Mechanosensitivity and Mechanotransduction: 4 (Mechanosensitivity in Cells and Tissues)
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Previous studies relied on several approaches to solve this problem. For example, Coste et al. Several other groups have tried developing alternative assays. Some have experimented with cells grown on flexible substrates that can be longitudinally or radially stretched to deliver mechanical stimuli Banes et al. Another approach involves adhering coated paramagnetic beads to specific targets and then applying localized pulling forces on molecules associated with the plasma membrane using electromagnets.
Wu et al. Although both the flexible substrate and attached magnetic bead techniques hold promise, further engineering is required to adapt them for screening. Xu et al. They engineered a platform that applied shear stress to cells grown in microwell plates via an array of vibrating pins dipped into the wells. Each vibrating pin was coupled to a solenoid, and a subwoofer driver controlled their stimulation frequency, amplitude, and duty cycle.
The vibrations generated by each pin mechanically perturbed the media covering the cells in the assay plate, creating a disturbed flow. Cellular responses to this shear stress was recorded optically through the bottom of the plate by using a calcium-sensitive dye to monitor intracellular calcium concentration in real time before, during, and after controlled delivery of mechanical stimuli. Because cancer cells grow well in culture and are dysregulated such that many genes are being ectopically expressed, Xu et al. They excluded lines expressing the known mechanoreceptors Piezo1 or Piezo2 to generate a pool of 25 cell lines.
Of these, two produced calcium responses to shear stress, with transients from the breast cancer cell line MDA-MB being particularly robust.
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Their assumption was that the unknown mechanoreceptor would be localized to the plasma membrane and contain at least two transmembrane domains. Bioinformatic analyses yielded a list of 2, potential target genes that met their criteria. A quick back-of-the-envelope calculation to estimate controls and combinations of multiple siRNAs per gene allows one to appreciate the transformative nature of the ability to screen in microwell plates. All aspects of the experiment, including liquid handling, siRNA screening, and plate reading could be automated.
Mechanosensitive mechanisms in transcriptional regulation | Journal of Cell Science
Of all the genes tested, knockdown of the G protein—coupled orphan receptor GPR68 significantly and repeatedly diminished the calcium influx after shear stress was applied. Critically, transfection of normally mechano-insensitive HEK cells with this GPCR resulted in calcium responses in the disturbed shear stress assay, indicating that expression of this GPCR was sufficient to endow mechanosensitivity.
Perhaps most impressive was the amplitude of the mechanical responses seen in responses to laminar flow a different type of mechanical stimulation after both human and mouse GPR68 expression. In contrast, neither expression of the mechanosensitive Piezo1 nor the osmosensitive TRPV4 resulted in calcium responses to laminar flow above baseline. Even more surprising, overexpression of other GPCRs in HEK cells, including those previously proposed to be mechanosensors, did not result in calcium transients to shear force.
Collectively, these findings provide the most compelling evidence to date for a bona fide mechanosensitive GPCR and, by doing so, open the door to several interesting questions. How does force activate GPR68? Preliminary findings from Xu et al.
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Several histidine residues that have been shown to be required for proton sensitivity Ludwig et al. Notably, mutations of the histidine residues required for pH sensing also blocked sensitivity to shear stress. Alternatively, it remains possible that shear stress liberates protons locally and the receptor is indirectly responding to mechanical stimulation.
Because protons are difficult to track using common biochemical, electrophysiological, and structural methods, it will take future studies to work out the details. That said, it is tempting to speculate that the electrostatic interactions perturbed by protons could be similarly affected by mechanical stress.
Pharmacological tests by Xu et al. Notably, removing extracellular calcium reduced the responses from GPRexpressing cells, so it would be very interesting to determine whether TRPC channels are coexpressed with GPR68 and mediate these effects. In addition, Xu et al. Thus, it seems likely that the remaining calcium signal is a result of IP3-induced release of intracellular calcium stores, as indicated by the blocking effect of thapsigargin. Here again, there are several potential downstream effectors, such as Stim and Orai Putney and Tomita, ; Vashisht et al. One of the most exciting aspects of identifying GPR68 as a novel mechanoreceptor is that it offers opportunities to explain unknown physiological mechanisms.
As a first step, Xu et al. They identified GPR68 in several tissues throughout the body, with the highest expression found in immune cell populations from the spleen. It will be fascinating to uncover how GPR68 contributes to immune function and whether the knockout mice are immunocompromised. Recently, it was suggested that mechanotransduction is important for antibody T-cell recognition and that the functional contribution of the mechanosensitive ion channel Piezo1 may be important for normal immune responses Liu et al.
Furthermore, mechanical shear stress may increase the chemical sensitivity of T-cell receptors Lee et al. Because GPCR68 was also observed abundantly in the spleen, it could function in parallel with Piezo channels in modulating the immune response. In addition to the spleen, Xu et al.
Indeed, primary cultures of GPRpositive endothelial cells show calcium transients in response to shear stress that are attenuated in the absence of the receptor. Furthermore, they found that flow-mediated dilation of third-order branches of mesenteric arteries were dependent on Gpr Intriguingly, application of Ogerin, a molecule previously identified as a selective positive allosteric modulator of GPR68 Huang et al. This ganglion contains the cell bodies of the sensory neurons that are the primary sensors for environmental and internal stimuli.
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Notably, the majority of DRG neurons detect mechanical stimuli, and previous work established that Piezo2 is required for touch discrimination, vibration sensitivity, and proprioception in mice and humans Ranade et al. However, other types of mechanosensitivity, such as those that respond to mechanical pain, are largely Piezo2 independent. Is GPR68 expressed in nociceptors, and is it important for pain responses?
Tissue acidosis modulates the function of several ion channels known to be important for pain, including acid-sensing ion channels Bohlen et al. In summary, Xu et al. Looking forward, we anticipate that the development of new screening assays that use other types of mechanical stimulation, such as compression tensile forces or membrane stretch, will yield the discovery of even more receptors.
Furthermore, many of the well understood mechanosensory mechanisms involve protein complexes Scholz et al. Lastly, the assay used by Xu et al.
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We already know of mechanosensitive ion channels selective for potassium that would not have been found with this approach. The continuous improvement of fluorescent indicators for other messenger pathways offers a wealth of new possibilities for future screens. November 4, Volume , No. Skip to main content. Lam , Alexander T. Ruby M. DOI: Acknowledgments The authors declare no competing financial interests. Lesley C.
Anson served as editor. OpenUrl CrossRef. Bagriantsev , S. Ang , A. Gallardo-Godoy , K. Clark , M. Arkin , A. Renslo , and D. Minor Jr. A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels. ACS Chem. Banes , A. Gilbert , D.
Taylor , and O. A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro.
Shear elegance: A novel screen uncovers a mechanosensitive GPCR
Cell Sci. Bhattacharya , M.
Bautista , K. Haeberle , E. Lumpkin , and D.