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

Abstract

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.

Bacterial Inclusion Bodies: Discovering Their Better Half

“…Restriction of aggregates to the pole was also observed during treatment with substances disrupting the protein motive force, suggesting an energy independent process of aggregation [20]. Energy-independent polar localization was also confirmed by others [30] but also convincing opposite results were obtained [15]. Thus, it is not absolutely clear if active ATP-dependent transport of smaller particles is required or if the fluidizing properties of active metabolism are responsible for the polar preference of protein aggregates [16].…”

“…There are claims that transport of aggregates to the poles is energy-driven as studies on reassembly of pressure dissociated IBs revealed that confinement to the pole is caused by the presence of the nucleoid but reassembly of smaller aggregates into large IBs does not occur in energy and nutrient depleted cells [16]. However, there are also reports stating that exclusion of aggregates from mid-cell to the pole is energy-free, and simply results from nucleoid exclusion [17,20,[28][29][30]. Restriction of aggregates to the pole was also observed during treatment with substances disrupting the protein motive force, suggesting an energy independent process of aggregation [20].…”

“…Thus, it is not absolutely clear if active ATP-dependent transport of smaller particles is required or if the fluidizing properties of active metabolism are responsible for the polar preference of protein aggregates [16]. IBs and aggregation foci are not only found at the polar sites but also in mid-cells presumably at the place of future septation sites [20,30,31]. The major disaggregating chaperones (DnaK, ClpB) also co-localize at the poles [20] and are required for disaggregation of polar aggregates [15].…”

Molecular simulations of cellular processes

“…Computer simulations are often integrated with experiments performed in vivo to provide a microscopic interpretation of cellular phenomena (Hihara et al 2012;Coquel et al 2013;Di Rienzo et al 2014;Earnest et al 2017). Although powerful to predict macromolecule behavior, this technique, defined as the Bcomputational microscope^by Schulten (Lee et al 2009), has some limitations (Takada 2012;Piana et al 2014;Ivani et al 2016;Song et al 2017;Wang et al 2017a).…”

Localized mRNA translation and protein association

“…Experimental studies of subcellular localization of RNAs and proteins have induced the development of spatiotemporal models of gene expression. A few available models are focused on protein aggregation and nucleoid occlusion [19][20][21]. Protein localization near the transcription region was described as a consequence of diffusion within and exchange between the condensed chromosomal DNA and an extrachromosomal area [9,22].…”