Effects of membrane viscoelasticity on the red blood cell dynamics in a microcapillary.

The mechanical properties of red blood cells (RBCs) play key roles in their biological functions in microcirculation. In particular, RBCs must deform significantly to travel through microcapillaries with sizes comparable with or even smaller than their own. Although the dynamics of RBCs in microcapillaries have received considerable attention, the effect of membrane viscoelasticity has been largely overlooked. In this work, we present a computational study based on the boundary integral method and thin-shell mechanics to examine how membrane viscoelasticity influences the dynamics of RBCs flowing through straight and constricted microcapillaries. Our results reveal that the cell with a viscoelastic membrane undergoes substantially different motion and deformation compared with results based on a purely elastic membrane model. Comparisons with experimental data also suggest the importance of accounting for membrane viscoelasticity to properly capture the transient dynamics of an RBC flowing through a microcapillary. Taken together, these findings demonstrate the significant effects of membrane viscoelasticity on RBC dynamics in different microcapillary environments. The computational framework also lays the groundwork for more accurate quantitative modeling of the mechanical response of RBCs in their mechanotransduction process in subsequent investigations.

Physical memory of astrocytes.

Traumatic brain injury (TBI) is a major risk factor for development of neurodegenerative disorders later in life. Short, repetitive, mechanical impacts can lead to pathology that appears days or months later. The cells have a physical "memory" of mechanical events. The origin of this memory is not known. To examine the properties of this memory, we used a microfluidic chip to apply programmed fluid shear pulses to adherent adult rat astrocytes. These caused a transient rise in intracellular Ca2+. In response to repeated stimuli, 6 to 24 hrs apart, the Ca2+ response increased. This effect lasted longer than 24 hrs. The Ca2+ responses were more sensitive to the number of repetitions than to the rest time between stimuli. We found that inhibiting the Ca2+ influx during conditioning stimulus did not eliminate the stress potentiation, suggesting that mechanical deformation during the primary injury is accountable for the later response. The mechanical mechanism that triggers this long term "memory" may act by plastic deformation of the cytoskeleton.

Membrane tension.

The cell membrane serves as a barrier that restricts the rate of exchange of diffusible molecules. Tension in the membrane regulates many crucial cell functions involving shape changes and motility, cell signaling, endocytosis, and mechanosensation. Tension reflects the forces contributed by the lipid bilayer, the cytoskeleton, and the extracellular matrix. With a fluid-like bilayer model, membrane tension is presumed uniform and hence propagated instantaneously. In this review, we discuss techniques to measure the mean membrane tension and how to resolve the stresses in different components and consider the role of bilayer heterogeneity.

Adherent cell remodeling on micropatterns is modulated by Piezo1 channels.

Adherent cells utilize local environmental cues to make decisions on their growth and movement. We have previously shown that HEK293 cells grown on the fibronectin stripe patterns were elongated. Here we show that Piezo1 function is involved in cell spreading. Piezo1 expressing HEK cells plated on fibronectin stripes elongated, while a knockout of Piezo1 eliminated elongation. Inhibiting Piezo1 conductance using GsMTx4 or Gd3+ blocked cell spreading, but the cells grew thin tail-like extensions along the patterns. Images of GFP-tagged Piezo1 showed plaques of Piezo1 moving to the extrusion edges, co-localized with focal adhesions. Surprisingly, in non-spreading cells Piezo1 was located primarily on the nuclear envelope. Inhibiting the Rho-ROCK pathway also reversibly inhibited cell extension indicating that myosin contractility is involved. The growth of thin extrusion tails did not occur in Piezo1 knockout cells suggesting that Piezo1 may have functions besides acting as a cation channel.

Shear stress-induced nuclear shrinkage through activation of Piezo1 channels in epithelial cells.

The cell nucleus responds to mechanical cues with changes in size, morphology and motility. Previous work has shown that external forces couple to nuclei through the cytoskeleton network, but we show here that changes in nuclear shape can be driven solely by calcium levels. Fluid shear stress applied to MDCK cells caused the nuclei to shrink through a Ca2+-dependent signaling pathway. Inhibiting mechanosensitive Piezo1 channels through treatment with GsMTx4 prevented nuclear shrinkage. Piezo1 knockdown also significantly reduced the nuclear shrinkage. Activation of Piezo1 with the agonist Yoda1 caused similar nucleus shrinkage in cells not exposed to shear stress. These results demonstrate that the Piezo1 channel is a key element for transmitting shear force input to nuclei. To ascertain the relative contribution of Ca2+ to cytoskeleton perturbation, we examined F-actin reorganization under shear stress and static conditions, and showed that reorganization of the cytoskeleton is not necessary for nuclear shrinkage. These results emphasize the role of the mechanosensitive channels as primary transducers in force transmission to the nucleus.

Investigating the interaction of Grammostola rosea venom peptides and model lipid bilayers with solid-state NMR and electron microscopy techniques.

Spider venom contains a number of small peptides that can control the gating properties of a wide range of ion channels with high affinity and specificity. These ion channels are responsible for coordination and control of many bodily functions such as transducing signals into sensory functions, smooth muscle contractions as well as serving as sensors in volume regulation. Hence, these peptides have been the topic of many research efforts in hopes that they can be used as biomedical therapeutics. Several peptides are known to control the gating properties of ion channels by involving the lipid membrane. GsMTx4, originally isolated from the Chilean Rose tarantula (Grammostola rosea), is known to selectively inhibit mechanosensitive ion channels by partitioning into the lipid bilayer. To further understand this indirect gating mechanism, we investigated the interactions between native GsAF2, VsTx1 and a synthetic form of GsMTx4 with model DMPC lipid bilayers using 31P solid-state NMR, 13C CP-MAS NMR, NS-TEM and cryo-TEM. The results reveal that these inhibitor cystine knot peptides perforate the DMPC lipid vesicles similarly with some subtle differences and ultimately create small spherical vesicles and anisotropic cylindrical and discoidal vesicles at concentrations near 1.0-1.5 mol% peptide. The anisotropic components align with their long axes along the NMR static B0 magnetic field, a property that should be useful in future NMR structural investigations of these systems. These findings move us forward in our understanding of how these peptides bind and interact with the lipid bilayer.

Functional analyses of heteromeric human PIEZO1 Channels.

PIEZO1 and PIEZO2 are mechanosensitive channels (MSCs) important for cellular function and mutations in them lead to human disorders. We examined how functional heteromers form between subunits of PIEZO1 using the mutants E2117K, E2117D, and E2117A. Homomers of E2117K do not conduct. E2117A homomers have low conductance with rapid inactivation, and those of E2117D have high conductance with slow inactivation. Pairing E2117K with E2117D or E2117A with E2117D gave rise to new channel species representing heteromers with distinct conductances. Whole-cell currents from co-expression of E2117A and E2117D fit well with a linear-combination model of homomeric channel currents suggesting that functional channels do not form from freely-diffusing, randomly-mixed monomers in-vitro. Whole-cell current from coexpressed PIEZO1/PIEZO2 also fit as a linear combination of homomer currents. High-resolution optical images of fluorescently-tagged channels support this interpretation because coexpressed subunits segregate into discrete domains.

GsMTx4-D provides protection to the D2.mdx mouse.

Duchenne muscular dystrophy is a life-limiting muscle disease that has no current effective therapy. Despite mounting evidence that dysregulation of mechanosensitive ion channels is a significant contributor to dystrophy pathogenesis, effective pharmacologic strategies targeting these channels are lacking. GsMTx4, and its enantiomer GsMTx4-D, are peptide inhibitors of mechanosensitive channels with identical activity. In previous studies, acute in vitro application of GsMTx4 to dystrophic murine muscle effectively reduced the excess MSC dependent calcium influx linked to contraction-induced muscle damage. Here we sought to determine if in vivo treatment with GsMTx4-D proffered benefit in the D2.mdx mouse. GsMTx4-D showed a 1-week half-life when administered by subcutaneous injection over four weeks. Informed by these results, D2.mdx mice were then treated by a subcutaneous injection regimen of GsMTx4-D for six weeks followed by determination of muscle mass, muscle susceptibility to eccentric contraction injury and multiple histological indicators of disease progression. The mice showed a reduction in the loss of muscle mass and a decrease in susceptibility to contraction induced injury. These protective effects were realized without reduction in fibrosis, supporting a model where GsMTx4-D acts directly on muscle cells. We propose GsMTx4-D represents a promising new therapy to slow disease progression and may complement other therapies such as anti-inflammatory agents and gene-replacement strategies.

Cytoskeletal Contribution to Cell Stiffness Due to Osmotic Swelling; Extending the Donnan Equilibrium.

Cell volume regulation is commonly analyzed with a model of a closed semipermeable membrane filled with impermeant mobile solutes and the Donnan Equilibrium is used to predict the hydrostatic pressure. This traditional model ignores the fact that most cells are filled with a crosslinked cytoskeleton that is elastic and can be stretched or compressed like a sponge with no obvious need to move mobile solutes. However, calculations show that under osmotic stress, the elastic energy of the cytoskeleton is far greater than the elastic energy of the membrane. Here we expand the traditional Donnan model to include the elasticity of a cytoskeleton with fixed charges and show that cell stiffening happens without a membrane.

Enantiomeric Aß peptides inhibit the fluid shear stress response of PIEZO1.

Traumatic brain injury (TBI) elevates Abeta (Aß) peptides in the brain and cerebral spinal fluid. Aß peptides are amphipathic molecules that can modulate membrane mechanics. Because the mechanosensitive cation channel PIEZO1 is gated by membrane tension and curvature, it prompted us to test the effects of Aß on PIEZO1. Using precision fluid shear stress as a stimulus, we found that Aß monomers inhibit PIEZO1 at femtomolar to picomolar concentrations. The Aß oligomers proved much less potent. The effect of Aßs on Piezo gating did not involve peptide-protein interactions since the D and L enantiomers had similar effects. Incubating a fluorescent derivative of Aß and a fluorescently tagged PIEZO1, we showed that Aß can colocalize with PIEZO1, suggesting that they both had an affinity for particular regions of the bilayer. To better understand the PIEZO1 inhibitory effects of Aß, we examined their effect on wound healing. We observed that over-expression of PIEZO1 in HEK293 cells increased cell migration velocity ~10-fold, and both enantiomeric Aß peptides and GsMTx4 independently inhibited migration, demonstrating involvement of PIEZO1 in cell motility. As part of the motility study we examined the correlation of PIEZO1 function with tension in the cytoskeleton using a genetically encoded fluorescent stress probe. Aß peptides increased resting stress in F-actin, and is correlated with Aß block of PIEZO1-mediated Ca2+ influx. Aß inhibition of PIEZO1 in the absence of stereospecific peptide-protein interactions shows that Aß peptides modulate both cell membrane and cytoskeletal mechanics to control PIEZO1-triggered Ca2+ influx.

Heterogeneous Cytoskeletal Force Distribution Delineates the Onset Ca2+ Influx Under Fluid Shear Stress in Astrocytes.

Mechanical perturbations increase intracellular Ca2+ in cells, but the coupling of mechanical forces to the Ca2+ influx is not well understood. We used a microfluidic chamber driven with a high-speed pressure servo to generate defined fluid shear stress to cultured astrocytes, and simultaneously measured cytoskeletal forces using a force sensitive actinin optical sensor and intracellular Ca2+. Fluid shear generated non-uniform forces in actinin that critically depended on the stimulus rise time emphasizing the presence of viscoelasticity in the activating sequence. A short (ms) shear pulse with fast rise time (2 ms) produced an immediate increase in actinin tension at the upstream end of the cell with minimal changes at the downstream end. The onset of Ca2+ rise began at highly strained areas. In contrast to stimulus steps, slow ramp stimuli produced uniform forces throughout the cells and only a small Ca2+ response. The heterogeneity of force distribution is exaggerated in cells having fewer stress fibers and lower pre-tension in actinin. Disruption of cytoskeleton with cytochalasin-D (Cyt-D) eliminated force gradients, and in those cells Ca2+ elevation started from the soma. Thus, Ca2+ influx with a mechanical stimulus depends on local stress within the cell and that is time dependent due to viscoelastic mechanics.

Investigating the structural dynamics of the PIEZO1 channel activation and inactivation by coarse-grained modeling.

The PIEZO channels, a family of mechanosensitive channels in vertebrates, feature a fast activation by mechanical stimuli (eg, membrane tension) followed by a slower inactivation. Although a medium-resolution structure of the trimeric form of PIEZO1 was solved by cryo-electron microscopy (cryo-EM), key structural changes responsible for the channel activation and inactivation are still unknown. Toward decrypting the structural mechanism of the PIEZO1 activation and inactivation, we performed systematic coarse-grained modeling using an elastic network model and related modeling/analysis tools (ie, normal mode analysis, flexibility and hotspot analysis, correlation analysis, and cryo-EM-based hybrid modeling and flexible fitting). We identified four key motional modes that may drive the tension-induced activation and inactivation, with fast and slow relaxation time, respectively. These modes allosterically couple the lateral and vertical motions of the peripheral domains to the opening and closing of the intra-cellular vestibule, enabling external mechanical forces to trigger, and regulate the activation/inactivation transitions. We also calculated domain-specific flexibility profiles, and predicted hotspot residues at key domain-domain interfaces and hinges. Our results offer unprecedented structural and dynamic information, which is consistent with the literature on mutational and functional studies of the PIEZO channels, and will guide future studies of this important family of mechanosensitive channels.

Flow induced adherens junction remodeling driven by cytoskeletal forces.

Adherens junctions (AJs) are a key structural component for tissue organization and function. Under fluid shear stress, AJs exhibit dynamic assembly/disassembly, but how shear stress couples to AJs is unclear. In MDCK cells we measured simultaneously the forces in cytoskeletal α-actinin and the density and length of AJs using a genetically coded optical force sensor, actinin-sstFRET, and fluorescently labeled E-cadherin (E-cad). We found that shear stress of 0.74dyn/cm2 for 3h significantly enhanced E-cad expression at cell-cell contacts and this phenomenon has two phases. The initial formation of segregated AJ plaques coincided with a decrease in cytoskeletal tension, but an increase in tension was necessary for expansion of the plaques and the formation of continuous AJs in the later phase. The changes in cytoskeletal tension and reorganization appear to be an upstream process in response to flow since it occurred in both wild type and dominant negative E-cad cells. Disruption of F-actin with a Rho-ROCK inhibitor eliminated AJ growth under flow. These results delineate the shear stress transduction paths in cultured cells, which helps to understand pathology of a range of diseases that involve dysfunction of E-cadherin.

Unsupervised Idealization of Ion Channel Recordings by Minimum Description Length: Application to Human PIEZO1-Channels.

Researchers can investigate the mechanistic and molecular basis of many physiological phenomena in cells by analyzing the fundamental properties of single ion channels. These analyses entail recording single channel currents and measuring current amplitudes and transition rates between conductance states. Since most electrophysiological recordings contain noise, the data analysis can proceed by idealizing the recordings to isolate the true currents from the noise. This de-noising can be accomplished with threshold crossing algorithms and Hidden Markov Models, but such procedures generally depend on inputs and supervision by the user, thus requiring some prior knowledge of underlying processes. Channels with unknown gating and/or functional sub-states and the presence in the recording of currents from uncorrelated background channels present substantial challenges to such analyses. Here we describe and characterize an idealization algorithm based on Rissanen's Minimum Description Length (MDL) Principle. This method uses minimal assumptions and idealizes ion channel recordings without requiring a detailed user input or a priori assumptions about channel conductance and kinetics. Furthermore, we demonstrate that correlation analysis of conductance steps can resolve properties of single ion channels in recordings contaminated by signals from multiple channels. We first validated our methods on simulated data defined with a range of different signal-to-noise levels, and then showed that our algorithm can recover channel currents and their substates from recordings with multiple channels, even under conditions of high noise. We then tested the MDL algorithm on real experimental data from human PIEZO1 channels and found that our method revealed the presence of substates with alternate conductances.

Mechanical stress activates NMDA receptors in the absence of agonists.

While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca2+ entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca2+ influx. Extracellular Mg2+ at 2 mM did not significantly affect the shear induced Ca2+ influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.

GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels.

GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ∼20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between "deep" and "shallow" binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as "area reservoirs" leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.

GsMTx4-D is a cardioprotectant against myocardial infarction during ischemia and reperfusion.

GsMTx4 is a selective inhibitor of cationic mechanosensitive ion channels (MSCs) and has helped establish the role of MSCs in cardiac physiology. Inhomogeneous local mechanical stresses due to hypercontracture and swelling during ischemic reperfusion injury (IRI) likely induce elevated MSC activity that can contribute to cation imbalance. The aim of this study was to determine if the D enantiomer of GsMTx4 can act as a cardioprotectant in a mouse IRI model. Ischemia and reperfusion involved ligating a coronary artery followed by release of the ligature. GsMTx4-D was tested by either acute intravenous injection during the ischemic event or by two day pretreatment by intraperitoneal injection, both methods achieving similar results. Based on pharmacokinetic studies, GsMTx4-D dosage was set to achieve expected plasma concentrations between 50 and 5000nM and heart tissue concentrations between 1 and 200nM by intravenous injection. Relative to vehicle injected animals, GsMTx4-D reduced infarct area by ~40% for acute and pretreated animals for both 20 and 45min ischemic challenges. Many indicators of cardiac output were indistinguishable from sham-treated control hearts after GsMTx4-D treatment showing improvement at both 4 and 48h post ischemia, and premature ventricular beats immediately following reperfusion were also significantly reduced. To determine if GsMTx4-D cardioprotection could act directly at the level of cardiomyocytes, we tested its effects in vitro on indicators of IRI damage like cation influx and activation of inflammatory kinases in isolated myocytes cultured under hypoxic conditions. Hypoxia challenged cardiomyocytes treated with 10µM GsMTx4-D showed improved contractility and near normal contraction-related Ca(2+) influx. GsMTx4-D inhibited indicators of ischemic damage such as the apoptotic signaling system JNK/c-Jun, but also inhibited the energy response signaling system Akt kinase. We conclude that GsMTx4-D is a potent cardioprotectant in vivo that may act directly on cardiomyocytes and potentially be useful in multidrug strategies to treat IRI.

Adaptation Independent Modulation of Auditory Hair Cell Mechanotransduction Channel Open Probability Implicates a Role for the Lipid Bilayer.

The auditory system is able to detect movement down to atomic dimensions. This sensitivity comes in part from mechanisms associated with gating of hair cell mechanoelectric transduction (MET) channels. MET channels, located at the tops of stereocilia, are poised to detect tension induced by hair bundle deflection. Hair bundle deflection generates a force by pulling on tip-link proteins connecting adjacent stereocilia. The resting open probability (P(open)) of MET channels determines the linearity and sensitivity to mechanical stimulation. Classically, P(open) is regulated by a calcium-sensitive adaptation mechanism in which lowering extracellular calcium or depolarization increases P(open). Recent data demonstrated that the fast component of adaptation is independent of both calcium and voltage, thus requiring an alternative explanation for the sensitivity of P(open) to calcium and voltage. Using rat auditory hair cells, we characterize a mechanism, separate from fast adaptation, whereby divalent ions interacting with the local lipid environment modulate resting P(open). The specificity of this effect for different divalent ions suggests binding sites that are not an EF-hand or calmodulin model. GsMTx4, a lipid-mediated modifier of cationic stretch-activated channels, eliminated the voltage and divalent sensitivity with minimal effects on adaptation. We hypothesize that the dual mechanisms (lipid modulation and adaptation) extend the dynamic range of the system while maintaining adaptation kinetics at their maximal rates.

Human PIEZO1 Ion Channel Functions as a Split Protein.

PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the "split protein" is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone.