Determination of oligomeric states of proteins via dual-color colocalization with single molecule localization microscopy

Abstract

The oligomeric state of plasma membrane proteins is the result of the interactions between 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 oligomeric states 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 oligomeric states of proteins, subunits are labeled with fluorescent proteins that only emit light following activation or conversion at different wavelengths. Typically, individual molecules 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 recall rate 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 oligomeric state of plasma membrane proteins. 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 localizations 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 oligomeric state of two SLC26 multifunctional anion exchangers and to determine the oligomeric state of four members of the SLC17 family of organic anion transporters.

Oligomerization and cellular localization of SLC26A11

“…SLC26 anion exchangers form dimeric assemblies that can be demonstrated by structural studies (22,(43)(44)(45)(46)(47)(48) as well as by native gel electrophoresis (49). Since both experimental approaches do not exclude formation of higher oligomers, which are not sufficiently stable to tolerate solubilization/purification, we recently used a new dual-color colocalization (DCC) approach for the accurate determination of subunit stoichiometries of membrane proteins in situ (50,51). This approach also demonstrated that a dimeric assembly is the predominant subunit stoichiometry of SLC26 proteins, without indications of higher oligomeric states.…”

Dual-color Colocalization in Single-molecule Localization Microscopy to Determine the Oligomeric State of Proteins in the Plasma Membrane

“…Please consult with your local experts, if necessary, to determine the suitability of any available microscope. As a guide, the microscope we used in our original publication ( Tan et al, 2022 ) was outfitted with the following components ( Tang et al, 2015 ):…”

“…Furthermore, the binomial distributions of signals are often spuriously affected by a significant amount of background signals that are almost indistinguishable from true signals. We recently published a novel strategy, dual color colocalization (DCC) ( Tan et al, 2022 ), circumventing these problems, as illustrated in the Graphical overview. The DCC strategy uses two covalently linked fluorescent proteins, serving as the marker (M) and the indicator (F), fused to the protein of interest.…”

“…as part of Research Unit FOR 5046, project P4. We acknowledge the original research paper ( Tan et al, 2022 ), from which this protocol is derived, as the primary reference when additional information is needed.…”

Determination of oligomeric organization of membrane proteins from native membranes at nanoscale-spatial and single-molecule resolution

“…Yet determining the precise oligomeric status for membrane proteins and structural details underlying these assemblies remains a challenging problem. Membrane protein oligomeric states are commonly characterized in cells or on membrane-mimetic platforms by diffusion and fluctuation measurements using fluorescence correlation spectroscopy (FCS) 8,9 , subunit counting using photobleaching step analysis 10,11 , single-molecule localization microscopy (SMLM) 4,5,[12][13][14] , and single-particle tracking [15][16][17] . Cell-based readouts preserve the native membrane environment but often lack sufficient spatial and molecular resolution to distinguish genuine oligomeric assemblies from spatially-proximal colocalization 4,5,18,19 .…”