Simulation-based Reconstructed Diffusion unveils the effect of aging on protein diffusion in Escherichia coli.

We have developed Simulation-based Reconstructed Diffusion (SbRD) to determine diffusion coefficients corrected for confinement effects and for the bias introduced by two-dimensional models describing a three-dimensional motion. We validate the method on simulated diffusion data in three-dimensional cell-shaped compartments. We use SbRD, combined with a new cell detection method, to determine the diffusion coefficients of a set of native proteins in Escherichia coli. We observe slower diffusion at the cell poles than in the nucleoid region of exponentially growing cells, which is independent of the presence of polysomes. Furthermore, we show that the newly formed pole of dividing cells exhibits a faster diffusion than the old one. We hypothesize that the observed slowdown at the cell poles is caused by the accumulation of aggregated or damaged proteins, and that the effect is asymmetric due to cell aging. Author summary: Knowledge of the location and mobility of molecules in living cells is paramount to understand cellular processes, protein interactions, folding and function. However, accurately measuring protein mobility in small compartments, such as bacterial cells, is challenging due to various factors. These include the effects of boundaries and compartment geometry, as well as technical limitations like the properties of fluorophores, the diffraction limit of light, and the camera speed. In Escherichia coli cells, the poles are important regions where most of the cellular proteins are synthesized by ribosomes organized in polysomes. At the same time, aggregated or misfolded proteins accumulate at the cell poles, increasing the local macromolecular crowding. We have developed Simulation-based Reconstructed Diffusion to separate the boundary and geometry effects from crowding effects on the observed protein diffusion. Using this method, we investigated how the accumulation of misfolded or damaged proteins at the poles affects the lateral diffusion of various native proteins. We also observed an increase in apparent crowding in the older pole of dividing cells. We related differences in macromolecular crowding in the pole regions of E. coli to aging of the cells, which may impact cellular functions like they do in eukaryotic cells. [ABSTRACT FROM AUTHOR]