Single-molecule tracking to determine the abundances and stoichiometries of freely-diffusing protein complexes in living cells: Past applications and future prospects.

Most biological processes in living cells rely on interactions between proteins. Live-cell compatible approaches that can quantify to what extent a given protein participates in homo- and hetero-oligomeric complexes of different size and subunit composition are therefore critical to advance our understanding of how cellular physiology is governed by these molecular interactions. Biomolecular complex formation changes the diffusion coefficient of constituent proteins, and these changes can be measured using fluorescence microscopy-based approaches, such as single-molecule tracking, fluorescence correlation spectroscopy, and fluorescence recovery after photobleaching. In this review, we focus on the use of single-molecule tracking to identify, resolve, and quantify the presence of freely-diffusing proteins and protein complexes in living cells. We compare and contrast different data analysis methods that are currently employed in the field and discuss experimental designs that can aid the interpretation of the obtained results. Comparisons of diffusion rates for different proteins and protein complexes in intracellular aqueous environments reported in the recent literature reveal a clear and systematic deviation from the Stokes-Einstein diffusion theory. While a complete and quantitative theoretical explanation of why such deviations manifest is missing, the available data suggest the possibility of weighing freely-diffusing proteins and protein complexes in living cells by measuring their diffusion coefficients. Mapping individual diffusive states to protein complexes of defined molecular weight, subunit stoichiometry, and structure promises to provide key new insights into how protein-protein interactions regulate protein conformational, translational, and rotational dynamics, and ultimately protein function.

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