Super-resolution displacement mapping of unbound single molecules reveals nanoscale heterogeneities in intracellular diffusivity

Abstract

Intracellular diffusion underlies vital cellular processes. However, it remains difficult to elucidate how an average-sized protein diffuses inside the cell with good spatial resolution and sensitivity. Here we introduce single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanometer-scale mapping of intracellular diffusivity through the local statistics of instantaneous displacements of freely diffusing single molecules. We thus show that diffusion in the mammalian cytoplasm and nucleus to both be spatially heterogeneous at the nanoscale, and that such variations in local diffusivity correlate well with the ultrastructure of the actin cytoskeleton and the chromosome, respectively. Moreover, we identify the net charge of the diffuser as a key determinant of diffusion rate: intriguingly, the possession of positive, but not negative, net charges significantly impedes diffusion, and the exact degree of slowdown is determined by the specific subcellular environments. We thus open a new door to understanding the physical rules that govern the intracellular interactions between biomolecules at the nanoscale.