Biology subjects

Sukharev, S.

Publications and source records attributed to Sukharev, S..

9 recordsLinked to original sources

bioRxiv2026

When do bacterial mechanosensitive channels get activated?

Bacterial cell envelopes are complex multilayer structures that combine mechanical protection with a multitude of transport and sensory functions. Bacteria maintain substantial turgor pressures which is needed to withstand external forces, grow and maintain shape. An osmotic downshock can create fatal pressure levels. To survive, bacteria rapidly release osmolytes, mainly ions and small metabolites, through mechanosensitive channels (Mscs). Mscs are embedded in the plasma membrane (PM) and act as pressure/tension-activated valves, reducing pressure and preserving cell integrity. It is not well understood, however, how overall stresses are distributed among the layers of the cell envelope, and, in particular, when tension would be borne by the PM and felt by the channels. In this study, we numerically and analytically examine stress distributions within a two-layer model of the cell envelope under varying turgor pressure. We use experimentally determined parameters for PM and peptidoglycan (PG) elasticities, and include enzyme/antibiotic induced weakening of the PG. We introduce, as a critical parameter, a PM reservoir, without which an observed substantial area increase during the shock would not be possible without PM rupture. We used finite-element simulations to model cell swelling assuming linear elasticity of the envelope components, and analytical calculations to model nonlinear PG strain hardening. We analyzed tension sharing between PG and PM with varying PM reservoir sizes during osmotic downshocks. We estimate that an envelope area increase of slightly more than 50% would occur at a turgor pressure of ~11 atm and would be sufficient to fully unfold a 50% PM reservoir and also generate sufficient PM tension to activate mechanosensitive channels. A softening of the PG layer with lysozyme or {beta}-lactam antibiotics is predicted to increase cell expansion at a given turgor pressure and trigger earlier activation of mechanosensitive channels, leading to increased, deleterious metabolite loss.

biophysics↗

bioRxiv2025

Fluorescent peptides for membrane tension and domain structure reporting

Cell mechanics play significant roles in all aspects of cell function. While many types of fluorescent linear force sensors inserted in cellular fibrillar elements have been developed, few tools are available to track two-dimensional tension in cell membranes. Here, we present a novel principle for tension detection using a fluorescent probe based on the scaffold of the GsMTx4 peptide from Grammostola venom. Previously, we have shown that amphipathic GsMTx4 binds to lipids and inhibits mechanosensitive channels by inserting more deeply into the membrane at tensions near activation thresholds, thereby acting as a buffer clamping lateral pressure in the bilayer. We leverage this property of GsMTx4 to redistribute between the shallow and deep immersion states, thereby designing probes with a fluorescent moiety that increases quantum yield in nonpolar environments. GsMTx4 analogs carrying fluorescent groups at the two positions increase fluorescence intensity in osmotically shocked liposomes and aspirated giant vesicles in a near-linear fashion in response to physiological bilayer tensions. The responses show dependence on membrane composition, particularly lipid charge and the presence of lipid-ordering components, such as sphingomyelin and cholesterol. Langmuir compression isotherms recorded in the presence of NBD analogs indicated initial incorporation into the monolayer, followed by sharp expulsion at the monolayer-bilayer equivalence pressure, with correlated changes in monolayer compressibility and fluorescence, illustrating the basic principle of probe action. The probes show promise for monitoring tension in biological membranes at low, non-inhibitory concentrations. Experiments with native cell-derived membrane vesicles reveal heterogeneous baseline staining and tension responses, underscoring the probes selectivity for distinct membrane domains. SignificanceCell mechanics are crucial for all cell functions, including division, survival, migration, and differentiation. Although many versions of fluorescent linear force sensors have been developed for cytoskeletal and ECM elements, few tools exist to monitor two-dimensional tension in cell membranes. Many cells are motile, actively deforming their membrane, supported and driven by the underlying cytoskeleton. There is a two-order-of-magnitude discrepancy between membrane tension estimates from the tether formation technique and the tensions that activate common mechanosensitive channels in most cells. This discrepancy highlights the need for non-invasive membrane probes that can independently measure membrane tension, especially since it can be highly localized and dynamic. Here, we introduce such probes and a new principle for tension measurement.

biophysics↗

bioRxiv2023

MscS is a critical component for osmotic survival of Vibrio cholerae

Like other intestinal bacteria, the facultative pathogen Vibrio cholerae adapts to a wide range of osmotic environments. Under drastic osmotic down-shifts, Vibrio avoids mechanical rupture by rapidly releasing excessive metabolites through mechanosensitive (MS) channels that belong to two major types, low-threshold MscS and high-threshold MscL. To investigate each channels individual contribution to V. choleraes osmotic permeability response, we generated individual{Delta} mscS, {Delta}mscL, and double{Delta} mscL {Delta}mscS mutants in V. cholerae O395 and characterized their tension-dependent activation in patch-clamp experiments, as well as their millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models reflecting the kinetic competition of osmolyte release with water influx. Both mutants lacking MscS exhibited delayed osmolyte release kinetics and decreased osmotic survival rates compared to WT. The{Delta} mscL mutant showed comparable release kinetics to WT, but a higher osmotic survival, while {Delta}mscS had low survival, comparable to the double{Delta} mscL {Delta}mscS mutant. By analyzing release kinetics following rapid medium dilution, we illustrate the sequence of events and define the set of parameters that characterize discrete phases of the osmotic response. Osmotic survival rates are directly correlated to the extent and duration of cell swelling, the rate of osmolyte release and the onset time, and the completeness of the post-shock membrane resealing. Not only do the two channels interact functionally during the resealing phase, but there is also a compensatory up-regulation of MscS in the{Delta} mscL strain suggesting some transcriptional crosstalk. The data reveal the advantage of the low-threshold MscS channel in curbing tension surges, without which MscL becomes toxic, and the role of MscS in the proper termination of the osmotic permeability response in Vibrio.

biophysics↗

bioRxiv2023

MscS inactivation and recovery are slow voltage-dependent processes sensitive to interactions with lipids

Mechanosensitive channel MscS, the major bacterial osmolyte release valve, shows a characteristic adaptive behavior. With a sharp onset of activating tension, the channel population readily opens, but under prolonged action of moderate near-threshold tension, it inactivates. The inactivated state is non-conductive and tension-insensitive, which suggests that the gate gets uncoupled from the lipid-facing domains. The kinetic rates for tension-driven opening-closing transitions are 4-6 orders of magnitude higher than the rates for inactivation and recovery. Here we show that inactivation is augmented and recovery is slowed down by depolarization. Hyperpolarization, conversely, impedes inactivation and speeds up recovery. We then address the question of whether protein-lipid interactions may set the rates and influence voltage dependence of inactivation and recovery. Mutations of conserved arginines 46 and 74 anchoring the lipid-facing helices to the inner membrane leaflet to tryptophans do not change the closing transitions, but instead change the kinetics of both inactivation and recovery and essentially eliminate their voltage-dependence. Uncharged polar substitutions (S or Q) for these anchors produce functional channels but increase the inactivation and reduce the recovery rates. The data suggest that it is not the activation and closing transitions, but rather the inactivation and recovery pathways that involve substantial rearrangements of the protein-lipid boundary associated with the separation of the lipid-facing helices from the gate. The discovery that hyperpolarization robustly assists MscS recovery indicates that membrane potential can regulate osmolyte release valves by putting them either on the ready or standby mode depending on the cells metabolic state.

biophysics↗

bioRxiv2023

Tight hydrophobic core and flexible helices yield MscL with a high tension gating threshold and a membrane area mechanical strain buffer

The mechanosensitive (MS) channel of large conductance, MscL, is the high-tension threshold osmolyte release valve that limits turgor pressure in bacterial cells in the event of drastic hypoosmotic shock. Despite MscL from M. tuberculosis (TbMscL) being the first structurally characterized MS channel, its protective mechanism of activation at nearly-lytic tensions has not been fully understood. Here, we describe atomistic simulations of expansion and opening of wild-type (WT) TbMscL in comparison with five of its gain-of-function (GOF) mutants. We show that under far-field membrane tension applied to the edge of the periodic simulation cell, WT TbMscL expands into a funnel-like structure with trans-membrane helices bent by nearly 70 degrees, but does not break its hydrophobic seal within extended 20 s simulations. GOF mutants carrying hydrophilic substitutions in the hydrophobic gate of increasing severity (A20N, V21A, V21N, V21T and V21D) also quickly transition into funnel-shaped conformations but subsequently fully open within 1-8 us. This shows that solvation of the de-wetted (vapor-locked) constriction is the rate-limiting step in the gating of TbMscL preceded by area-buffering silent expansion. Pre-solvated gates in severe V21N and V21D mutants eliminate this barrier. We predict that the asymmetric shape-change of the periplasmic side of the channel during the silent expansion provides strain-buffering to the outer leaflet thus re-distributing the tension to the inner leaflet, where the gate resides.

biophysics↗

bioRxiv2020

Dissipation During the Gating Cycle of the Bacterial Mechanosensitive Ion Channel Approaches the Landauer's Limit

The Landauers principle sets a thermodynamic bound of kBT ln 2 on the energetic cost of erasing each bit of information. It holds for any memory device, regardless of its physical implementation. It was recently shown that carefully built artificial devices can saturate this bound. In contrast, biological computation-like processes, e.g., DNA replication, transcription and translation use an order of magnitude more than their Landauers minimum. Here we show that saturating the Landauer bound is nevertheless possible with biological devices. This is done using a mechanosensitive channel of small conductance (MscS) from E. coli as a memory bit. MscS is a fast-acting osmolyte release valve adjusting turgor pressure inside the cell. Our patch-clamp experiments and data analysis demonstrate that under a slow switching regime, the heat dissipation in the course of tension-driven gating transitions in MscS closely approaches its Landauers limit. We discuss the biological implications of this physical trait.

biophysics↗

bioRxiv2018

A bacterial-like mechanosensitive channel controls osmoregulation and infectivity in Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease, undergoes drastic morphological and biochemical modifications as it passes between hosts and transitions from extracellular to intracellular stages. The osmotic and mechanical aspects of these cellular transformations are not understood. Here we identify and characterize a novel mechanosensitive channel in T. cruzi (TcMscS) belonging to the superfamily of small conductance mechanosensitive channels (MscS). TcMscS is activated by membrane tension and forms a large pore permeable to anions, cations, and small osmolytes. The channel changes its location from the contractile vacuole complex in epimastigotes to the plasma membrane as the parasites develop into intracellular amastigotes. TcMscS knockout parasites show significant fitness defects, including increased cell volume, calcium dysregulation, impaired differentiation, and a dramatic decrease in infectivity. Our work provides mechanistic insights into components supporting pathogen adaptation inside the host thus opening the exploration of mechanosensation as a prerequisite of protozoan infectivity.

microbiology↗

bioRxiv2017

Recovery of equilibrium free energy from non-equilibrium thermodynamics with mechanosensitive ion channels in E. coli

Bacterial mechanosensitive channels are major players in cells ability to cope with hypo-osmotic stress. Excess turgor pressure due to fast water influx is reduced as the channels, triggered by membrane tension, open and release osmolytes. However, in vitro measurements of the free energy difference between the open and closed states of ion channels are challenging due to hysteresis effects and inactivation. Exploiting recent developments in statistical physics, we present a general formalism to extract the free energy difference between the closed and open states of mechanosensitive ion channels from non-equilibrium work distributions associated with the channels gating recorded in native patches under ramp stimulation protocols. We show that the work distributions obtained from the gating of MscS channels in E. coli membrane satisfy the strong symmetry relations predicted by the fluctuation theorems and recover the equilibrium free energy difference between the closed and open states of the channel within 1 kB T of its best estimate obtained from an independent experiment.

biophysics↗

bioRxiv2017

Spatiotemporal relationships defining the adaptive gating of the bacterial mechanosensitive channel MscS

Adaptive desensitization and inactivation are common properties of most ion channels and receptors. The mechanosensitive channel of small conductance MscS, which serves as a low-threshold osmolyte release valve in most bacteria, is unusual because it slowly inactivates not from the open, but from the resting state under moderate tensions. The manifestation of this mechanism is the channels ability to discriminate the rate of tension application, i.e., to ignore slow tension ramps but fully respond to abruptly applied stimuli. In this work, we present a reconstruction of the landscape for tension-dependent MscS transitions based on patch current kinetics recorded under specially designed pressure protocols. The data are analyzed with a three-state continuous time Markov model of gating, where the tension-dependent transition rates are governed by Arrhenius-type relations. The analysis provides assignments to the intrinsic opening, closing, inactivation, and recovery rates as well as their tension dependencies. These parameters, which define the spatial (areal) distances between the energy wells and the positions of barriers, describe the tension-dependent distribution of the channel population between the three states and quantitatively predict the experimentally observed dynamic pulse and ramp responses. Our solution also provides an analytic expression for the area of the inactivated state in terms of two experimentally accessible parameters: the tension at which inactivation probability is maximized, {gamma}*, and the midpoint tension for activation, {gamma}0.5. The analysis initially performed on Escherichia coli MscS shows its applicability to the previously uncharacterized MscS homolog from Pseudomonas aeruginosa. MscS inactivation minimizes metabolic losses during osmotic permeability response and thus contributes to the environmental fitness of bacteria.

biophysics↗