OSCA/TMEM63 mechanosensitive ion channels play critical physiological roles in plants and animals. These channels bear structural homology to the dual functional TMEM16 family, and OSCA1.2 was recently shown to form a lipid-lined ion conduction pathway in the open state. This raised the question of whether members of the OSCA/TMEM63 family may also function as mechanically activated lipid scramblases. Using a combination of in vitro and cellular assays with computational techniques, we show that phospholipids can be translocated through the open pores of OSCA1.1/1.2/2.2 and TMEM63A/B proteins, suggesting a dual ion channel and lipid scramblase function for members of this protein family. We characterize the effects of mutating key groove lining residues demonstrating that different residues form bottlenecks for lipids and ions respectively and show that cholesterol inhibits lipid scrambling by stabilizing the closed state and slowing translocation through the open pore. We show that lipid scrambling in TMEM63 proteins can be activated by mechanical forces in the membrane, making these mechanically activated lipid scramblases. Finally, we demonstrate that this activity is important for the mechanically induced morphological remodeling of biological membranes and the resilience of cells to high mechanical forces. OSCA/TMEM63 proteins act as both ion channels and mechanically activated lipid scramblases. They translocate lipids through open pores, with cholesterol inhibiting scrambling, aiding membrane remodeling and cell resilience under mechanical stress.

The tools for subjecting cells to high-magnitude shear stress within conventional cell culture dishes and well plates on an orbital shaking platform have remained unchanged for the past 30 years. Here, we develop a pipeline for creating custom cell culture dishes and well plates of arbitrary size and complexity using 3D printing technology. We describe two methods: direct 3D printing and reversible bonding of ACLAR film to 3D-printed structures. We show that the custom chambers support alignment of human aortic endothelial cells cultured under flow while capturing robust activation of extracellular signal-regulated kinase in a shear stress- and time-dependent manner. We show that shear stress regulates the post-translational modification of the shear-stress-sensitive mechanosensitive ion channel PIEZO1, resulting in an increase in N-linked glycosylation that may be relevant to the channel's ability to sense and respond to shear stress. We also developed the first scanning and transmission electron microscopy protocol compatible with cells mechanically stimulated on an orbital shaker and demonstrated another approach for customizing conventional labware. The simplicity of fabrication, cost-effectiveness of this pipeline, and the ability to process a large number of cells simultaneously for multiple downstream experimental end points mean that the methodology developed here is likely to be of broad utility in the in vitro study of endothelial mechanobiology.

PIEZO channels are critical for sensory mechanotransduction. While MyoD-family inhibitor proteins were identified as PIEZO1 auxiliary subunits, their broader regulatory roles, particularly in sensory cells, remained unclear. Here, we demonstrate native MDFIC and MDFI regulate endogenous PIEZO channel currents in various nonsensory cell types. However, neither MDFIC nor MDFI are expressed in primary sensory neurons. In these cell types, we identified an uncharacterized third member of this family, Mdfic2/Gm765, that shares the ability to physically bind to PIEZO1 and PIEZO2. MDFIC2 is selectively expressed in subsets of mechanosensitive neurons, including dorsal root ganglia, trigeminal ganglia, and vagal sensory neurons. Like its paralogues, MDFIC2 alters PIEZO1/2 mechanosensitivity and inactivation kinetics, converting them into high-threshold slowly inactivating mechanoreceptors. Extensive cryo-EM reveals a conserved binding pocket for these auxiliary subunits in the pore modules of both PIEZO1 and PIEZO2 mediated by the posttranslationally modified distal C termini of MyoD-family inhibitor proteins. This structural and functional characterization of MyoD-family inhibitor proteins as PIEZO1/2 channel auxiliary subunits offers insights into the mechanobiology of nonsensory and sensory cells.

Merkel cells are epithelial cells involved in the discrimination of light touch. Situated in the skin, these specialized cells decode mechanical cues through the mechanosensitive ion channel PIEZO2. Merkel cell carcinoma lines have been widely used as in vitro models for Merkel cells and like the native cell, they exhibit mechanically evoked currents. Herein, we show that unlike the native Merkel cell, which principally uses PIEZO2, mechanically evoked currents in the Merkel cell carcinoma line MCC13 are predominantly carried by PIEZO1, with variable contributions from PIEZO2. Slowly inactivating current types in these cells are carried by PIEZO1 in complex with auxiliary subunits MDFIC or MDFI. Moreover, PIEZO1 strongly influences the endogenous levels of MDFIC, a known transcriptional repressor, whereby the loss of PIEZO1 dramatically reduced cellular levels of MDFIC. This suggests that the removal of PIEZO1 from a cell will likely have influences on the cellular transcriptome beyond its canonical Ca2+-based signalling pathways. In conclusion, utilizing MCC13 cells as a simple in vitro model of native Merkel cell mechanotransduction should be carried out with caution.

PIEZO1 and PIEZO2 are mechanically activated ion channels central to touch, proprioception, and vascular development. Although purified PIEZO1 gates in lipid bilayers, cellular mechanotransduction relies on interacting proteins that tune channel sensitivity, kinetics, and spatial activation via three mechanisms: reshaping the membrane microenvironment, conveying forces via extracellular/intracellular tethers, or directly modifying gating energetics. We review PIEZO-associated proteins and propose a unified framework linking these mechanotransduction complexes to precision medicine.

Mechanosensitive PIEZO1 channels have emerged as key transducers of mechanical forces in the cardiovascular system. In cardiomyocytes, we previously showed that PIEZO1 decodes mechanical cues driving pressure-overload induced hypertrophy. However, conflicting reports exist on the influence of PIEZO1 on baseline cardiac function. Here we show that conditional deletion of Piezo1 from cardiomyocytes in adult mice results in premature mortality. The hearts from these mice exhibited signs of accelerated aging, including elevated markers of the senescence associated secretory phenotype, with significant blunting of the normal cardiac hypertrophic response to aging, associated with a reduction in the activation of the pro-hypertrophic Ca2+/calmodulin-dependent protein kinase II (CaMKII). Functionally, aged-Piezo1 KO mice exhibited impaired cardiac relaxation due to altered cellular Ca2+ handling kinetics. Young adult Piezo1 KO mice exhibited a normal resting heart rate but developed significant progressive sinus bradycardia and cardiac fibrotic remodelling with aging, which was most prominent in the right atrium, where Piezo1 expression is highest in the healthy heart. Mechanistically, loss of PIEZO1 was associated with a marked reduction in the anti-fibrotic molecule, atrial natriuretic peptide (ANP). Moreover, in vivo, ANP release instigated by atrial stretch was markedly blunted in conditional Piezo1 KO mice, providing a plausible and long sought-after mechanism for the link between mechanical stretch and ANP release. Taken together, our data show that PIEZO1 is a crucial homeostatic molecule during cardiac aging, enabling adaptation to an aging tissue microenvironment. ### Competing Interest Statement The authors have declared no competing interest.

PIEZO1 is a mechanically activated ion channel essential for mechanotransduction and downstream signaling in almost all organ systems. Western blotting is commonly used to study the expression, stability, and post-translational modifications of proteins. However, as a large transmembrane protein, PIEZO1 contains extensive hydrophobic regions and undergoes post-translational modifications that increase its propensity for nonspecific protein-protein interactions. As a result, conventional sample preparation methods seem unsuitable for PIEZO1. For example, heating and sonicating transmembrane proteins exposes hydrophobic regions, leading to aggregation, improper detergent interactions, and loss of solubility, ultimately compromising their detection in western blots. To address these challenges, we developed a western blot protocol optimized for human PIEZO1 by preparing lysates consistently at lower temperatures and incorporating strong reducing and alkylation reagents into the western blot lysis buffer to ensure proper protein solubilization and minimal cross-linking. Using the same antibody, we also developed an immunoprecipitation protocol with optimized detergents to maintain the solubilization of native human PIEZO1, enabling the discovery of a new family of auxiliary subunits. Key features • Simple modifications to the standard RIPA buffer prevent protein aggregates of large transmembrane proteins. • Minimal protein degradation and cross-linking by modifying cell lysis conditions and protein extraction process. • Clear separation of glycosylated and non-glycosylated PIEZO1 by SDS-PAGE.

YnaI is a member of the family of bacterial MscS (mechanosensitive channel of small conductance)-like channels. Channel gating upon hypoosmotic stress and the role of lipids in this process have been extensively studied for MscS, but are less well understood for YnaI, which features two additional transmembrane helices. Here, we combined cryogenic electron microscopy, molecular dynamics simulations and patch-clamp electrophysiology to advance our understanding of YnaI. The two additional helices move the lipid-filled hydrophobic pockets in YnaI further away from the lipid bilayer and change the function of the pocket lipids from being a critical gating element in MscS to being more of a structural element in YnaI. Unlike MscS, YnaI shows pronounced gating hysteresis and remains open to a substantially lower membrane tension than is needed to initially open the channel. Thus, at near-lytic membrane tension, both MscL and YnaI will open, but while MscL has a large pore and must close quickly to minimize loss of essential metabolites, YnaI only conducts ions and can thus remain open for longer to continue to facilitate pressure equilibration across the membrane.

Transient receptor potential (TRP) channels are implicated in a wide array of mechanotransduction processes. However, a question remains whether TRP channels directly sense mechanical force, thus acting as primary mechanotransducers. We use several recent examples to demonstrate the difficulty in definitively ascribing mechanosensitivity to TRP channel subfamilies. Ultimately, despite being implicated in an ever-growing list of mechanosignalling events in most cases limited robust or reproducible evidence supports the contention that TRP channels act as primary transducers of mechanical forces. They either (i) possess unique and as yet unspecified structural or local requirements for mechanosensitivity; or (ii) act as mechanoamplifiers responding downstream of the activation of a primary mechanotransducer that could include Ca2+-permeable mechanosensitive (MS) channels or other potentially unidentified mechanosensors.

Integrin-mediated focal adhesions form the link between the extracellular matrix (ECM) and the cytoskeleton and are the primary subcellular sites for sensing substrate stiffness and ECM composition. These mechanical properties of the cellular micro-environment are key modulators of cardiac fibroblast activation and subsequent differentiation into myofibroblasts, and downstream cardiac remodeling. PIEZO1, a calcium permeable ion channel, is a well-established key sensor of mechanical forces in the cardiovascular system. In this study, we aim to understand whether PIEZO1influences cardiac fibroblast phenotype and function. Using total internal reflection fluorescence (TIRF) microscopy, we show enrichment of PIEZO1 in focal adhesions in both primary human and mouse cardiac fibroblasts, and in a knock-in fibroblast line expressing a PIEZO1-HaloTag fusion protein. Live imaging followed by single particle tracking of the HaloTag showed PIEZO1 being more stable in focal adhesions compared to the rest of the membrane. Using a transiently expressed genetically encoded focal adhesion localized calcium-sensor (GCaMP7s-Paxillin-mScarletI), we confirmed that PIEZO1 is critical for calcium transients present at focal adhesions. PIEZO1 knock-down using siRNA or knock-out using CRISPR/Cas9 modified the fibroblasts focal adhesion sizes, cell spread area, whole-cell stiffness (measured using atomic force microscopy) and cell migratory capacity, all of which are processes closely linked to cytoskeletal organization. Furthermore, PIEZO1 levels were responsive to substrate stiffness and ECM density. Chemical activation of PIEZO1 using the agonist Yoda-1 drastically altered its sub-cellular localization and stimulated fibroblast activation/differentiation, in a similar fashion to TGF-beta1, including actin cytoskeleton remodeling and increased cellular contractility. These results implicate PIEZO1 channels in fibroblast activation mediated via focal adhesion signaling.