Drosophila Mechanotransduction—Linking Proteins and Functions

The sensation of touch, gravity, and sound all rely on dedicated ion channels that transduce mechanical stimulus forces into electrical response signals. The functional workings and molecular identities of these mechanotransducer channels are little understood. Recent work shows that the mechanotransducers for fly and vertebrate hearing share equivalent gating mechanisms, whereby this mechanism can be probed non-invasively in the mechanics of the Drosophila ear. Here, we describe how this mechanics can be used to evaluate the roles of identified proteins in the process of mechanosensation and, specifically, their contributions to mechanotransduction.     

Stretch-activated whole cell currents in adult rat cardiac myocytes

Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 microM Gd(3+) but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately -6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 - 5.0 SL (microm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.     

Mechanosensitive channels: what can they do and how do they do it?

While mechanobiological processes employ diverse mechanisms, at their heart are force-induced perturbations in the structure and dynamics of molecules capable of triggering subsequent events. Among the best characterized force-sensing systems are bacterial mechanosensitive channels. These channels reflect an intimate coupling of protein conformation with the mechanics of the surrounding membrane; the membrane serves as an adaptable sensor that responds to an input of applied force and converts it into an output signal, interpreted for the cell by mechanosensitive channels. The cell can exploit this information in a number of ways: ensuring cellular viability in the presence of osmotic stress and perhaps also serving as a signal transducer for membrane tension or other functions. This review focuses on the bacterial mechanosensitive channels of large (MscL) and small (MscS) conductance and their eukaryotic homologs, with an emphasis on the outstanding issues surrounding the function and mechanism of this fascinating class of molecules.     

Bubbles, gating, and anesthetics in ion channels

We suggest that bubbles are the bistable hydrophobic gates responsible for the on-off transitions of single channel currents. In this view, many types of channels gate by the same physical mechanism—dewetting by capillary evaporation—but different types of channels use different sensors to modulate hydrophobic properties of the channel wall and thereby trigger and control bubbles and gating. Spontaneous emptying of channels has been seen in many simulations. Because of the physics involved, such phase transitions are inherently sensitive, unstable threshold phenomena that are difficult to simulate reproducibly and thus convincingly. We present a thermodynamic analysis of a bubble gate using morphometric density functional theory of classical (not quantum) mechanics. Thermodynamic analysis of phase transitions is generally more reproducible and less sensitive to details than simulations. Anesthetic actions of inert gases—and their interactions with hydrostatic pressure (e.g., nitrogen narcosis)—can be easily understood by actions on bubbles. A general theory of gas anesthesia may involve bubbles in channels. Only experiments can show whether, or when, or which channels actually use bubbles as hydrophobic gates: direct observation of bubbles in channels is needed. Existing experiments show thin gas layers on hydrophobic surfaces in water and suggest that bubbles nearly exist in bulk water.     

Effects of fish size, time‐to‐fatigue and turbulence on swimming performance: a case study of Galaxias maculatus

A simple relationship for the inanga Galaxias maculatus swimming velocity is suggested and tested in low and high turbulence channels. The relationship connects the swimming velocity with fish Reynolds and Froude numbers and can be used in both ecological analysis ( e.g . habitat requirements) and management strategies ( e.g . fishways design). Contrary to some previous studies and intuition, effects of turbulence on swimming performance appeared to be negligible. The most likely explanation for this result relates to mechanics of fish–turbulence interactions that may be dependent on both the turbulence scales and energy. The data suggest that future studies of turbulence effects on fish behaviour should involve, in addition to turbulence energetics, consideration of fish dimensions in relation to the spectrum of turbulence scales.     

Cellular Pressure and Volume Regulation and Implications for Cell Mechanics

In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death, and migration. Volume and shape regulation also directly impacts the mechanics of cells and tissues. Here, we develop a mathematical model of cellular volume and pressure regulation, incorporating essential elements such as water permeation, mechanosensitive channels, active ion pumps, and active stresses in the cortex. The model can fully explain recent experimental data, and it predicts cellular volume and pressure for several models of cell cortical mechanics. Moreover, we show that when cells are subjected to an externally applied load, such as in an atomic force microscopy indentation experiment, active regulation of volume and pressure leads to a complex cellular response. Instead of the passive mechanics of the cortex, the observed cell stiffness depends on several factors working together. This provides a mathematical explanation of rate-dependent response of cells under force.     

Cellular Pressure and Volume Regulation and Implications for Cell Mechanics

In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death, and migration. Volume and shape regulation also directly impacts the mechanics of cells and tissues. Here, we develop a mathematical model of cellular volume and pressure regulation, incorporating essential elements such as water permeation, mechanosensitive channels, active ion pumps, and active stresses in the cortex. The model can fully explain recent experimental data, and it predicts cellular volume and pressure for several models of cell cortical mechanics. Moreover, we show that when cells are subjected to an externally applied load, such as in an atomic force microscopy indentation experiment, active regulation of volume and pressure leads to a complex cellular response. Instead of the passive mechanics of the cortex, the observed cell stiffness depends on several factors working together. This provides a mathematical explanation of rate-dependent response of cells under force.     

Brownian dynamics simulations of the recognition of the scorpion toxin P05 with the small-conductance calcium-activated potassium channels

The recognition of the scorpion toxin P05 and the small-conductance, calcium-activated potassium (SK) channels, rsk1, rsk2, and rsk3, has been studied by means of the Brownian dynamics (BD) method. All of the 25 available structures of P05 in the RCSB Protein Data Bank determined by NMR were considered during the simulation, which indicated that the conformation of P05 affects both the recognition and binding between the two proteins significantly. Comparing the top four high-frequency structures of P05 binding to the SK channels, we found that the rsk2 channel, with high frequencies and lowest electrostatic interaction energies (E (int)(ES)), is the most favorable for P05 binding, while rsk3 is intermediate, and rsk1 is the least favorable. Among the 25 structures of P05, the 13th structure docks into the binding site of the rsk2 channel with the highest probability and most favorable electrostatic interactions. From the P05-rsk2 channel binding model, we identified the residues critical for the recognition of these two proteins through triplet contact analyses. P05 locates around the extracellular mouth of the SK channels and contacts the SK channels using its alpha-helix rather than beta-sheets. The critical triplet contacts for recognition between P05 and the rsk2 channel are Arg6 (P05)-Asp364 (SK), Arg7 (P05)-Asn368 (SK), and Arg13 (P05)-Asp341 (SK). The structure of the P05-rsk2 complex with the most favorable electrostatic interaction energy was further refined by molecular mechanics, showing that six hydrogen bonding interactions exist between P05 and the rsk2 channel: one hydrogen bond is formed between Arg6 (P05) and Asp364(D) (rsk2); Arg7 (P05) forms three hydrogen bonds with Asp341(B) (rsk2)) and Asp364(C) (rsk2); two hydrogen bonds are formed by Arg13 (P05) with Asp341(A) (rsk2) and Asp364(B) (rsk2). The simulation results are in good agreement with the previous molecular biological experiments and can explain the binding phenomena between P05 and SK channels at the level of molecular structure. The consistency between the results of the BD simulations and the experimental data indicated that our 3D model of the P05-rsk2 channel complex is reasonable and can be employed in further biological studies, such as rational design of the novel therapeutic agents blocking the small-conductance, calcium-activated and apamin-sensitive potassium channels, and for mutagenesis studies in both toxins and SK channels. In particular, both the BD simulations and the molecular mechanics refinements indicate that residue Asp364 of the rsk2 channel is critical for its recognition and binding functionality towards P05. This phenomenon has not been appreciated in the previous mutagenesis experiments, indicating that this might be a new clue for further functional study of SK channels.     

ENaCs and ASICs as therapeutic targets

The epithelial Na(+) channel (ENaC) and acid-sensitive ion channel (ASIC) branches of the ENaC/degenerin superfamily of cation channels have drawn increasing attention as potential therapeutic targets in a variety of diseases and conditions. Originally thought to be solely expressed in fluid absorptive epithelia and in neurons, it has become apparent that members of this family exhibit nearly ubiquitous expression. Therapeutic opportunities range from hypertension, due to the role of ENaC in maintaining whole body salt and water homeostasis, to anxiety disorders and pain associated with ASIC activity. As a physiologist intrigued by the fundamental mechanics of salt and water transport, it was natural that Dale Benos, to whom this series of reviews is dedicated, should have been at the forefront of research into the amiloride-sensitive sodium channel. The cloning of ENaC and subsequently the ASIC channels has revealed a far wider role for this channel family than was previously imagined. In this review, we will discuss the known and potential roles of ENaC and ASIC subunits in the wide variety of pathologies in which these channels have been implicated. Some of these, such as the role of ENaC in Liddle's syndrome are well established, others less so; however, all are related in that the fundamental defect is due to inappropriate channel activity.     

Bilayer-mediated clustering and functional interaction of MscL channels

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.     

Transient polarization currents in the squid giant axon.

It has been repeatedly noted that the change of conformation of the molecules that serve as the ion-selective channels for sodium and potassium conductance in the nerve membrane will be accompanied by a change in the dipole moment of the molecule. This time-dependent change of dipole moment will produce transient currents in the membrane. The canonical form for these currents is determined with conventional statistical mechanics formalism. It is pointed out that the voltage dependence of the conductance channel conductance determines the free energy of the system to within a factor that is an unknown function of the voltage. Since the dipole currents do not depend on this unknown function, they are completely determined 0y the observed properties of the conductance system. The predicted properties of these dipole currents, their time constants and strengths, are calculated. By using the observed properties of gating currents, the density of the sodium channels is computed. The predicted properties of the dipole currents are found to compare satisfactorily with the observed properties of gating currents.     

Entropic tension in crowded membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function in vivo. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than [Formula: see text] in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.     

Mechanics as a Means of Information Propagation in Development

New research demonstrates that mechanics can serve as a means of information propagation in developing embryos. Historically, the study of embryonic development has had a dichotomy between morphogens and pattern formation on the one hand and morphogenesis and mechanics on the other. Secreted signals are the preeminent means of information propagation between cells and used to control cell fate, while physical forces act downstream or in parallel to shape tissue morphogenesis. However, recent work has blurred this division of function by demonstrating that mechanics can serve as a means of information propagation. Adhesive or repulsive interactions can propagate through a tissue as a wave. These waves are rapid and directional and can be used to control the flux of cells through a developmental trajectory. Here, two examples are reviewed in which mechanics both guides and mediates morphogenesis and two examples in which mechanics intertwines with morphogens to regulate cell fate.     

Autoregulation and conduction of vasomotor responses in a mathematical model of the rat afferent arteriole

We have formulated a mathematical model for the rat afferent arteriole (AA). Our model consists of a series of arteriolar smooth muscle cells and endothelial cells, each of which represents ion transport, cell membrane potential, and gap junction coupling. Cellular contraction and wall mechanics are also represented for the smooth muscle cells. Blood flow through the AA lumen is described by Poiseuille flow. The AA model's representation of the myogenic response is based on the hypothesis that changes in hydrostatic pressure induce changes in the activity of nonselective cation channels. The resulting changes in membrane potential then affect calcium influx through changes in the activity of the voltage-gated calcium channels, so that vessel diameter decreases with increasing pressure values. With this configuration, the model AA maintains roughly stable renal blood flow within a physiologic range of blood flow pressure. Model simulation of vasoconstriction initiated from local stimulation also agrees well with findings in the experimental literature, notably those of Steinhausen et al. (Steinhausen M, Endlich K, Nobiling R, Rarekh N, Schütt F. J Physiol 505: 493-501, 1997), which indicated that conduction of vasoconstrictive response decays more rapidly in the upstream flow direction than downstream. The model can be incorporated into models of integrated renal hemodynamic regulation.     

Simulations of neuromuscular control in lamprey swimming.

The neuronal generation of vertebrate locomotion has been extensively studied in the lamprey. Models at different levels of abstraction are being used to describe this system, from abstract nonlinear oscillators to interconnected model neurons comprising multiple compartments and a Hodgkin-Huxley representation of the most relevant ion channels. To study the role of sensory feedback by simulation, it eventually also becomes necessary to incorporate the mechanical movements in the models. By using simplifying models of muscle activation, body mechanics, counteracting water forces, and sensory feedback through stretch receptors and vestibular organs, we have been able to close the feedback loop to enable studies of the interaction between the neuronal and the mechanical systems. The neuromechanical simulations reveal that the currently known network is sufficient for generating a whole repertoire of swimming patterns. Swimming at different speeds and with different wavelengths, together with the performance of lateral turns can all be achieved by simply varying the brainstem input. The neuronal mechanisms behind pitch and roll manoeuvres are less clear. We have put forward a 'crossed-oscillators' hypothesis where partly separate dorsal and ventral circuits are postulated. Neuromechanical simulations of this system show that it is also capable of generating realistic pitch turns and rolls, and that vestibular signals can stabilize the posture during swimming.     

Voltage gating of VDAC is regulated by nonlamellar lipids of mitochondrial membranes

Evidence is accumulating that lipids play important roles in permeabilization of the mitochondria outer membrane (MOM) at the early stage of apoptosis. Lamellar phosphatidylcholine (PC) and nonlamellar phosphatidylethanolamine (PE) lipids are the major membrane components of the MOM. Cardiolipin (CL), the characteristic lipid from the mitochondrial inner membrane, is another nonlamellar lipid recently shown to play a role in MOM permeabilization. We investigate the effect of these three key lipids on the gating properties of the voltage-dependent anion channel (VDAC), the major channel in MOM. We find that PE induces voltage asymmetry in VDAC current-voltage characteristics by promoting channel closure at cis negative applied potentials. Significant asymmetry is also induced by CL. The observed differences in VDAC behavior in PC and PE membranes cannot be explained by differences in the insertion orientation of VDAC in these membranes. Rather, it is clear that the two nonlamellar lipids affect VDAC gating. Using gramicidin A channels as a tool to probe bilayer mechanics, we show that VDAC channels are much more sensitive to the presence of CL than could be expected from the experiments with gramicidin channels. We suggest that this is due to the preferential insertion of VDAC into CL-rich domains. We propose that the specific lipid composition of the mitochondria outer membrane and/or of contact sites might influence MOM permeability by regulating VDAC gating.     

细胞结构动力学——实时调控细胞力学的因素分析

机械力普遍存在于活细胞的生命活动中,而细胞内力学活动必须依赖骨架结构传递,这种独特的力学形式被称为细胞结构力学.单位时间内细胞结构力学变化受多因素调控,如外力、渗透压、动力分子、张力敏感性离子通道、胞内力学感受器及骨架组装等,构成了细胞结构动力学研究的重要内容.基于荧光共振能量转移(FRET)原理开发的荧光张力探针能整合到细胞骨架内,将细胞结构力学变化转化为光学信号,可能带来细胞力学研究的革命.随着细胞结构动力学研究内容的不断深入,特别是太空时代细胞力学稳态的打破,细胞结构动力学将在生命及医学研究领域显露出越来越重要的地位.     

The ion channels to cytoskeleton connection as potential mechanism of mechanosensitivity

As biological force-sensing systems mechanosensitive (MS) ion channels present the best example of coupling molecular dynamics of membrane proteins to the mechanics of the surrounding cell membrane. In animal cells MS channels have over the past two decades been very much in focus of mechanotransduction research. In recent years this helped to raise awareness of basic and medical researchers about the role that abnormal MS channels may play in the pathophysiology of diseases, such as cardiac hypertrophy, atrial fibrillation, muscular dystrophy or polycystic kidney disease. To date a large number of MS channels from organisms of diverse phylogenetic origins have been identified at the molecular level; however, the structure of only few of them has been determined. Although their function has extensively been studied in a great variety of cells and tissues by different experimental approaches it is, with exception of bacterial MS channels, very little known about how these channels sense mechanical force and which cellular components may contribute to their function. By focusing on MS channels found in animal cells this article discusses the ways in which the connections between cytoskeleton and ion channels may contribute to mechanosensory transduction in these cells. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Properties of Large-Conductance Cationic Channels in the Neuronal Nuclear Envelope

The genetic apparatus of the nucleus of eukaryotic cells is surrounded by the nuclear envelope containing ion channels with different biophysical properties. The channels responsible for calcium release from the nuclear envelope into the intranuclear space have attracted special attention. As we found, a great number of large-conductance ion channels selective with respect to monovalent cations and impermeable for bivalent cations are present in all membranes where inositol trisphosphate receptors are expressed. We showed that channels of this type are insensitive to blockers of potassium channels and, according to their properties, cannot be attributed to one well-known type of ion channels or another. In addition, since their blockers remain unknown, these channels at present cannot be isolated and cloned. Channels of this type deserve further investigation; we hypothesize that large-conductance cationic channels can be involved in the control of the duration of Ca²⁺ release from the calcium store.     

A finite element model of myocardial infarction using a composite material approach

Computational models are effective tools to study cardiac mechanics under normal and pathological conditions. They can be used to gain insight into the physiology of the heart under these conditions while they are adaptable to computer assisted patient-specific clinical diagnosis and therapeutic procedures. Realistic cardiac mechanics models incorporate tissue active/passive response in conjunction with hyperelasticity and anisotropy. Conventional formulation of such models leads to mathematically-complex problems usually solved by custom-developed non-linear finite element (FE) codes. With a few exceptions, such codes are not available to the research community. This article describes a computational cardiac mechanics model developed such that it can be implemented using off-the-shelf FE solvers while tissue pathologies can be introduced in the model in a straight-forward manner. The model takes into account myocardial hyperelasticity, anisotropy, and active contraction forces. It follows a composite tissue modeling approach where the cardiac tissue is decomposed into two major parts: background and myofibers. The latter is modelled as rebars under initial stresses mimicking the contraction forces. The model was applied in silico to study the mechanics of infarcted left ventricle (LV) of a canine. End-systolic strain components, ejection fraction, and stress distribution attained using this LV model were compared quantitatively and qualitatively to corresponding data obtained from measurements as well as to other corresponding LV mechanics models. This comparison showed very good agreement.