Differences In Mechanosensitivity Research Articles

“Force-From-Lipids” Dependence of the MscCG Mechanosensitive Channel Gating on Anionic Membranes

Mechanosensory transduction in Corynebacterium glutamicum plays a major role in glutamate efflux for industrial MSG, whose production depends on the activation of MscCG-type mechanosensitive channels. Dependence of the MscCG channel activation by membrane tension on the membrane lipid content has to date not been functionally characterized. Here, we report the MscCG channel patch clamp recording from liposomes fused with C. glutamicum membrane vesicles as well as from proteoliposomes containing the purified MscCG protein. Our recordings demonstrate that mechanosensitivity of MscCG channels depends significantly on the presence of negatively charged lipids in the proteoliposomes. MscCG channels in liposome preparations fused with native membrane vesicles exhibited the activation threshold similar to the channels recorded from C. glutamicum giant spheroplasts. In comparison, the activation threshold of the MscCG channels reconstituted into azolectin liposomes was higher than the activation threshold of E. coli MscL, which is gated by membrane tension close to the bilayer lytic tension. The spheroplast-like activation threshold was restored when the MscCG channels were reconstituted into liposomes made of E. coli polar lipid extract. In liposomes made of polar lipids mixed with synthetic phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the activation threshold of MscCG was significantly reduced compared to the activation threshold recorded in azolectin liposomes, which suggests the importance of anionic lipids for the channel mechanosensitivity. Moreover, the micropipette aspiration technique combined with patch fluorometry demonstrated that membranes containing anionic phosphatidylglycerol are softer than membranes containing only polar non-anionic phosphatidylcholine and phosphatidylethanolamine. The difference in mechanosensitivity between C. glutamicum MscCG and canonical MscS of E. coli observed in proteoliposomes explains the evolutionary tuning of the force from lipids sensing in various bacterial membrane environments.

3D hydrogel-based microcapsules as an in vitro model to study tumorigenicity, cell migration and drug resistance

In this work, we analyzed the reliability of alginate-gelatin microcapsules as artificial tumor model. These tumor-like scaffolds are characterized by their composition and stiffness (∼25 kPa), and their capability to restrict -but not hinder- cell migration, proliferation and release from confinement.Hydrogel-based microcapsules were initially utilized to detect differences in mechano-sensitivity between MCF7 and MDA-MB-231 breast cancer cells, and the endothelial cell line EA.hy926. Additionally, we used RNA-seq and transcriptomic methods to determine how the culture strategy (i.e. 2D v/s 3D) may pre-set the expression of genes involved in multidrug resistance, being then validated by performing cytotoxicological tests and assays of cell morphology. Our results show that both breast cancer cells can generate elongated multicellular spheroids inside the microcapsules, prior being released (mimicking intravasation stages), a behavior which was not observed in endothelial cells. Further, we demonstrate that cells isolated from 3D scaffolds show resistance to cisplatin, a process which seems to be strongly influenced by mechanical stress, instead of hypoxia. We finally discuss the role played by aneuploidy in malignancy and resistance to anticancer drugs, based on the increased number of polynucleated cells found within these microcapsules.Overall, our outcomes demonstrate that alginate-gelatin microcapsules represent a simple, yet very accurate tumor-like model, enabling us to mimic the most relevant malignant hints described in vivo, suggesting that confinement and mechanical stress need to be considered when studying pathogenicity and drug resistance of cancer cells in vitro. Statement of significanceIn this work, we analyzed the reliability of alginate-gelatin microcapsules as an artificial tumor model. These scaffolds are characterized by their composition, elastic properties, and their ability to restrict cell migration, proliferation, and release from confinement. Our results demonstrate four novel outcomes: (i) studying cell migration and proliferation in 3D enabled discrimination between malignant and non-pathogenic cells, (ii) studying the cell morphology of cancer aggregates entrapped in alginate-gelatin microcapsules enabled determination of malignancy degree in vitro, (iii) determination that confinement and mechanical stress, instead of hypoxia, are required to generate clones resistant to anticancer drugs (i.e. cisplatin), and (iv) evidence that resistance to anticancer drugs could be due to the presence of polynucleated cells localized inside polymer-based artificial tumors.

Fear Extinction-Based Inter-Individual and Sex Differences in Pain-Related Vocalizations and Anxiety-like Behaviors but Not Nocifensive Reflexes.

Inter-individual and sex differences in pain responses are recognized but their mechanisms are not well understood. This study was intended to provide the behavioral framework for analyses of pain mechanisms using fear extinction learning as a predictor of phenotypic and sex differences in sensory (mechanical withdrawal thresholds) and emotional-affective aspects (open field tests for anxiety-like behaviors and audible and ultrasonic components of vocalizations) of acute and chronic pain. In acute arthritis and chronic neuropathic pain models, greater increases in vocalizations were found in females than males and in females with poor fear extinction abilities than females with strong fear extinction, particularly in the neuropathic pain model. Female rats showed higher anxiety-like behavior than males under baseline conditions but no inter-individual or sex differences were seen in the pain models. No inter-individual and sex differences in mechanosensitivity were observed. The data suggest that vocalizations are uniquely suited to detect inter-individual and sex differences in pain models, particularly in chronic neuropathic pain, whereas no such differences were found for mechanosensitivity, and baseline differences in anxiety-like behaviors disappeared in the pain models.

Heterogeneous role of integrins in fibroblast response to small cyclic mechanical stimulus generated by a nanoporous gold actuator

It is important to understand the effects of mechanical stimulation on cell behaviors for homeostasis. Many studies have been performed on cell responses to mechanical stimuli, but the mechanosensing mechanism is still under debate. In the present study, experiments employing molecular dynamics (MD) simulations concerning the effects of cyclic mechanical stimulus on cell proliferation were performed based on the hypothesis that mechanosensing depends on integrin types. We used a nanoporous gold (NPG) actuator to prevent transfer of a mechanical stimulus via molecules other than integrins. Surprisingly, a small cyclic strain of only 0.5% enhanced the proliferation of fibroblasts. α5β1 and αvβ3 integrins showed high sensitivity to the mechanical stimulus, whereas α1β1 and α2β1 integrins exhibited low mechanosensitivity. The MD simulations showed that different conformational changes of the integrin headpiece induced by binding to the ECM led to a difference in mechanosensitivity between αI and αI-less integrin types. Thus, the present study provides evidence to support the hypothesis and suggests the mechanism for the heterogeneous roles of integrins in mechanosensing.

The effect of neurodynamic technique of tibial nerve on range of motion, pain, and mechanosensitivity of the lower extremity in healthy individuals: A preliminary study

The aim of this study was to determine the effects of neurodynamic technique of tibial nerve on range of motion, pain, and mechanosensitivity of the lower extremity in healthy individuals. The study was a non-equivalent, one-group, and pre-post test design. Nineteen healthy adults participated in the study and conducted neurodynamic techniques of the tibial nerve. The outcome measures included range of motion, pain, and mechanosensitivity measured by electromyography during a straight leg raise test. Surface electromyography data were collected from the biceps femoris, medial gastrocnemius, lateral gastrocnemius, and tibialis anterior. There was a significant difference in range of motion and pain between the pre-test and the post-test. There was a non-significant difference in mechanosensitivity between the pre-test and the post-test. Therefore, we concluded that a neurodynamic technique of the tibial nerve reduced pain and increased range of motion in healthy adults. This neurodynamic technique is an effective intervention for improvement of lower limb pain and range of motion.

Neurodynamic tests for patellofemoral pain syndrome: a pilot study

BackgroundPatellofemoral pain syndrome (PFPS) is a common musculoskeletal disorder. There is little consensus on the etiology, but one explanatory model suggests that PFPS can be caused by referred pain. Neurodynamic tests are used to explore the mechanosensitivity of peripheral nerves, and previous studies have shown a relationship between increased mechanosensitivity and anterior knee pain by using the femoral slump test (FST). Previously the prone knee bend test (PKB) does not appear to have been included. The main purpose of this pilot study was to examine whether there was an identifiable difference in mechanosensitivity between left and right sides that could be identified using both the PKB and FST tests for the femoral nerve in patients with unilateral PFPS.MethodsThis cross-sectional pilot study tested 12 patients with unilateral PFPS for altered mechanosensitivity using both PKB and FST. The pain-free knee was used as a control. The selected test procedures were similar to those clinicians use in everyday practice.Results8 and 4 of the 12 patients were found to have increased levels of mechanosensitivity in the PFPS leg using the PKB and FST, respectively. Both tests provoked stronger pain in the leg with PFPS compared with the asymptomatic leg (p < 0.05 Wilcoxon Signed Rank Test). The symptoms were more often located in the anterior knee, with structural differentiation by neck flexion appearing to increase the symptoms more when testing the leg with PFPS.ConclusionsAlthough the reliability of the tests is unknown and the study sample size was small, the PKB and FST test procedures used in clinical practice appear capable of revealing altered mechanosensitivity in unilateral PFPS patients. The PKB test appears to detect mechanosensitivity in more patients than the FST. We recommend including both tests in future larger blinded controlled studies which should also assess reliability of the tests.Trial registrationISRCTN 12473526. Registered 20 May 2015, retrospectively registered.

Degeneration of the osteocyte network in the C57BL/6 mouse model of aging.

Age-related bone loss and associated fracture risk are major problems in musculoskeletal health. Osteocytes have emerged as key regulators of bone mass and as a therapeutic target for preventing bone loss. As aging is associated with changes in the osteocyte lacunocanalicular system, we focused on the responsible cellular mechanisms in osteocytes. Bone phenotypic analysis was performed in young-(5mo) and aged-(22mo) C57BL/6 mice and changes in bone structure/geometry correlated with alterations in osteocyte parameters determined using novel multiplexed-3D-confocal imaging techniques. Age-related bone changes analogous to those in humans were observed, including increased cortical diameter, decreased cortical thickness, reduced trabecular BV/TV and cortical porosities. This was associated with a dramatic reduction in osteocyte dendrite number and cell density, particularly in females, where osteocyte dendricity decreased linearly from 5, 12, 18 to 22mo and correlated significantly with cortical bone parameters. Reduced dendricity preceded decreased osteocyte number, suggesting dendrite loss may trigger loss of viability. Age-related degeneration of osteocyte networks may impair bone anabolic responses to loading and gender differences in osteocyte cell body and lacunar fluid volumes we observed in aged mice may lead to gender-related differences in mechanosensitivity. Therapies to preserve osteocyte dendricity and viability may be beneficial for bone health in aging.

The mechano-sensitivity of cardiac ATP-sensitive potassium channels is mediated by intrinsic MgATPase activity

Cardiac ATP-sensitive K+ (KATP) channel activity plays an important cardio-protective role in regulating excitability in response to metabolic stress. Evidence suggests that these channels are also mechano-sensitive and therefore may couple KATP channel activity to increased cardiac workloads. However, the molecular mechanism that couples membrane stretch to channel activity is not currently known. We hypothesized that membrane stretch may alter the intrinsic MgATPase activity of the cardiac KATP channel resulting in increased channel activation. The inside-out patch-clamp technique was used to record single-channel and macroscopic recombinant KATP channel activity in response to membrane stretch elicited by negative pipette pressure. We found that stretch activation requires the presence of the SUR subunit and that inhibition of MgATPase activity with either the non-hydrolysable ATP analog AMP-PNP or the ATPase inhibitor BeFx significantly reduced the stimulatory effect of stretch. We employed a point mutagenic approach to determine that a single residue (K1337) in the hairpin loop proximal to the major MgATPase catalytic site in the SUR2A subunit is responsible for the difference in mechano-sensitivity between SUR2A and SUR1 containing KATP channels. Moreover, using a double cysteine mutant substitution in the hairpin loop region revealed the importance of a key residue-residue interaction in this region that transduces membrane mechanical forces into KATP channel stimulation via increases in channel MgATPase activity. With respect to KATP channel pharmacology, glibenclamide, but not glicalizide or repaglinide, was able to completely inhibit KATP channel mechano-sensitivity. In summary, our results provide a highly plausible molecular mechanism by which mechanical membrane forces are rapidly converted in changes in KATP channel activity that have implications for our understanding of cardiac KATP channels in physiological or pathophysiological settings that involve increased workload.

Microstome–macrostome transformation in the polymorphic ciliateTetrahymena voraxleads to mechanosensitivity associated with prey-capture behaviour

Ciliates feed by phagocytosis. Some ciliate species, such as Tetrahymena vorax, are polymorphic, a strategy that provides more flexible food utilization. Cells of the microstomal morph of T. vorax feed on bacteria, organic particles and organic solutes in a non-selective manner, whereas macrostome cells are predators that consume specific prey ciliates. In the present study, we investigated a possible correlation between phagocytosis and mechanosensitivity in macrostome T. vorax. Microstome cells seem to be insensitive to mechanical stimulation whereas macrostome cells depolarise in response to mechanical stimulation of the anterior part of the cell. The amplitude of the receptor potential induced by either a prey ciliate or a 5 μm push by a glass needle was sufficient to elicit a regenerative Ca²⁺ spike. The difference in mechanosensitivity of the two forms correlates with the swimming behaviour when hitting an obstacle; microstome cells swim alongside the obstacle whereas macrostome cells swim backwards, turn and resume forward swimming. Macrostome cells prevented from backward swimming and the subsequent turn failed to capture prey cells in their pouch. Macrostome cells consumed heterospecific prey ciliates preferentially over conspecific microstome cells. This selectivity is not due to electrical membrane responses elicited by physical contact. Both microstome and macrostome cells accumulated in an area containing putative substances released from heterospecific prey ciliates, but the substances did not elicit any electrophysiological membrane responses. We conclude that the mechanosensitivity of macrostome cells is associated with the prey-capture behaviour, whereas the selective phagocytosis is probably due to chemo-attraction to heterospecific prey ciliates.

Mechanosensing of stem bending and its interspecific variability in five neotropical rainforest species.

In rain forests, sapling survival is highly dependent on the regulation of trunk slenderness (height/diameter ratio): shade-intolerant species have to grow in height as fast as possible to reach the canopy but also have to withstand mechanical loadings (wind and their own weight) to avoid buckling. Recent studies suggest that mechanosensing is essential to control tree dimensions and stability-related morphogenesis. Differences in species slenderness have been observed among rainforest trees; the present study thus investigates whether species with different slenderness and growth habits exhibit differences in mechanosensitivity. Recent studies have led to a model of mechanosensing (sum-of-strains model) that predicts a quantitative relationship between the applied sum of longitudinal strains and the plant's responses in the case of a single bending. Saplings of five different neotropical species (Eperua falcata, E. grandiflora, Tachigali melinonii, Symphonia globulifera and Bauhinia guianensis) were subjected to a regimen of controlled mechanical loading phases (bending) alternating with still phases over a period of 2 months. Mechanical loading was controlled in terms of strains and the five species were subjected to the same range of sum of strains. The application of the sum-of-strain model led to a dose-response curve for each species. Dose-response curves were then compared between tested species. The model of mechanosensing (sum-of-strain model) applied in the case of multiple bending as long as the bending frequency was low. A comparison of dose-response curves for each species demonstrated differences in the stimulus threshold, suggesting two groups of responses among the species. Interestingly, the liana species B. guianensis exhibited a higher threshold than other Leguminosae species tested. This study provides a conceptual framework to study variability in plant mechanosensing and demonstrated interspecific variability in mechanosensing.

Calcium response in single osteocytes to locally applied mechanical stimulus: Differences in cell process and cell body

It is proposed that osteocytes embedded in the bone matrix have the ability to sense deformation and/or damage to the matrix and to feed these mechanical signals back to the adaptive bone remodeling process. When osteoblasts differentiate into osteocytes during the bone formation process, they change their morphology to a stellate form with many slender processes. This characteristic cell shape may underlie the differences in mechanosensitivity between the cell processes and cell body. To elucidate the mechanism of cellular response to mechanical stimulus in osteocytes, we investigated the site-dependent response to quantitatively controlled local mechanical stimulus in single osteocytes isolated from chick embryos, using the technique of calcium imaging. A mechanical stimulus was applied to a single osteocyte using a glass microneedle targeting a microparticle adhered to the cell membrane by modification with a monoclonal antibody OB7.3. Application of the local deformation induced calcium transients in the vicinity of the stimulated point and caused diffusive wave propagation of the calcium transient to the entire intracellular region. The rate of cell response to the stimulus was higher when applied to the cell processes than when applied to the cell body. In addition, a large deformation was necessary at the cell body to induce calcium transients, whereas a relatively small deformation was sufficient at the cell processes, suggesting that the mechanosensitivity of the cell processes was higher than that of the cell body. These results suggest that the cell shape with slender processes contributes to the site-dependent mechanosensitivity in osteocytes.

Chemosensitivity and Mechanosensitivity of Nociceptors from Incised Rat Hindpaw Skin

The authors have demonstrated a decrease in pH in the incisional wound environment, suggesting a possible contribution of low pH to postsurgical pain. In this study, the authors characterized the acid-responsiveness of nociceptors innervating the plantar aspect of the rat hind paw 1 day after plantar incision and compared this to plantar skin from unincised control rats. Using the rat glabrous in vitro skin-tibial nerve preparation, afferent nerve activities from single mechanosensitive nociceptors were recorded. Differences in mechanosensitivity, spontaneous activity, and chemosensitivity of units were evaluated. For chemosensitivity, acid-responsiveness of nociceptors to lactic acid (pH 5.5 to 6.5) was studied. C-fibers showed dose-dependent, sustained responses to lactic acid. A greater proportion of C-fibers from 2 mm or less from the incision was activated by pH 6.0 lactic acid (52.9%) compared to control (14.3%). Total evoked potentials during acid exposure were greater in C-fibers innervating 2 mm or less from the incision compared to those in unincised skin. The prevalence of acid responses and total evoked potentials during acid exposure in C-fibers innervating more than 2 mm from the incision were not different from control. Few A-fibers responded to lactic acid, with a range of pH 5.5 to 6.5 in both incision and control groups. Increased spontaneous activity and mechanosensitivity were also evident. C-fibers in the vicinity of the incision showed qualitatively and quantitatively greater chemosensitivity to pH 6.0 lactic acid compared to control. This change was localized to 2 mm or less from the incision, suggesting increased chemosensitivity of nociceptive C-fibers 1 day after plantar incision.

The morphology of the pit organs and lateral line canal neuromasts of Mustelus antarcticus (Chondrichthyes: Triakidae)

The pit organs (free neuromasts) of sharks are part of the lateral line sensory system, but there is still confusion about their exact morphology and function(s). This is partly because of reported physiological differences between the pit organs and the lateral line canal neuromasts, and partly because the morphology of pit organs has not been adequately documented. To compare their morphology, the pit organs and canal neuromasts of the gummy shark Mustelus antarcticus (Chondrichthyes: Triakidae) were examined using transmission and scanning electron microscopy. Both pit organs and canal neuromasts had hair cells with the `staircase' arrangement of sensory hairs (stereovilli) characteristic of vertebrate mechanoreceptors. Stereovilli bundles of different sizes were distributed haphazardly throughout the pit organs and canal neuromasts. The density of hair cells was similar in the pit organs and canal neuromasts, but differences in the overall size and/or shape of the sensory epithelia might account for some of the reported differences in mechanosensitivity.

Distribution of mechanoreceptor channels in theParamecium surface membrane

Different sites of theParamecium surface were mechanically stimulated with a fine-tipped glass stylus. Ciliated and deciliated specimens showed a decrease in depolarizing mechanosensitivity and a subsequent increase in hyperpolarizing mechanosensitivity, when the site of stimulation was shifted from the anterior to the posterior end of the cell. Maximal depolarizing sensitivity was observed slightly posterior to the front end of the cell. Specimens without cilia failed to produce action potentials, but no differences in mechanosensitivity were detected. Stimuli given to dorsal surfaces produced larger hyperpolarizations, or smaller depolarizations, than stimuli applied to ventral surfaces at the same latitude. In voltage-clamped cells posterior stimulation elicited outward receptor currents with 5 ms half-time of decay. Inward receptor currents following anterior stimulation decayed with half-times between 20 and 70 ms. Reversal potentials of the receptor currents became more positive, when more anterior surfaces were stimulated. Measurements of the reversal potentials in solutions with varied K and Ca concentrations showed that the posterior receptor currents are exclusively carried by K, while the anterior receptor currents are the sum of K and Ca currents. Ca and K mechanoreceptor currents cancel out in the midposterior region of the cell. Quantitative evaluations of the data suggest that the receptor potentials inParamecium occur due to activated Ca and K mechanoreceptor channels distributing over the somatic cell surface in the manner of overlapping gradients.