combination of image correlation spectroscopy, image cross-correlation spectroscopy, image triple auto-correlation spectroscopy and image triple cross-correlation spectroscopy.