UC Berkeley

Intracellular diffusion defines the kinetics and timescales of biological processes. However, due to the fast motion of biomolecules and the sub-micron scale of intracellular structures, elucidating how unbound molecules diffuse through the cell is challenging. Here, I introduce a recently developed techniques, single-molecule displacement/diffusivity mapping (SMdM), which capitalizes on super-resolution capabilities of single-molecule localization microscopy (SMLM), while uniquely maps out the motion and diffusivity of freely diffusing molecules at the nanoscale.In this dissertation, I present my recent efforts to transform SMdM into a powerful quantification tool by leveraging high-throughput single-molecule statistics. I first investigated enzymatic diffusion during catalysis challenging existing theories by utilizing high statistics of sub-millisecond displacements of unhindered single enzymes freely diffusing in common buffers to quantify D for four enzymes under catalytic turnovers to show no significant changes in D during catalysis, and additionally formulated how ∼ ±1% D precisions can be achieved. By similarly leveraging high-throughput single-molecule statistics to determine D, in the subsequent study, I showed that SMdM can characterize D up to 350 μm2/s for water-soluble dyes and small-molecule metabolites. I unveil that small-molecule intracellular diffusion is dominated by vast regions of high diffusivity ~60-70% of that in vitro, lifting a paradoxical speed limit of intracellular diffusion as suggested by previous studies. Next, I uncover strong in vitro charge-driven protein-protein interactions replicating the sign-asymmetric charge effects on intracellular diffusivity discovered in mammalian cells. By converting between D to molecular weight, we were provide novel insight into how positively charged protein at the limit of low diffuser concentration avoid aggregation and coacervation inside of cytoplasm, by modulating the concentration of negatively charged protein. Here we showed the formation in situ that positively charged diffusers drag a single monolayer of negatively charged macromolecules forming a net negatively charged complex, shedding new insight into mechanisms that drive charged-based diffusion interactions inside of the cell. In collaboration with Rebecca Heald’s group, I work with Xenopus laevis extracts to study how both charge and molecular size impacts diffusivity of macromolecules in membrane-less cytoplasm for a wide range of protein and demonstrate that negatively charged macromolecules diffuse approximately 45% slower in extract, but positively charged macromolecules diffuse substantially slower. We then demonstrate how nonspecific RNA interactions drive the solubilization of positively charged macromolecules, where depletion of RNA resulted in release of positively charged ribosomes that resulted in cytoplasmic aggregation. Lastly, we show that the filamentous actin cytoskeleton induces macromolecular crowding demonstrating nanoscale intracellular structures shape diffusion processes inside of the cell. Collectively, by uniquely mapping out fast diffusion at the single-molecule and super-resolution levels, my research sheds new insight on the fundamental laws governing molecular transport within living cells.