Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations

Abstract

The cytoplasm is a complex, crowded, actively-driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem-cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nano-rheology on genetically encoded multimeric 40-nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of the fission yeast Schizosaccharomyces pombe. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the average particle diffusivity for each individual cell spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger Simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume an equally wide intra- and inter-cellular variation in cytoplasmic viscosity. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, cytoskeletal effects, cell cycle stage and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not “well-mixed” but represents a highly heterogeneous environment in which subcellular components at the 40-nm size-scale experience dramatically different effective viscosities within an individual cell, as well as in different cells in the population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.Graphical abstract

Protein homeostasis from diffusion-dependent control of protein synthesis and degradation

“…Similarly high variability has been reported for the diffusion of genetically-encoded nanoparticles expressed in S. pombe (Garner et al, 2023), which is thought to reflect the heterogeneity of the cytoplasmic environment.…”

Diffusive lensing as a mechanism of intracellular transport and compartmentalization

“…The complex, heterogeneous milieu of the cellular interior has been recently shown to cause heterogeneous diffusion (Garner et al, 2022; Huang et al, 2021; Parry et al, 2014; Śmigiel et al, 2022; Xiang et al, 2020), yet the consequences of such inhomogeneity on compartmentalization and mesoscale molecular dynamics have remained unclear. Through agent-based modeling of diffusion, we have shown that heterogenous viscosity can lead to simulated particle trajectories converging into viscous hotspots, causing the accumulation of diffusing particles into membraneless compartments defined by the higher-viscosity zones.…”

“…Such expressways and their associated fluxes may impact reaction kinetics by altering substrate turnover rates, congruent with the model of unusual transport processes potentially modifying reaction kinetics (Bénichou et al, 2010). In the context of subcellular viscous regions (Garner et al, 2022), cells may compensate for geometry-imposed constraints on packing density and size of these regions by altering the viscosity ratio (against the bulk milieu) instead. To map the detailed effects of inhomogeneous viscosity on reaction rates, however, our work suggests that a key prerequisite is to chart a suitable set of metaparameters that provide an adequate description of inhomogeneous diffusion (Jin and Verkman, 2007), as a one-parameter description relying exclusively on the average diffusion coefficient is insufficient.…”

“…Diffusion is a fundamental phenomenon of transport at scales ranging from atoms to galaxies. In cells, diffusion of individual components occurs in a complex, crowded milieu (Ellis, 2001;Luby-Phelps, 1999;van den Berg et al, 2017) that demonstrates position-dependent diffusivity (Berret, 2016;Garner et al, 2022;Śmigiel et al, 2022;Xiang et al, 2020). Diffusion can occur within or between cellular compartments, where concentrated components carry out chemical reactions.…”

Cell wall damage increases macromolecular crowding effects in the Escherichia coli cytoplasm

“…Cell components compartmentalize by demixing or preferential interactions in which protein crowders assemble with other biomolecules or membranes. Indeed, diffusion experiments have shown significant spatial heterogeneity in prokaryotes and eukaryotes 8,9 . Crowders captured in large complexes crowd less effectively because a crowder needs to diffuse to generate the depletion force, and complexation reduces the crowder number density.…”