Single molecule imaging of membrane protein interactions and dynamics in living cells

Orateur : Max ULBRICH (Centre for Biological Signalling Studies, University Freiburg, Germany)

Many ion channels and receptors exhibit a heteromeric composition with different subunit types assembled in a defined or random fashion. Biochemical methods have certain limitations that can make it difficult to determine the exact number and the stoichiometry of the subunits in a membrane protein. With the recent development of highly sensitive cameras, it became possible to record from single fluorescent molecules within cells in a quantitative manner. We demonstrate that direct imaging of single membrane proteins in their native environment, the membrane of a living cell, allows to determine the multimerization state of a protein with high confidence. Initially we developed a method to count the number of subunits by counting the bleaching steps of GFP fused to the protein of interest. The observation of several different fluorescent proteins at the same time allows us to decipher complex assembly mechanisms. A particular challenge is the analysis of GPCR interactions because the equilibrium between monomers and dimers is dynamic and finely tuned and is postulated to be activation dependent for several clinically highly relevant receptors. Specific and effective labeling with multiple color tags is a prerequisite for the quantitative determination of this interaction, and we show how to tackle the problem of expressing multiple proteins at very low but equal levels in mammalian expression systems.

Télécharger dans mon agenda