Tackling the puzzle of protein reaction-diffusion inside living cells with sub-5-nm localization.

Abstract

The interior of the cells where most proteins perform their function, is a mixture of thousands of components, but by no means is it a “well-mixed” reactor. While diffusion of proteins can both assist and hinder the formation of heterogeneous environments in cells, the reactions of proteins also affect their tumbling and diffusion. How are protein diffusion and reaction coupled in the living cell? Using Minflux, we can answer this question at the single-molecule level. Minflux provides imaging and tracking at a resolution of 2-3 nm in three dimensions, with minimal bleaching, and high enough speed to distinguish the diffusion of a protein bound vs. unbound in a complex or free vs. stuck onto surfaces inside the cell. Here we study a protein-RNA complex in live cells: U1A-SL2 RNA, whose binding affinity and dynamics have been well studied in vitro and at the ensemble level in human cells. To obtain the best super-resolution tracking in living cells, we first optimized the labeling dyes from twelve options; only one binding partner needs to be labeled at a time to detect changes in its diffusion. Bound and unbound protein populations can be differentiated and spatially located with respect to specific regions in the cell.