Localization of Protein Aggregation in Escherichia coli Is Governed by Diffusion and Nucleoid Macromolecular Crowding Effect

Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of “soft” intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.Author Summary: Localization of proteins to specific positions inside bacteria is crucial to several physiological processes, including chromosome organization, chemotaxis or cell division. Since bacterial cells do not possess internal sub-compartments (e.g., cell organelles) nor vesicle-based sorting systems, protein localization in bacteria must rely on alternative mechanisms. In many instances, the nature of these mechanisms remains to be elucidated. In Escherichia coli, the localization of aggregates of misfolded proteins at the poles or the center of the cell has recently been linked to aging. However, the molecular mechanisms governing this localization of the protein aggregates remain controversial. To identify these mechanisms, we have devised an integrated strategy combining innovative experimental and modeling approaches. Our results show the importance of the increased macromolecular crowding in the nucleoids, the regions within the cell where the bacterial chromosome preferentially condensates. They indicate that a purely diffusive pattern of aggregates mobility combined with nucleoid occlusion underlies their accumulation in polar and mid-cell positions.