Mechanosensory Processes Research Articles

Dystroglycan-HSPG interactions provide synaptic plasticity and specificity.

This study examined the roles of the laminin and proteoglycan receptor dystroglycan (DG) in extracellular matrix stabilization and cellular mechanosensory processes conveyed through communication between the extracellular matrix (ECM) and cytoskeleton facilitated by DG. Specific functional attributes of HS-proteoglycans (HSPGs) are conveyed through interactions with DG and provide synaptic specificity through diverse interactions with an extensive range of cell attachment and adaptor proteins which convey synaptic plasticity. HSPG-DG interactions are important in phototransduction and neurotransduction and facilitate retinal bipolar-photoreceptor neuronal signaling in vision. Besides synaptic stabilization, HSPG-DG interactions also stabilize basement membranes and the ECM and have specific roles in the assembly and function of the neuromuscular junction. This provides neuromuscular control of muscle systems that control conscious body movement as well as essential autonomic control of diaphragm, intercostal and abdominal muscles and muscle systems in the face, mouth and pharynx which assist in breathing processes. DG is thus a multifunctional cell regulatory glycoprotein receptor and regulates a diverse range of biological and physiological processes throughout the human body. The unique glycosylation of the αDG domain is responsible for its diverse interactions with ECM components in cell-ECM signaling. Cytoskeletal cell regulatory switches assembled by the βDG domain in its role as a nuclear scaffolding protein respond to such ECM cues to regulate cellular behavior and tissue homeostasis thus DG has fascinating and diverse roles in health and disease.

An inhibitory acetylcholine receptor gates context-dependent mechanosensory processing in C. elegans

An animal's current behavior influences its response to sensory stimuli, but the molecular and circuit-level mechanisms of this context-dependent decision-making are not well understood. Caenorhabditis elegans are less likely to respond to a mechanosensory stimulus by reversing if the stimuli is received while the animal turns. Inhibitory feedback from turning associated neurons are needed for this gating. But until now, it has remained unknown precisely where in the circuit gating occurs and which specific neurons and receptors receive inhibition from the turning circuitry. Here, we use genetic manipulations, single-cell rescue experiments, and high-throughput closed-loop optogenetic perturbations during behavior to reveal the specific neuron and receptor responsible for receiving inhibition and altering sensorimotor processing. Our measurements show that an inhibitory acetylcholine-gated chloride channel comprising LGC-47 and ACC-1 expressed in neuron type RIM disrupts mechanosensory evoked reversals during turns, presumably in response to inhibitory signals from turning-associated neuron SAA.

The structure of the Caenorhabditis elegans TMC-2 complex suggests roles of lipid-mediated subunit contacts in mechanosensory transduction

Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily conserved family of membrane proteins whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila. TMC1 and TMC2 are components of ion channel complexes, but the molecular features that tune these complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here, we present the single-particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans. The complex is composed of two copies of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex highlights conserved protein-lipid interactions, as well as a π-helical structural motif in the pore-forming helices, that together suggest a mechanism for TMC-mediated mechanosensory transduction.

Inhibitory feedback from the motor circuit gates mechanosensory processing in Caenorhabditis elegans.

Animals must integrate sensory cues with their current behavioral context to generate a suitable response. How this integration occurs is poorly understood. Previously, we developed high-throughput methods to probe neural activity in populations of Caenorhabditis elegans and discovered that the animal's mechanosensory processing is rapidly modulated by the animal's locomotion. Specifically, we found that when the worm turns it suppresses its mechanosensory-evoked reversal response. Here, we report that C. elegans use inhibitory feedback from turning-associated neurons to provide this rapid modulation of mechanosensory processing. By performing high-throughput optogenetic perturbations triggered on behavior, we show that turning-associated neurons SAA, RIV, and/or SMB suppress mechanosensory-evoked reversals during turns. We find that activation of the gentle-touch mechanosensory neurons or of any of the interneurons AIZ, RIM, AIB, and AVE during a turn is less likely to evoke a reversal than activation during forward movement. Inhibiting neurons SAA, RIV, and SMB during a turn restores the likelihood with which mechanosensory activation evokes reversals. Separately, activation of premotor interneuron AVA evokes reversals regardless of whether the animal is turning or moving forward. We therefore propose that inhibitory signals from SAA, RIV, and/or SMB gate mechanosensory signals upstream of neuron AVA. We conclude that C. elegans rely on inhibitory feedback from the motor circuit to modulate its response to sensory stimuli on fast timescales. This need for motor signals in sensory processing may explain the ubiquity in many organisms of motor-related neural activity patterns seen across the brain, including in sensory processing areas.

Generation of a whole-brain hemodynamic response function and sex-specific differences in cerebral processing of mechano-sensation in mice detected by BOLD fMRI.

BOLD fMRI has become a prevalent method to study cerebral sensory processing in rodent disease models, including pain and mechanical hypersensitivity. fMRI data analysis is frequently combined with a general-linear-model (GLM) -based analysis, which uses the convolution of a hemodynamic response function (HRF) with the stimulus paradigm. However, several studies indicated that the HRF differs across species, sexes, brain structures, and experimental factors, including stimulation modalities or anesthesia, and hence might strongly affect the outcome of BOLD analyzes. While considerable work has been done in humans and rats to understand the HRF, much less is known in mice. As a prerequisite to investigate mechano-sensory processing and BOLD fMRI data in male and female mice, we (1) designed a rotating stimulator that allows application of two different mechanical modalities, including innocuous von Frey and noxious pinprick stimuli and (2) determined and statistically compared HRFs across 30 brain structures and experimental conditions, including sex and, stimulus modalities. We found that mechanical stimulation lead to brain-wide BOLD signal changes thereby allowing extraction of HRFs from multiple brain structures. However, we did not find differences in HRFs across all brain structures and experimental conditions. Hence, we computed a whole-brain mouse HRF, which is based on 88 functional scans from 30 mice. A comparison of this mouse-specific HRF with our previously reported rat-derived HRF showed significantly slower kinetics in mice. Finally, we detected pronounced differences in cerebral BOLD activation between male and female mice with mechanical stimulation, thereby exposing divergent processing of noxious and innocuous stimuli in both sexes.

Labeling PIEZO2 activity in the peripheral nervous system

Sensory neurons detect mechanical forces from both the environment and internal organs to regulate physiology. PIEZO2 is a mechanosensory ion channel critical for touch, proprioception, and bladder stretch sensation, yet its broad expression in sensory neurons suggests it has undiscovered physiological roles. To fully understand mechanosensory physiology, we must know where and when PIEZO2-expressing neurons detect force. The fluorescent styryl dye FM 1-43 was previously shown to label sensory neurons. Surprisingly, we find that the vast majority of FM 1-43 somatosensory neuron labeling in mice invivo is dependent on PIEZO2 activity within the peripheral nerve endings. We illustrate the potential of FM 1-43 by using it to identify novel PIEZO2-expressing urethral neurons that are engaged by urination. These data reveal that FM 1-43 is a functional probe for mechanosensitivity via PIEZO2 activation invivo and will facilitate the characterization of known and novel mechanosensory processes in multiple organ systems.

The role of mechanobiology in bone and cartilage model systems in characterizing initiation and progression of osteoarthritis

Multifaceted changes in the mechanobiological environment of skeletal joints, at multiple length scales, are central to the development of diseases-like osteoarthritis (OA). Recent evidence demonstrates related mechanical alterations in both bone and cartilage tissues, with crosstalk between the tissues being an important factor in acute and chronic degenerative processes. However, recapitulating multicellular tissue systems in the laboratory to study the entire osteochondral unit remains challenging. Thus, the development of accurate and reproducible OA model systems and the selection of the most suitable model for individual experimental approaches are critical. This review first discusses recent progress in understanding mechanosensory processes in healthy and osteoarthritic joints. Subsequently, we review advancements in the development of in vitro and ex vivo model systems ranging from 2D monocultures through to joint organ-on-a-chip models. Use of these systems allows for the study of multiple cell types in controlled, reproducible, and dynamic environments, which can incorporate precisely controlled mechanical and biochemical stimuli, and biophysical cues. The way in which these models have, and will continue to, improve our ability to recapitulate complex mechanical/paracrine signaling pathways in osteochondral tissues is then discussed. As the accuracy of model systems advances, they will have a significant impact on both our understanding of the pathobiology of OA and in identifying and screening therapeutic targets to improve treatment of this complex disease.

Perpendicular alignment of lymphatic endothelial cells in response to spatial gradients in wall shear stress

One-way valves in the lymphatic system form from lymphatic endothelial cells (LECs) during embryonic development and are required for efficient tissue drainage. Although fluid flow is thought to guide both valve formation and maintenance, how this occurs at a mechanistic level remains incompletely understood. We built microfluidic devices that reproduce critical aspects of the fluid flow patterns found at sites of valvulogenesis. Using these devices, we observed that LECs replicated aspects of the early steps in valvulogenesis: cells oriented perpendicular to flow in the region of maximum wall shear stress (WSS) and exhibited enhanced nuclear localization of FOXC2, a transcription factor required for valvulogenesis. Further experiments revealed that the cell surface protein E-selectin was required for both of these responses. Our observations suggest that spatial gradients in WSS help to demarcate the locations of valve formation, and implicate E-selectin as a component of a mechanosensory process for detecting WSS gradients.

Mammalian TRP Ion Channels are Insensitive to Membrane Stretch

TRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading, and detection of cerebrospinal fluid flow. However, it is unclear in many instances whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six subfamilies. We found that these TRP channels were insensitive to short membrane stretch in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and thus they are more likely activated by cytoplasmic tethers or downstream components and act as amplifiers of cellular mechanosensory signaling cascades.

Model of the Nerve Impulse with Account of Mechanosensory Processes: Stationary Solutions.

Mechanotransduction refers to a physiological process by which mechanical forces, such as pressures exerted by ionized fluids on cell membranes and tissues, can trigger excitations of electrical natures that play important role in the control of various sensory (i.e. stimuli-responsive) organs and homeostasis of living organisms. In this work, the influence of mechanotransduction processes on the generic mechanism of the action potential is investigated analytically, by considering a mathematical model that consists of two coupled nonlinear partial differential equations. One of these two equations is the Korteweg-de Vries equation governing the spatio-temporal evolution of the density difference between intracellular and extracellular fluids across the nerve membrane, and the other is Hodgkin-Huxley cable equation for the transmembrane voltage with a self-regulatory (i.e. diode-type) membrane capacitance. The self-regulatory feature here refers to the assumption that membrane capacitance varies with the difference in density of ion-carrying intracellular and extracellular fluids, thus ensuring an electromechanical feedback mechanism and consequently an effective coupling of the two nonlinear equations. The exact one-soliton solution to the density-difference equation is obtained in terms of a pulse excitation. With the help of this exact pulse solution the Hodgkin-Huxley cable equation is shown to transform, in steady state, to a linear eigenvalue problem some bound states of which can be obtained exactly. Few of such bound-state solutions are found analytically.

Mammalian TRP ion channels are insensitive to membrane stretch.

ABSTRACTTRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading and detection of cerebrospinal fluid flow. However, in many instances it is unclear whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six mammalian subfamilies. We found that these TRP channels were insensitive to short membrane stretches in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that, when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and, thus, are more likely to be activated by cytoplasmic tethers or downstream components and to act as amplifiers of cellular mechanosensory signaling cascades.

OSCA/TMEM63 are an Evolutionarily Conserved Family of Mechanically Activated Ion Channels.

Mechanically activated (MA) ion channels convert physical forces into electrical signals, and are essential for eukaryotic physiology. Despite their importance, few bona-fide MA channels have been described in plants and animals. Here, we show that various members of the OSCA and TMEM63 family of proteins from plants, flies, and mammals confer mechanosensitivity to naïve cells. We conclusively demonstrate that OSCA1.2, one of the Arabidopsis thaliana OSCA proteins, is an inherently mechanosensitive, pore-forming ion channel. Our results suggest that OSCA/TMEM63 proteins are the largest family of MA ion channels identified, and are conserved across eukaryotes. Our findings will enable studies to gain deep insight into molecular mechanisms of MA channel gating, and will facilitate a better understanding of mechanosensory processes in vivo across plants and animals.

TRPM7 controls mesenchymal features of breast cancer cells by tensional regulation of SOX4

Mechanically induced signaling pathways are important drivers of tumor progression. However, if and how mechanical signals affect metastasis or therapy response remains poorly understood. We previously found that the channel-kinase TRPM7, a regulator of cellular tension implicated in mechano-sensory processes, is required for breast cancer metastasis in vitro and in vivo. Here, we show that TRPM7 contributes to maintaining a mesenchymal phenotype in breast cancer cells by tensional regulation of the EMT transcription factor SOX4. The functional consequences of SOX4 knockdown closely mirror those produced by TRPM7 knockdown. By traction force measurements, we demonstrate that TRPM7 reduces cytoskeletal tension through inhibition of myosin II activity. Moreover, we show that SOX4 expression and downstream mesenchymal markers are inversely regulated by cytoskeletal tension and matrix rigidity. Overall, our results identify SOX4 as a transcription factor that is uniquely sensitive to cellular tension and indicate that TRPM7 may contribute to breast cancer progression by tensional regulation of SOX4.

Studying Mechanosensitivity of Two-Pore Domain K+ Channels in Cellular and Reconstituted Proteoliposome Membranes.

Mechanical force sensation is fundamental to a wide breadth of biology from the classic senses of touch, pain, hearing, and balance to less conspicuous sensations of proprioception, blood pressure, and osmolarity and basic aspects of cell growth, differentiation, and development. These diverse and essential systems use force-gated (or mechanosensitive) ion channels that convert mechanical stimuli into cellular electrical signals. TRAAK, TREK1, and TREK2 are K+-selective ion channels of the two-pore domain K+ (K2P) family that are mechanosensitive: they are gated open by increasing membrane tension. TRAAK and TREK channels are thought to play roles in somatosensory and other mechanosensory processes in neuronal and non-neuronal tissues. Here, we present protocols for three assays to study mechanical activation of these channels in cell membranes: (1) cell swelling, (2) cell poking, and (3) patched membrane stretching. Patched membrane stretching is also applicable to the study of mechanosensitive K2P channel activity in a cell-free system and a procedure for proteoliposome reconstitution and patching is also presented. These approaches are alsoreadily applicable to the study of other mechanosensitive ion channels.

Active Mechanisms of Vibration Encoding and Frequency Filtering in Central Mechanosensory Neurons

To better understand biophysical mechanisms of mechanosensory processing, we investigated two cell types in the Drosophila brain (A2 and B1 cells) that are postsynaptic to antennal vibration receptors. A2 cells receive excitatory synaptic currents in response to both directions of movement: thus, twice per vibration cycle. The membrane acts as a low-pass filter, so that voltage and spiking mainly track the vibration envelope rather than individual cycles. By contrast, B1 cells are excited by only forward or backward movement, meaning they are sensitive to vibration phase. They receive oscillatory synaptic currents at the stimulus frequency, and they bandpass filter these inputs to favor specific frequencies. Different cells prefer different frequencies, due to differences in their voltage-gated conductances. Both Na+ and K+ conductances suppress low-frequency synaptic inputs, so cells with larger voltage-gated conductances prefer higher frequencies. These results illustrate how membrane properties and voltage-gated conductances can extract distinct stimulus features into parallel channels.

Origin of the Force: The Force-From-Lipids Principle Applied to Piezo Channels.

Piezo channels are a ubiquitously expressed, principal type of molecular force sensor in eukaryotes. They enable cells to decode a myriad of physical stimuli and are essential components of numerous mechanosensory processes. Central to their physiological role is the ability to change conformation in response to mechanical force. Here we discuss the evolutionary origin of Piezo in relation to other MS channels in addition to the force that gates Piezo channels. In particular, we discuss whether Piezo channels are inherently mechanosensitive in accordance with the force-from-lipid paradigm which has been firmly established for bacterial MS channels and two-pore domain K+ (K2P) channels. We also discuss the evidence supporting a reliance on or direct interaction with structural scaffold proteins of the cytoskeleton and extracellular matrix according to the force-from-filament principle. In doing so, we explain the false dichotomy that these distinctions represent. We also discuss the possible unifying models that shed light on channel mechanosensitivity at the molecular level.

Peripheral Mechanosensory Neuron Dysfunction Underlies Tactile and Behavioral Deficits in Mouse Models of ASDs

Patients with autism spectrum disorders (ASDs) commonly experience aberrant tactile sensitivity, yet the neural alterations underlying somatosensory dysfunction and the extent to which tactile deficits contribute to ASD characteristics are unknown. We report that mice harboring mutations in Mecp2, Gabrb3, Shank3, and Fmr1 genes associated with ASDs in humans exhibit altered tactile discriminationand hypersensitivity to gentle touch. Deletion of Mecp2 or Gabrb3 in peripheral somatosensoryneurons causes mechanosensory dysfunction through loss of GABAA receptor-mediated presynaptic inhibition of inputs to the CNS. Remarkably, tactile defects resulting from Mecp2 or Gabrb3 deletion in somatosensory neurons during development, but not in adulthood, cause social interaction deficits and anxiety-like behavior. Restoring Mecp2 expression exclusively in the somatosensory neurons of Mecp2-null mice rescues tactile sensitivity, anxiety-like behavior, and social interaction deficits, but not lethality, memory, or motor deficits. Thus, mechanosensory processing defects contribute to anxiety-like behavior and social interaction deficits in ASD mouse models. PAPERCLIP.

Mechanosensing Defects in Nuclear Envelope Related Disorders

Recent data indicate that mutations in nuclear envelope genes cause both defects in mechanotransduction signaling and force transmission across the nuclear envelop. A central role in this mechanosensory process has been attributed to A-type lamins, which together with the LInker of the Nucleoskeleton and Cytoskeleton (LINC)-complex enables force transmission across the nuclear envelop. Whereas a basic picture is that extracellular mechanical forces are transmitted from outside the cell to the nucleus, we hypothesize that mutations in A-type lamin (LMNA) or in nesprin can affect the mechanical transmission from the nuclear envelop to the extracellular matrix. Human myoblasts with either LMNA or nesprin-1 mutations were cultured on soft substrates (12 kPa) and compared to control (WT) myoblasts. On soft surface, LMNA and nesprin-1 mutated myoblasts exhibited enlarged and increased number of focal adhesions, increased stress fibers and enlarged cell spreading area compared with WT. Further, nesprin-1 mutant exerted significantly higher traction forces on the substrate compared with WT myoblasts. These abnormalities were greatly reduced after treatment with Y27632, or SU6656, which inhibit Rho-associated protein kinase or the Src family kinase respectively, suggesting an abnormal activation of Rho-dependent Src pathway in mutant cells. Treatment with the MLCK inhibitor ML7 also significantly reduced the spreading area in mutant cells but without modifying the number and distribution of perinuclear actin stress fibers. We concluded that the integrity of the LINC complex and the lamina is required for proper regulation of the cytoplasmic actin contractility in soft substrates, through an apparent Src-dependent ROCK pathway.

Ca2+ imaging of cricket protocerebrum responses to air current stimulation

Crickets (Gryllus bimaculatus) use the cercal sensory system at the rear of the abdomen to detect air currents and direct predator avoidance behavior. Sensory information regarding the direction and dynamic properties of air currents is processed within the terminal abdominal ganglion, and conveyed by ascending giant interneurons (GIs) to higher centers including the brain. However, the brain region responsible for decoding cercal sensory information has not yet been identified, nor the response properties within the brain characterized. In this study, we performed in vivo Ca2+ imaging to investigate wind-evoked neural activities within the cricket protocerebrum. Ca2+ responses to air current stimuli were observed at peripheral regions of the ventrolateral neuropile (VLNP) where projection of GIs’ axon terminals has been observed in larvae. The wind-evoked Ca2+ response had temporal dynamics and directional sensitivity that varied with different recorded regions displaying transient or sustained Ca2+ increases. Individual cells showed Ca2+ elevation in response to air currents from a specific angle, while stimuli from a different angle evoked decreased signals. Removing the antennae reduced the air-current-evoked responses in VLNP, suggesting contribution of sensory inputs from antennae in addition to the cercal inputs. The VLNP is presumably an integrative center for mechanosensory processing from antennae and cerci where directional information is primarily decoded by protocerebral neurons.

Comparative immunolocalisation of fibrillin-1 and perlecan in the human foetal, and HS-deficient hspg2 exon 3 null mutant mouse intervertebral disc

The aim of this study was to examine the comparative localisations of fibrillin-1 and perlecan in the foetal human, wild-type C57BL/6 and HS-deficient hspg2Δ³⁻/Δ³⁻ exon 3 null mouse intervertebral disc (IVD) using fluorescent laser scanning confocal microscopy. Fibrillin-1 fibrils were prominent components of the outer posterior and anterior annulus fibrosus (AF) of the foetal human IVD. Finer fibrillin-1 fibrils were evident in the inner AF where they displayed an arcade-type arrangement in the developing lamellae. Relatively short but distinct fibrillin-1 fibrils were evident in the central region of the IVD and presumptive cartilaginous endplate and defined the margins of the nuclear sheath in the developing nucleus pulposus (NP). Fibrillin-1 was also demonstrated in the AF of C57BL/6 wild-type mice but to a far lesser extent in the HS-deficient hspg2Δ³⁻/Δ³⁻ exon 3 null mouse. This suggested that the HS chains of perlecan may have contributed to fibrillin-1 assembly or its deposition in the IVD. The cell-matrix interconnections provided by the fibrillin fibrils visualised in this study may facilitate communication between disc cells and their local biomechanical microenvironment in mechanosensory processes which regulate tissue homeostasis. The ability of fibrillin-1 to sequester TGF-β a well-known anabolic growth factor in the IVD also suggests potential roles in disc development and/or remodelling.