Biology subjects

Linnik, D.

Publications and source records attributed to Linnik, D..

3 recordsLinked to original sources

bioRxiv2026

Single-protein diffusion in micrometer-size vesicles is governed by viscosity, surface charge, and membrane interactions

Giant unilamellar vesicles (GUVs) are a powerful model system for studying protein dynamics in cell-sized compartments with a precisely tunable physicochemical environment. Here, we apply Single-Molecule Displacement Mapping (SMdM) to quantify how luminal viscosity, ionic strength, membrane lipid composition, and protein surface charge of soluble proteins govern intraluminal single-molecule diffusion. Increasing viscosity led to a predictable decrease in the diffusion coefficient. However, spatially resolved diffusion measurements reveal reduced protein mobility near the membrane, suggesting additional membrane-proximal interactions not captured by particle-based simulations. We observe long-range (~1 um) effects of membrane charge on protein mobility that extend well beyond classical Debye screening, which may reflect shape fluctuations of the GUV membrane. For the membrane-associated protein LactC2, we resolve two distinct diffusing populations within a single vesicle, corresponding to free and membrane-bound states. Our workflow provides a quantitative framework for probing single-protein mobility in vesicle-based synthetic cells. These results advance our understanding of how molecular crowding and transient membrane interactions shape protein diffusion within cell-sized compartments, with possible implications for metabolic organization.

biophysics↗

bioRxiv2025

Condition-dependent, amorphous protein agglomerates control cytoplasmic rheology

Molecular crowding in the bacterial cytoplasm restricts the diffusion of large molecules, impacting cellular processes. However, how nutrient availability influences cytoplasmic rheology is not well understood. With single-particle tracking in Escherichia coli, we observed a threefold variation in the diffusion of a 40-nm particle across exponential growth conditions. Previously suggested determinants of rheology did not account for this variation; instead, we found a strong anticorrelation between the diffusion coefficient and the abundance of amino acid metabolism proteins, persisting upon genetic perturbations and showing that lower diffusion is associated with increased viscoelasticity. Photoactivated light microscopy revealed that some amino acid metabolism proteins form clusters. Electron microscopy showed that these proteins could form amorphous agglomerates at physiological concentrations in vitro, likely driven by their low intrinsic disorder, high compactness and hydropathy score. These findings show that protein agglomerates regulate cytoplasmic rheology in a condition-dependent manner, suggesting an underappreciated level of cytoplasmic organization. HighlightsO_LIDiffusion of 40-nm particles varies threefold across growth conditions in E. coli C_LIO_LICytoplasmic diffusion inversely correlates with COG-E protein abundance COG-E proteins form agglomerates that increase cytoplasmic viscoelasticity C_LIO_LIProtein compactness and hydrophobicity predict condition-dependent crowding effects C_LI

biophysics↗

bioRxiv2023

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 infer 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. We find that this observation 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.

biophysics↗