Transduction Of Mechanical Forces Research Articles

Mechanosensitive channel MscL gating transitions coupling with constriction point shift.

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.

Mechanosensitive Channels in Lung Health and Disease.

The lung is an inherently mechanosensitive organ, where cells of the airway and parenchyma experience a range of mechanical forces throughout life including shear, stretch, and compression, in both health and disease. In this regard, pediatric and adult lung diseases such as wheezing and asthma, bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis (PF) all involve macroscopic and cellular changes to the mechanical properties of the bronchial airways and/or parenchyma to varying extents. Accordingly, understanding how mechanical forces are sensed in the lung, and the responses of cells and tissues in the context of normal development and health versus disease conditions becomes highly relevant. There is increasing recognition that transduction of mechanical forces into cellular responses involves a number of channels, some of which are inherently mechanosensitive. Such channels trigger mechanotransduction pathways that may further mediate cellular remodeling, inflammation, and other pathophysiologic mechanisms in response to stretch, stiffness, and inflammatory cascades. Two particularly important channel families have emerged in pulmonary pathophysiology: the transient receptor potential vanilloid family with focus on member TRPV4 and the recently identified Piezo (PZ) channels. Here, we explore current understanding of the contributions of TRPV4 and PZ channels in lung health and disease states, focusing on the interactions between these mechanosensitive channels and their local environment including immune cells, the extracellular matrix, and cellular cytoskeletal elements. We further discuss potential areas for future research to better understand the impact of mechanical channels on pulmonary health and disease. © 2023 American Physiological Society. Compr Physiol 13:5157-5178, 2023.

Physical forces guide curvature sensing and cell migration mode bifurcating.

The ability of cells to sense and adapt to curvy topographical features has been implicated in organ morphogenesis, tissue repair, and tumor metastasis. However, how individual cells or multicellular assemblies sense and differentiate curvatures remains elusive. Here, we reveal a curvature sensing mechanism in which surface tension can selectively activate either actin or integrin flows, leading to bifurcating cell migration modes: focal adhesion formation that enables cell crawling at convex front edges and actin cable assembly that pulls cells forward at concave front edges. The molecular flows and curved front morphogenesis are sustained by coordinated cellular tension generation and transmission. We track the molecular flows and mechanical force transduction pathways by a phase-field model, which predicts that multicellular curvature sensing is more efficient than individual cells, suggesting collective intelligence of cells. The unique ability of cells in curvature sensing and migration mode bifurcating may offer insights into emergent collective patterns and functions of living active systems at different length scales.

De Novo Cloning and Functional Characterization of a Mechanosensitive Piezo-Like Ion Channel in the Crayfish.

Mechanosensitive ion channels are the principal elements in the transduction of mechanical force to neural activity. To date, considerably fewer studies have been published about the molecular and structural properties of mechanosensitive channels. Piezo channels are the only ion channel family in eukaryotes which is selectively gated by the membrane tension. Piezo channels have been described in mammals and some other eukaryotes. However, not much information is available for the crustaceans. Conventional cloning methods were used to clone the putative PIEZO channel mRNA in crayfish ganglia samples. HEK293T cells were transfected by the plasmid of the cloned gene for functional studies. The CDS of the mRNA translated into the protein sequence and three-dimensional structure of the channel has been calculated. An mRNA, 9378 bp, was firstly cloned from crayfish which codes a 2674 residues protein. The cloned sequence is similar to the piezo channel mRNAs reported in the other species. The sequence of the coded protein has been analyzed, and some functional domains have been identified. A three-dimensional structure of the coded protein was successfully calculated in reference to mouse piezo 1 channel protein data. A plasmid with a fluorescent protein indicator was synthesized for heterologous expression in HEK293T cells. The evoked calcium response to mechanical stimulation was not different from those observed in the control cells. However, the transfected cells were more sensitive to the gating modifier YODA-1. Based on the apparent similarity in sequence, structure and functional properties to other known piezo channels, it has been proposed that cloned mRNA may code a piezo-like ion channel in crayfish.

A cooperative network at the nuclear envelope counteracts LINC-mediated forces during oogenesis in C. elegans

Oogenesis involves transduction of mechanical forces from the cytoskeleton to the nuclear envelope (NE). In Caenorhabditis elegans , oocyte nuclei lacking the single lamin protein LMN-1 are vulnerable to collapse under forces mediated through LINC (linker of nucleoskeleton and cytoskeleton) complexes. Here, we use cytological analysis and in vivo imaging to investigate the balance of forces that drive this collapse and protect oocyte nuclei. We also use a mechano-node-pore sensing device to directly measure the effect of genetic mutations on oocyte nuclear stiffness. We find that nuclear collapse is not a consequence of apoptosis. It is promoted by dynein, which induces polarization of a LINC complex composed of Sad1 and UNC-84 homology 1 (SUN-1) and ZYGote defective 12 (ZYG-12). Lamins contribute to oocyte nuclear stiffness and cooperate with other inner nuclear membrane proteins to distribute LINC complexes and protect nuclei from collapse. We speculate that a similar network may protect oocyte integrity during extended oocyte arrest in mammals.

The glycocalyx and calcium dynamics in endothelial cells.

The endothelial glycocalyx is a dynamic surface layer composed of proteoglycans, glycoproteins, and glycosaminoglycans with a key role in maintaining endothelial cell homeostasis. Its functions include the regulation of endothelial barrier permeability and stability, the transduction of mechanical forces from the vascular lumen to the vessel walls, serving as a binding site to multiple growth factors and vasoactive agents, and mediating the binding of platelets and the migration of leukocytes during an inflammatory response. Many of these processes are associated with changes in intracellular calcium levels that may occur through mechanisms that alter calcium entry in the endothelium or the release of calcium from the endoplasmic reticulum. Whether the endothelial glycocalyx can regulate calcium dynamics in endothelial cells is unresolved. Interestingly, during cardiovascular disease progression, changes in calcium dynamics are observed in association with the degradation of the glycocalyx and with changes in barrier permeability and vascular reactivity. Herein, we aim to provide a summarized overview of what is known regarding the role of the glycocalyx as a regulator of endothelial barrier and vascular reactivity during homeostatic and pathological conditions and to provide a perspective on how such processes may relate to calcium dynamics in endothelial cells, exploring a possible connection between components of the glycocalyx and calcium-sensitive pathways in the endothelium.

Exosome-mediated transduction of mechanical force regulates prostate cancer migration via microRNA

Physical cues in the extracellular microenvironment regulate cancer cell metastasis. Functional microRNA (miRNA) carried by cancer derived exosomes play a critical role in extracellular communication between cells and the extracellular microenvironment. However, little is known about the role of exosomes loaded miRNAs in the mechanical force transmission between cancer cells and extracellular microenvironment. Herein, our results suggest that stiff extracellular matrix (ECM) induced exosomes promote cancer cell migration. The ECM mechanical force regulated the exosome miRNA cargo of prostate cancer cells. Exosome miRNAs regulated by the ECM mechanical force modulated cancer cell metastasis by regulating cell motility, ECM remodeling and the interaction between cancer cells and nerves. Focal adhesion kinase mediated-ECM mechanical force regulated the intracellular miRNA expression, and F-actin mediate-ECM mechanical force regulated miRNA packaging into exosomes. The above results demonstrated that the exosome miRNA cargo promoted cancer metastasis by transmitting the ECM mechanical force. The ECM mechanical force may play multiple roles in maintaining the microenvironment of cancer metastasis through the exosome miRNA cargo.

Phosphoinositide Signaling and Mechanotransduction in Cardiovascular Biology and Disease.

Phosphoinositides, which are membrane-bound phospholipids, are critical signaling molecules located at the interface between the extracellular matrix, cell membrane, and cytoskeleton. Phosphoinositides are essential regulators of many biological and cellular processes, including but not limited to cell migration, proliferation, survival, and differentiation, as well as cytoskeletal rearrangements and actin dynamics. Over the years, a multitude of studies have uniquely implicated phosphoinositide signaling as being crucial in cardiovascular biology and a dominant force in the development of cardiovascular disease and its progression. Independently, the cellular transduction of mechanical forces or mechanotransduction in cardiovascular cells is widely accepted to be critical to their homeostasis and can drive aberrant cellular phenotypes and resultant cardiovascular disease. Given the versatility and diversity of phosphoinositide signaling in the cardiovascular system and the dominant regulation of cardiovascular cell functions by mechanotransduction, the molecular mechanistic overlap and extent to which these two major signaling modalities converge in cardiovascular cells remain unclear. In this review, we discuss and synthesize recent findings that rightfully connect phosphoinositide signaling to cellular mechanotransduction in the context of cardiovascular biology and disease, and we specifically focus on phosphatidylinositol-4,5-phosphate, phosphatidylinositol-4-phosphate 5-kinase, phosphatidylinositol-3,4,5-phosphate, and phosphatidylinositol 3-kinase. Throughout the review, we discuss how specific phosphoinositide subspecies have been shown to mediate biomechanically sensitive cytoskeletal remodeling in cardiovascular cells. Additionally, we discuss the direct interaction of phosphoinositides with mechanically sensitive membrane-bound ion channels in response to mechanical stimuli. Furthermore, we explore the role of phosphoinositide subspecies in association with critical downstream effectors of mechanical signaling in cardiovascular biology and disease.

Piezo2 Mediates Low-Threshold Mechanically Evoked Pain in the Cornea.

Mammalian Piezo2 channels are essential for transduction of innocuous mechanical forces by proprioceptors and cutaneous touch receptors. In contrast, mechanical responses of somatosensory nociceptor neurons evoking pain, remain intact or are only partially reduced in Piezo2-deficient mice. In the eye cornea, comparatively low mechanical forces are detected by polymodal and pure mechanosensory trigeminal ganglion neurons. Their activation always evokes ocular discomfort or pain and protective reflexes, thus being a unique model to study mechanotransduction mechanisms in this particular class of nociceptive neurons. Cultured male and female mouse mechano- and polymodal nociceptor corneal neurons display rapidly, intermediately and slowly adapting mechanically activated currents. Immunostaining of the somas and peripheral axons of corneal neurons responding only to mechanical force (pure mechano-nociceptor) or also exhibiting TRPV1 (transient receptor potential cation channel subfamily V member 1) immunoreactivity (polymodal nociceptor) revealed that they express Piezo2. In sensory-specific Piezo2-deficient mice, the distribution of corneal neurons displaying the three types of mechanically evoked currents is similar to the wild type; however, the proportions of rapidly adapting neurons, and of intermediately and slowly adapting neurons were significantly reduced. Recordings of mechano- and polymodal-nociceptor nerve terminals in the corneal surface of Piezo2 conditional knock-out mice revealed a reduced number of mechano-sensitive terminals and lower frequency of nerve terminal impulse discharges under mechanical stimulation. Eye blinks evoked by von Frey filaments applied on the cornea were lower in Piezo2-deficient mice compared with wild type. Together, our results provide direct evidence that Piezo2 channels support mechanically activated currents of different kinetics in corneal trigeminal neurons and contributes to transduction of mechanical forces by corneal nociceptors.SIGNIFICANCE STATEMENT The cornea is a richly innervated and highly sensitive tissue. Low-threshold mechanical forces activate corneal receptors evoking discomfort or pain. To examine the contribution of Piezo2, a low-threshold mechanically activated channel, to acute ocular pain, we characterized the mechanosensitivity of corneal sensory neurons. By using Piezo2 conditional knock-out mice, we show that Piezo2 channels, present in the cell body and terminals of corneal neurons, are directly involved in acute corneal mechano-nociception. Inhibition of Piezo2 for systemic pain treatment is hindered because of its essential role for mechano-transduction processes in multiple body organs. Still, topical modulation of Piezo2 in the cornea may be useful to selectively relief unpleasant sensations and pain associated with mechanical irritation accompanying many ocular surface disorders.

Optical Rotation and Thermometry of Laser Tweezed Silicon Nanorods.

Optical rotation of laser tweezed nanoparticles offers a convenient means for optical to mechanical force transduction and sensing at the nanoscale. Plasmonic nanoparticles are the benchmark system for such studies, but their rapid rotation comes at the price of high photoinduced heating due to Ohmic losses. We show that Mie resonant silicon nanorods with characteristic dimensions of ∼220 × 120 nm2 can be optically trapped and rotated at frequencies up to 2 kHz in water using circularly polarized laser light. The temperature excess due to heating from the trapping laser was estimated by phonon Raman scattering and particle rotation analysis. We find that the silicon nanorods exhibit slightly improved thermal characteristics compared to Au nanorods with similar rotation performance and optical resonance anisotropy. Altogether, the results indicate that silicon nanoparticles have the potential to become the system of choice for a wide range of optomechanical applications at the nanoscale.

Role and mechanism of micro-energy treatment in regenerative medicine.

With the continuous integration and intersection of life sciences, engineering and physics, the application for micro-energy in the basic and clinical research of regenerative medicine (RM) has made great progress. As a key target in the field of RM, stem cells have been widely used in the studies of regeneration. Recent studies have shown that micro-energy can regulate the biological behavior of stem cells to repair and regenerate injured organs and tissues by mechanical stimulation with appropriate intensity. Integrins-mediated related signaling pathways may play important roles in transducing mechanical force about micro-energy. However, the complete mechanism of mechanical force transduction needs further research. The purpose of this article is to review the biological effect and mechanism of micro-energy treatment on stem cells, to provide reference for further research.

Monitoring Biochemical and Structural Changes in Human Periodontal Ligaments during Orthodontic Treatment by Means of Micro-Raman Spectroscopy.

The aim of the study was to examine the biochemical and structural changes occurring in the periodontal ligament (PDL) during orthodontic-force application using micro-Raman spectroscopy (-RS). Adolescent and young patients who needed orthodontic treatment with first premolar extractions were recruited. Before extractions, orthodontic forces were applied using a closed-coil spring that was positioned between the molar and premolar. Patients were randomly divided into three groups, whose extractions were performed after 2, 7, and 14 days of force application. From the extracted premolars, PDL samples were obtained, and a fixation procedure with paraformaldehyde was adopted. Raman spectra were acquired for each PDL sample in the range of 1000–3200 cm and the more relevant vibrational modes of proteins (Amide I and Amide III bands) and CH and CH modes were shown. Analysis indicated that the protein structure in the PDL samples after different time points of orthodontic-force application was modified. In addition, changes were observed in the CH and CH high wavenumber region due to local hypoxia and mechanical force transduction. The reported results indicated that -RS provides a valuable tool for investigating molecular interchain interactions and conformational modifications in periodontal fibers after orthodontic tooth movement, providing quantitative insight of time occurring for PDL molecular readjustment.

Force transduction by cadherin adhesions in morphogenesis.

Mechanical forces drive the remodeling of tissues during morphogenesis. This relies on the transmission of forces between cells by cadherin-based adherens junctions, which couple the force-generating actomyosin cytoskeletons of neighboring cells. Moreover, components of cadherin adhesions adopt force-dependent conformations that induce changes in the composition of adherens junctions, enabling transduction of mechanical forces into an intracellular response. Cadherin mechanotransduction can mediate reinforcement of cell–cell adhesions to withstand forces but also induce biochemical signaling to regulate cell behavior or direct remodeling of cell–cell adhesions to enable cell rearrangements. By transmission and transduction of mechanical forces, cadherin adhesions coordinate cellular behaviors underlying morphogenetic processes of collective cell migration, cell division, and cell intercalation. Here, we review recent advances in our understanding of this central role of cadherin adhesions in force-dependent regulation of morphogenesis.

Single Plasmonic Nanosprings for Visualizing Reactive-Oxygen-Species-Activated Localized Mechanical Force Transduction in Live Cells.

Mechanical force signaling in cells has been regarded as the biological foundation of various important physiological functions. To understand the nature of these biological and physiological processes, imaging and determining the mechanical signal transduction dynamics in live cells are required. Herein, we proposed a strategy to determine mechanical force as well as its changes with single-particle dark-field spectral microscopy by using a single plasmonic nanospring as a mechanical sensor, which can transfer force-induced molecular extension/compression into spectral responses. With this robust plasmonic nanospring, we achieved the visualization of activation of localized mechanical force transduction in single live cells triggered by reactive-oxygen-species (ROS) stimulation. The successful demonstration of a biochemical ROS signal to mechanical signal conversion suggested this strategy is promising for studying mechanical force signaling and regulation in live biological systems.

BioSpotlight / Citations

BioTechniquesVol. 61, No. 2 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114439AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit A reality check for dsRNAsA number of popular methods and commercial kits for single-step isolation of high-quality RNA use a concentrated solution of guanidinium thiocyanate (GuSCN)–a powerful chaotropic agent—to denature cellular proteins and inactivate RNases. Besides acting as a robust protein denaturant, a less well-known effect of concentrated GuSCN solutions is to promote DNA—DNA, RNA—DNA, and RNA—RNA hybridization. In this issue of BioTechniques, Triin Mölder and Mart Speek at the Tallinn University of Technology provide evidence suggesting that use of concentrated GuSCN during isolation of cellular RNA causes the formation of double-stranded RNAs (dsRNAs) from complementary single-stranded RNAs (ssRNAs). The authors demonstrated that incubation in a concentrated GuSCN solution of low concentrations of two in vitro—transcribed RNAs with a region of complementarity catalyzed their hybridization into dsRNA, in the absence or presence of total cellular RNA. This suggests the need to carefully verify that any putative novel dsRNAs identified from RNA samples isolated by GuSCN extraction are not actually artifacts caused by this effect.See “Accelerated RNA—RNA hybridization by concentrated guanidinium thiocyanate solution in single-step RNA isolation”Indenting multiple cellsStudies of the response of cells to mechanical force transduction have relied on instruments such as atomic force microscopes (AFMs) or cytoindentors, but these tools can only indent single cells one-by-one, which makes the collection of data from a sufficient number of cells a tedious and time-consuming affair. To overcome this problem, researchers at McGill University led by Claire Brown and Sabrina Leslie describe in this month's issue their development of a high-throughput, parallelized cytoindentor for the simultaneous local compression of multiple live cells. They modified their previously described convex lens—induced confinement (CLiC) set-up by adhering a film patterned with microposts to a microscope lens. This lens is then gradually lowered onto multiple cells maintained in a stage-top incubator to indent micrometer-sized areas on each cell while allowing imaging of those cells. By functionalizing the micropost arrays with an extracellular matrix protein such as fibronectin, it was also possible to pull on the cells. This novel parallelized cytoindentor will greatly facilitate studies of mechanobiology in living cells.See “Parallelized cytoindentation using convex micropatterned surfaces”Protein interactions: limiting diffusion on arraysProtein arrays are important tools for those exploring protein—protein interactions on a large scale. These arrays can be spotted with purified proteins, or cell-free protein synthesis can be performed on the arrays. Protein synthesis approaches such as the nucleic acid programmable protein array (NAPPA) streamline the building of large arrays, but they also produce large, diffuse protein spots that limit array density. Yazaki et al. solve this problem with their newly devised HaloTag- NAPPA technology, which produces higher-density protein spotting. To accomplish this, they exchanged the C-terminal GST fusion used in the original NAPPA for a HaloTag fusion that can be irreversibly bound to ligands co-spotted on the array. HaloTag-NAPPA captured synthesized proteins with significantly less diffusion compared to GST fusion proteins. To test their method, the authors spotted 12,000 Arabidopsis clones onto arrays, synthesized proteins, and then examined protein— protein interactions with 38 plant transcription factors. The new approach identified novel interactions with high sensitivity and low background.Yazaki, J. et al. 2016. Mapping transcription factor interactome networks using HaloTag protein arrays. Proc. Natl. Acad. Sci. USA. DOI: 10.1073/pnas.1603229113Gene expression profiling: it's all about locationProfiling gene expression is a powerful approach for understanding basic biology and human disease. Now, Stahl et al. introduce a new method that could significantly expand the scope of gene expression profiling by adding spatial resolution within tissue samples. In “spatial transcriptomics,” histological samples are placed onto slides with reverse transcription primers possessing positional barcodes immobilized on the slide surface. Following permeabilization, reverse transcription reagents are placed onto the tissues, where cDNA is synthesized and left coupled to the oligonucleotides on the slide. Once the tissue is enzymatically removed, the cDNAs are collected and sequenced, akin to traditional gene expression profiling. After sequencing, the locations of cDNA transcripts can be mapped back to the tissue using the incorporated positional barcodes. By adding only a few additional steps, scientists will now be able to obtain spatial information by transcriptional profiling, thus greatly enhancing gene expression data sets.Stahl, P.L., et al. 2016. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science. 353(6294):78-82.FiguresReferencesRelatedDetails Vol. 61, No. 2 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print August 2016 Information© 2016 Author(s)PDF download

A role for actin polymerization in persistent pulmonary hypertension of the newborn.

Persistent pulmonary hypertension of the newborn (PPHN) is defined as the failure of normal pulmonary vascular relaxation at birth. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial myocytes, resulting in elevation of smooth muscle α-actin and γ-actin content in elastic and resistance pulmonary arteries in PPHN compared with age-matched controls. This review examines the original histological characterization of PPHN with attention to cytoskeletal structural remodeling and actin isoform abundance, reviews the existing evidence for understanding the biophysical and biochemical forces at play during neonatal circulatory transition, and specifically addresses the role of the cortical actin architecture, primarily identified as γ-actin, in the transduction of mechanical force in the hypoxic PPHN pulmonary circuit.

Piezo2 is the major transducer of mechanical forces for touch sensation in mice.

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

Using injectoporation to deliver genes to mechanosensory hair cells.

Mechanosensation, the transduction of mechanical force into electrochemical signals, allows organisms to detect touch and sound, to register movement and gravity, and to sense changes in cell volume and shape. The hair cells of the mammalian inner ear are the mechanosensors for the detection of sound and head movement. The analysis of gene function in hair cells has been hampered by the lack of an efficient gene transfer method. Here we describe a method termed injectoporation that combines tissue microinjection with electroporation to express cDNAs and shRNAs in mouse cochlear hair cells. Injectoporation allows for gene transfer into dozens of hair cells, and it is compatible with the analysis of hair cell function using imaging approaches and electrophysiology. Tissue dissection and injectoporation can be carried out within a few hours, and the tissue can be cultured for days for subsequent functional analyses.

Mechanochemistry with metallosupramolecular polymers.

The transduction of mechanical force into useful chemical reactions is an emerging design approach to impart soft materials with new functions. Here, we report that mechanochemical transductions can be achieved in metallosupramolecular polymers. We show that both reversible and irreversible reactions are possible and useful to create mechanically responsive materials that display new functions. The metallopolymer studied was a cross-linked network assembled from a europium salt and a telechelic poly(ethylene-co-butylene) with 2,6-bis(1'-methylbenzimidazolyl)pyridine (Mebip) ligands at the termini. The Eu(3+) complexes serve both as mechanically responsive binding motifs and as built-in optical probes that can monitor the extent of (dis)assembly due to their characteristic photoluminescent properties. Indeed, dose-dependent and reversible metal-ligand dissociation occurs upon exposure to ultrasound in solution. The absence of ultrasound-induced dissociation of a low-molecular weight model complex and in-depth studies of temperature effects confirm that the dissociation is indeed the result of mechanical activation. The influence of the strength of the metal-ligand interactions on the mechanically induced dissociation was also explored. Metallopolymers in which the Mebip ligands were substituted with more strongly coordinating dipicolinate (dpa) ligands do not dissociate upon exposure to ultrasound. Finally, we show that mechanochemical transduction in metallosupramolecular polymers is also possible in the solid state. We demonstrate mending of damaged objects through ultrasound as well as mechanochromic behavior based on metal-exchange reactions in metallopolymers imbibed with an auxiliary metal salt.

Hemodynamic shear stress regulates the apelin/APJ system in human endothelial cells

Introduction: The apelin/APJ system is being considered to play a crucial role in cardiovascular function. Namely, the isoforms apelin-12 and apelin-13 seem to be most active in the cardio vasculature. Application of fluid shear stress to endothelial cells elicits the formation of NO via PI3K/AKT signaling and phosphorylation of the endothelial NO synthase (eNOS). We suppose that the apelin/APJ system is involved in the transduction of mechanical forces into intracellular signaling. Methods: We applied hemodynamic shear stress (1.5 dyne/cm2 for venous, 20 dyne/cm2 for arterial conditions) on HUVEC, HCAEC, and HCASMC using a parallel plate flow chamber. Cells were stimulated with apelin-12 and apelin-13. Static controls were kept under similar conditions. After shear stress exposure cells were harvested and prepared for real-time qPCR and Western Blot analysis or fixed and stained for anti-APJ (IF). The supernatant was collected for nitrite measurements. Additionally, HUVEC were transfected with APJ siRNA. Results: After application of shear stress, apelin gene and protein expression were regulated in ECs only. In HUVEC, expression of apelin was increased by 40% under low shear stress, whereas under high shear stress conditions apelin expression decreased by 50% (p<0.01). Interestingly, HCAEC showed an opposite behaviour (p<0.001). Furthermore, in ECs APJ gene and protein expression increased after exposure to shear stress (p<0.05), which could be confirmed by IF. Incubation of HUVEC with apelin-12 and -13 revealed a dose-dependent increase of NO production only after incubation with apelin-12 (P<0.01). Western Blot Analysis underlined apelin isoform dependent cellular signaling. Incubation with apelin-12 for up to 15 min lead to a significant increase in PI3 kinase and Akt phosphorylation under static and dynamic conditions (p=0.02), whereas incubation with apelin-13 had no significant influence on PI3k/Akt signalling and lead to phosphorylation of Erk 1/2 instead. In order to demonstrate shear stress dependency of the apelin/APJ system, we transfected HUVEC with APJ siRNA and exposed them to our standard flow conditions. Transfection with APJ siRNA lead to a loss of shear stress dependent eNOS up-regulation and adhesion. Conclusion: Our results show for the first time a shear stress regulated adjustment of the Apelin/APJ system in human endothelial cells depending on arterial or venous flow conditions. This is confirmed by an enhanced eNOS expression and NO production. Interestingly, apelin-12 and apelin-13 activate different signaling pathways in HUVEC and give rise for various biological responses.