Biology subjects

Volkov, I. L.

Publications and source records attributed to Volkov, I. L..

4 recordsLinked to original sources

bioRxiv2026

Recovering membrane interaction kinetics of single molecules from 3D tracking data

Interactions between cytosolic biomolecules and the bacterial inner membrane are fundamental to many cellular processes, yet directly measuring their binding kinetics in living cells remains challenging. Conventional two-dimensional single-molecule tracking analyses can be insufficient, particularly when membrane association does not markedly alter the diffusion rate. Here, we present a method to recover membrane interaction kinetics from three-dimensional single-molecule trajectories in rod-shaped bacteria. Using simulated 3D tracking data, we identify membrane-associated motion by quantifying how well short trajectory segments follow the circular curvature of the cell membrane. The resulting measure is further analyzed using a hidden Markov modeling framework, enabling robust discrimination between cytosolic and membrane-bound states and capturing the dynamics of state transitions without requiring diffusion-rate changes or direct colocalization with membrane markers. This work establishes a general framework for extracting membrane interaction kinetics from 3D single-molecule tracking data in live bacteria, and highlights the value of realistic microscopy simulations for quantitative interpretation and systematic bias assessment.

biophysics↗

bioRxiv2024

A microfluidic platform for in situ studies of bacteria electroporation

Electroporation of dye-labelled bio-molecules has proven to be a valuable alternative to fluorescent protein fusion for single-molecule tracking in living cells. However, control over cell viability, electroporation efficiency and environment conditions before, during and after electroporation is difficult to achieve in bulk experiments. Here, we present a microfluidic platform capable of single-cell electroporation with in situ microscopy and demonstrate delivery of DNA into bacteria. Via real time observation of the electroporation process, we find that the effect of electrophoresis plays an important role when performing electroporation in a miniaturized platform and show that its undesired action can be balanced by using bipolar electrical pulses. We suggest that a low temperature of the sample during electroporation is important for cell viability due to temperature-dependant viscoelastic properties of the cell membrane. We further found that the presence of low conductive liquid between cells and the electrodes leads to a voltage divider effect which strongly influences the success of on-chip electroporation. Finally, we conclude that electroporation is intrinsically a highly stochastic process that is difficult to fully control via external parameters and envision that the microfluidic system presented here, capable of single-cell read-out, can be used for further fundamental studies to increase our understanding of the electroporation process.

bioengineering↗

bioRxiv2024

Dynamic binding of the bacterial chaperone Trigger factor to translating ribosomes

The bacterial chaperone Trigger factor (TF) binds to ribosome-nascent chain complexes (RNCs) and co-translationally aids the folding of proteins in bacteria. Decades of studies have given a broad, but often conflicting, description of the substrate specificity of TF, its RNC-binding dynamics, and competition with other RNC-binding factors, such as the Signal Recognition Particle (SRP). Previous RNC-binding kinetics experiments were conducted on stalled RNCs in reconstituted systems, and consequently, may not represent the interaction of TF with ribosomes translating mRNA in the cytoplasm of the cell. Here, we used single-particle tracking (SPT) to measure TF binding to actively translating ribosomes inside living Escherichia coli. In cells, TF displays two distinct binding modes -- long (ca 1 s) target-specific RNC binding, and shorter (ca 50 ms) sampling of non-target RNCs. RNC binding events are interrupted only by transient excursions to a freely diffusing state (ca 40 ms). We also show that TF competes with SRP for RNC binding in vivo, and in doing so, tunes the binding selectivity of SRP.

biophysics↗

bioRxiv2020

Direct measurements of mRNA translation kinetics in living cells

Ribosome mediated mRNA translation is central to life as we know it. The cycle of translation has, however, not been characterized in a living cell. Here we have developed a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding an mRNA and translating it, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in elongation at any particular time, and that neither of the subunits, in general, continues translation from one open reading frame to the next on a poly-cistronic mRNA. Furthermore, we find that a variety of previously published orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs, with the rate of translation initiation only modestly affected. Hence, our results suggest that other mRNA elements than the SD sequence play major roles in directing the ribosome to the correct translation start sites.

molecular biology↗