Retrieving the intracellular topology from multi-scale protein mobility mapping in living cells

In living cells, most proteins diffuse over distances of micrometres within seconds. Protein translocation is constrained due to the cellular organization into subcompartments that impose diffusion barriers and guide enzymatic activities to their targets. Here, we introduce an approach to retrieve structural features from the scale-dependent mobility of green fluorescent protein monomer and multimers in human cells. We measure protein transport simultaneously between hundreds of positions by multi-scale fluorescence cross-correlation spectroscopy using a line-illuminating confocal microscope. From these data we derive a quantitative model of the intracellular architecture that resembles a random obstacle network for diffusing proteins. This topology partitions the cellular content and increases the dwell time of proteins in their local environment. The accessibility of obstacle surfaces depends on protein size. Our method links multi-scale mobility measurements with a quantitative description of intracellular structure that can be applied to evaluate how drug-induced perturbations affect protein transport and interactions. Numerous obstacles posed by cellular subcompartments and structures constrain protein transport in the cell. Here, Baum et al. map the intracellular topology from a diffusing protein’s point of view by measuring the diffusive movements of fluorescently labelled reporter proteins in living cells on multiple time and length scales.