Determination of protein stoichiometries via dual-color colocalization with single molecule localization microscopy

Abstract

The stoichiometry of plasma membrane protein complexes is a result of the interactions between the individual proteins and an important determinant of their function. Most approaches used to address this question rely on extracting these complexes from their native environment, which may disrupt weaker interactions. Therefore, microscopy techniques have been increasingly used in recent years to determine protein stoichiometries in situ. Classical light microscopy suffers from insufficient resolution, but super-resolution methods such as single molecule localization microscopy (SMLM) can circumvent this problem. When using SMLM to determine protein stoichiometries, subunits are labeled with fluorescent proteins that only emit light following activation or conversion at different wavelengths. Typically, individual signals are counted based on a binomial distribution analysis of emission events detected within the same diffraction-limited volume. This strategy requires low background noise, a high detection efficiency for the fluorescent tag and intensive post-imaging data processing. To overcome these limitations, we developed a new method based on SMLM to determine the stoichiometry of plasma membraneproteins. Our dual-color colocalization (DCC) approach allows for accurate in situ counting even with low efficiencies of fluorescent protein detection. In addition, it is robust in the presence of background signals and does not require temporal clustering of signals from individual proteins within the same diffraction limited volume, which greatly simplifies data acquisition and processing. We used DCC-SMLM to resolve the controversy surrounding the stoichiometries of two SLC26 multifunctional anion exchangers and to determine the stoichiometries of four members of the SLC17 family of organic anion transporters.