Biology subjects

Anishkin, A.

Publications and source records attributed to Anishkin, A..

8 recordsLinked to original sources

bioRxiv2026

Desensitization, inactivation, and the tension-proof safety mechanism of inactivated MscS

MscS is the main low-threshold tension-activated osmolyte release valve in bacteria. Working alone or together with high-threshold MscL, it regulates turgor and protects cells from mechanical rupture during osmotic down-shock. The channel exhibits complex adaptive behavior, including desensitization and full inactivation, both of which occur at relatively low sub-lytic tension. There is debate over whether the commonly observed non-conductive state of MscS with the tension-sensing helices splayed away from the gate corresponds to the closed or inactivated state. In this work, using specialized pressure protocols in patch-clamp electrophysiology, we highlight the difference between reversible adaptation (desensitization) and inactivation. We show that inactivated channels cannot be reactivated with high tension, up to the limit of patch stability. This aligns with cryo-EM studies by Zhang et al. (Nature, 2021, 590:509-5018), who applied extreme tension to the splayed nanodisc-reconstituted MscS (PDB 6VYK) by depleting lipids with cyclodextrin, and observed a new flattened but apparently non-conductive structure (PDB 6VYM). To characterize these two states, we performed a steered Molecular Dynamics simulation from the initial splayed structure to the flattened conformation, confirming that they are connected through a smooth conformational pathway and remain largely dehydrated and entirely non-conductive throughout the transition. The data show that the initial splayed conformation meets all the criteria of the inactivated state, distorting but not opening under extreme tension. By combining patch-clamp experiments with simulations based on cryo-EM data, we demonstrate that inactivated MscS resists activation, thereby maintaining the membrane barrier when tension exceeds the activation threshold. SIGNIFICANCEBacterial energetics, which relies on the electrochemical proton gradient across the inner membrane as an intermediary, conflicts with the presence of a dense population of highly conductive mechano-activated channels in the same cytoplasmic membrane, which must be proton-tight. Moreover, the tension activation threshold for the common MscS channel is low and can be easily exceeded by fluctuations in the concentrations of internal or external osmolytes. In this paper, we describe the important adaptive inactivation of MscS at tensions near its activation threshold, and specifically, the complete resistance of inactivated MscS to opening at any tension. The data show another layer of tight regulation of MscS residing in the energy-coupling membranes.

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↗

bioRxiv2024

Turgor and the conformational pathway for MscS recovery

The bacterial mechanosensitive channel MscS is an adaptive osmolyte release valve that cycles between closed, open, and inactivated states. Since some of these conformations are stable only in the lipid environment under specific conditions, the structures that are currently available cannot explain the entire functional cycle. Previous patch-clamp characterization has provided insights into the missing functional state by estimating protein expansion areas associated with the closed-to-open and closed-to-inactivated transitions and indicating that the closed state must be the most compact. In this paper, we model the conformational transition of MscS from the splayed conformation with the uncoupled gate to the putative compact closed state. The compaction pathway revealed in preliminary extrapolated motion simulations (ExMoS) involved an upward sliding motion of the internal TM3 barrel inside the outer sheath formed by TM1-TM2 helical pairs. This move leads to several structural changes: (1) the relocation of the characteristic kink at G113 to a new position at G121, (2) the establishment of the hydrophobic TM2-TM3 contact, (3) a new pattern of interactions with membrane lipids, and (4) the formation of stabilizing salt bridges between TM1-TM2 loops and the cytoplasmic cage domain. In the intact bacterial cell, the driving force for this upward motion is likely to be turgor pressure normal to the plane of the membrane acting on the upper hemisphere of the cage domain from the inside. Under continuing lipid synthesis in the inner leaflet of the plasma membrane, turgor pressure is also predicted to maximize the lateral pressure of lipids in the membrane, thus driving MscS compaction. Steered simulations were performed on the splayed state to mimic these effects by applying normal forces to the upper part of the cage domain and by applying lateral compression to the TM1-TM2 pairs, emulating the pressure of lipids. The structure arrived at the predicted compact state of the channel. This state was critically stabilized by displacing non-bilayer lipids from the TM2-TM3 crevices into the bilayer. We propose that the energized metabolic state of the cell generating high turgor and promoting lipid synthesis should strongly favor the compact closed state of MscS. The normal forces pressing the dome of the cage domain against the membrane may provide a common recovery mechanism for the entire family of MscS-like channels found exclusively in organisms with walled cells, which evolved to function under turgor pressure. A conversion of turgor into membrane tension under hypoosmotic cytoplasm swelling and peptidoglycan expansion will drive opposite processes of opening followed by adaptive MscS closure and inactivation.

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

Mechanosensitive channel MscS is critical for termination of the bacterial hypoosmotic permeability response

Free-living microorganisms are subjected to drastic changes in osmolarity. To avoid lysis under sudden osmotic down-shock, bacteria quickly expel small metabolites through the tension-activated channels MscL, MscS, and MscK. We examined five chromosomal knockout strains, {Delta}mscL, {Delta}mscS, a double knockout {Delta}mscS {Delta}mscK, and a triple knockout {Delta}mscL {Delta}mscS {Delta}mscK in comparison to the wild-type parental strain. Stopped-flow experiments confirmed that both MscS and MscL mediate fast osmolyte release and curb cell swelling, but osmotic viability assays indicated that they are not equivalent. MscS alone was capable of rescuing the cell population, but in some strains MscL did not rescue and additionally became toxic in the absence of both MscS and MscK. Furthermore, MscS was upregulated in the {Delta}mscL strain, suggesting either a cross-talk between the two genes/proteins or the influence of cell mechanics on mscS expression. The data shows that for the proper termination of the permeability response, the high-threshold (MscL) and the low-threshold (MscS/MscK) channels must act sequentially. In the absence of low-threshold channels, at the end of the release phase, MscL should stabilize membrane tension at around 10 mN/m. Patch-clamp protocols emulating the tension changes during the release phase indicated that the non-inactivating MscL, residing at its own tension threshold, flickers and produces a protracted leakage. The MscS/MscK population, when present, stays open at this stage to reduce tension below the MscL threshold and silence the large channel. When MscS reaches its own threshold, it inactivates and thus ensures proper termination of the hypoosmotic permeability response. This functional interplay between the high- and low-threshold channels is further supported by the compromised osmotic survival of bacteria expressing non-inactivating MscS mutants. Summary (for the table of contents)The kinetics of hypotonic osmolyte release from E. coli is analyzed in conjunction with bacterial survival. It is shown that MscL, the high-threshold emergency release valve, rescues bacteria from down-shocks only in the presence of MscS, MscK or other low-threshold channels that are necessary to pacify MscL at the end of the release phase.

biophysics↗

bioRxiv2020

Mechanical Activation of MscL Revealed by a Locally Distributed Tension Molecular Dynamics Approach

Membrane tension perceived by mechanosensitive (MS) proteins mediates cellular responses to mechanical stimuli and osmotic stresses, and it also guides multiple biological functions including cardiovascular control and development. In bacteria, MS channels function as tension-activated pores limiting excessive turgor pressure, with MscL (MS channel of large conductance) acting as an emergency release valve preventing cell lysis. Previous attempts to simulate gating transitions in MscL by either directly applying steering forces to the protein or by increasing the whole system tension were not fully successful and often disrupted the integrity of the system. We present a novel locally distributed tension molecular dynamics (LDT-MD) simulation method that allows application of forces continuously distributed among lipids surrounding the channel using a specially constructed collective variable. We report reproducible and reversible transitions of MscL to the open state with measured parameters of lateral expansion and conductivity that exactly satisfy experimental values. The LDT-MD method enables exploration of the MscL gating process with different pulling velocities and variable tension asymmetry between the inner and outer membrane leaflets. We use LDT-MD in combination with well-tempered metadynamics to reconstruct the tension-dependent free energy landscape for the opening transition in MscL. SIGNIFICANCEMembrane-embedded mechanosensitive (MS) proteins are essential for numerous biological functions including cardiovascular control and development, osmotic regulation, touch and pain sensing. In this work, we present a novel molecular dynamics simulation method that allows rapid and systematic exploration of structure, dynamics, and energetics of the mechanical transduction process in MS proteins under tightly controlled local tension distributed in the lipid rim around the protein. We provide a detailed description of the gating transition for the tension-activated bacterial mechanosensitive channel of large conductance, MscL, which is the best characterized channel of this type. MscL functions as a tension-activated emergency osmolyte release valve that limits excessive turgor pressure, prevents cell lysis and thus imparts environmental stability to most free-living bacteria.

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↗