ABSTRACT
The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-2160 to +1800 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary.
ABSTRACT
Faster-growing cells must synthesize proteins more quickly. Increased ribosome abundance only partly accounts for increases in total protein synthesis rates. The productivity of individual ribosomes must increase too, almost doubling by an unknown mechanism. Prior models point to diffusive transport as a limiting factor but raise a paradox: faster-growing cells are more crowded, yet crowding slows diffusion. We suspected that physical crowding, transport, and stoichiometry, considered together, might reveal a more nuanced explanation. To investigate, we built a first-principles physics-based model of Escherichia coli cytoplasm in which Brownian motion and diffusion arise directly from physical interactions between individual molecules of finite size, density, and physiological abundance. Using our microscopically detailed model, we predicted that physical transport of individual ternary complexes accounts for ~80% of translation elongation latency. We also found that volumetric crowding increases during faster growth even as cytoplasmic mass density remains relatively constant. Despite slowed diffusion, we predicted that improved proximity between ternary complexes and ribosomes wins out, illustrating a simple physics-based mechanism for how individual elongating ribosomes become more productive. We speculate that crowding imposes a physical limit on growth rate and undergirds cellular behavior more broadly. Unfitted colloidal-scale modeling offers systems biology a complementary "physics engine" for exploring how cellular-scale behaviors arise from physical transport and reactions among individual molecules. IMPORTANCE Ribosomes are the factories in cells that synthesize proteins. When cells grow faster, there are not enough ribosomes to keep up with the demand for faster protein synthesis without individual ribosomes becoming more productive. Yet, faster-growing cells are more crowded, seemingly making it harder for each ribosome to do its work. Our computational model of the physics of translation elongation reveals the underlying mechanism for how individual ribosomes become more productive: proximity and stoichiometry of translation molecules overcome crowding. Our model also suggests a universal physical limitation of cell growth rates.
