Biology subjects

Cox, C. D.

Publications and source records attributed to Cox, C. D..

14 recordsLinked to original sources

bioRxiv2026

Aging activates latent enhancers that can be reversed withexercise

Aging profoundly alters the genome's regulatory landscape, but the properties of age-acquired enhancers, including how they are selected and sustained in aged animals are unclear. We used single-cell multiomics in the mouse heart to identify age-responsive cis-regulatory elements and endurance exercise to understand which of these elements can be attenuated. Aging increased AP-1 engagement at more than 100,000 regulatory loci across cardiac cell types. Exercise opposed most of these gains. Despite low endogenous accessibility, age-gained enhancer sequences drove reporter activity comparable to stable elements, revealing substantial latent transcriptional potential. Local sequence features distinguished responsiveness to aging and exercise. Reporter perturbations implicated sequence outside the AP-1 core, and mutagenesis localized informative positions to dispersed segments of dynamic enhancers. These enhancers were IL-1-sensitive and were preferentially responsive to neutralization of the cytokine in vivo. AP-1 inhibition further opposed age-associated transcription. Together, these findings support a model in which aging engages latent cell-type specific enhancers that are sustained in an active state by ongoing trans signaling. Aging therefore changes the deployment of existing regulatory information, while the acquired activity remains responsive to changes in signaling even in old age.

molecular biology↗

bioRxiv2025

TMEM63 proteins act as mechanically-activated cholesterol modulated lipid scramblases contributing to membrane mechano-resilience

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 the first identified 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.

biophysics↗

bioRxiv2025

Deletion of PIEZO1 in adult cardiomyocytes accelerates cardiac aging and causes premature death

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.

biophysics↗

bioRxiv2024

Metabotropic NMDA Receptor Signaling Contributes to Sex Differences in Synaptic Plasticity and Episodic Memory

Men generally outperform women on encoding spatial components of episodic memory whereas the reverse holds for semantic elements. Here we show that female mice outperform males on tests for non-spatial aspects of episodic memory ("what", "when"), suggesting that the human findings are influenced by neurobiological factors common to mammals. Analysis of hippocampal synaptic plasticity mechanisms and encoding revealed unprecedented, sex-specific contributions of non-classical metabotropic NMDA receptor (NMDAR) functions. While both sexes used non-ionic NMDAR signaling to trigger actin polymerization needed to consolidate long-term potentiation (LTP), NMDAR GluN2B subunit antagonism blocked these effects in males only and had the corresponding sex-specific effect on episodic memory. Conversely, blocking estrogen receptor alpha eliminated metabotropic stabilization of LTP and episodic memory in females only. The results show that sex differences in metabotropic signaling critical for enduring synaptic plasticity in hippocampus have significant consequences for encoding episodic memories.

neuroscience↗

bioRxiv2022

3D spheroid-microvasculature-on-a-chip for tumor-endothelium mechanobiology interplay

In the final step of cancer metastasis, tumor cells become lodged in a distant capillary bed, where they can undergo extravasation and form a secondary tumor. While increasing evidence suggests blood/lymphatic flow and shear stress play a critical role in the tumor extravasation process, there is a lack of systematic and biomechanical approaches to recapitulate sophisticated 3D microtissue interactions within the controllable hydrodynamic microenvironment. Here, we report a simple-to-use 3D spheroid-microvasculature-on-a-chip (SMAC) model. Under static and controlled flow conditions, the SMAC recapitulates the biomechanical crosstalk between heterogeneous tumor spheroids and the endothelium in a high-throughput and quantitative manners. As an in vitro metastasis mechanobiology model, we discover 3D spheroid-induced endothelial compression and cell-cell junction degradation in the process of tumor migration and expansion. Lastly, we examine the shear stress effects on the endothelial orientation, polarization as well as the tumor spheroid expansion. Taken together, our SMAC model offers a miniaturized, cost-efficient and versatile platform for future investigation on metastasis mechanobiology, enhanced permeability and retention effect and even personalized therapeutic evaluation.

bioengineering↗

bioRxiv2022

Membrane curvature governs the distribution of Piezo1 in cellulo

Piezo1 is a bona fide mechanosensitive ion channel ubiquitously expressed in mammalian cells. The distribution of Piezo1 within a cell is essential for various biological processes including cytokinesis, cell migration, and wound healing. However, the underlying principles that guide the subcellular distribution of Piezo1 remain largely unexplored. Here, we demonstrate that membrane curvature serves as a key regulator of the spatial distribution of Piezo1 in the plasma membrane of living cells. Piezo1 depletes from highly curved membrane protrusions such as filopodia and enriches to nanoscale membrane invaginations. Quantification of the curvaturedependent sorting of Piezo1 directly reveals the in situ nano-geometry of the Piezo1-membrane complex. Piezo1 density on filopodia increases upon activation, independent of Ca2+, suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that diminishes with channel activation.

biophysics↗

bioRxiv2020

Modified N-linked glycosylation status predicts trafficking defective human Piezo1 channel mutations

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like all membrane proteins, they pass through biosynthetic quality control in the endoplasmic reticulum and Golgi that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the cap region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for higher-order glycosylation in the propeller regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated protein. The higher order glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.

biophysics↗

bioRxiv2020

Biomechanical characterization of endothelial cells exposed to shear stress using acoustic force spectroscopy

Characterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness and contractile prestress of living endothelial cells were determined by Acoustic Force Spectroscopy (AFS) focusing on the displacement of functionalized microspheres located at the cell-cell periphery. The specific configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow, reaching shear stresses in the physiological range (i.e. 8 dyne cm-2) within 48 hours of barrier function maturation. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was applied in a single measurement revealing a force-dependent apparent stiffness in the kPa range. Moreover, our results show that different degrees of stiffening, defining the elastic behavior of the cell under different experimental conditions, i.e. static and dynamic, are caused by different levels of contractile prestress in the cytoskeleton, and are modulated by shear stress-mediated junction development and stabilization at cell borders. These results demonstrate that the AFS is capable of fast and high-throughput force measurements of adherent cells under conditions mimicking their native microenvironment, and thus revealing the shear stress dependence of mechanical properties of neighbouring endothelial cells.

bioengineering↗

bioRxiv2020

An acoustic platform for single-cell, high-throughput measurements of the viscoelastic properties of cells

Cellular processes including adhesion, migration and differentiation are governed by the distinct mechanical properties of each cell. Importantly, the mechanical properties of individual cells can vary depending on local physical and biochemical cues in a time-dependent manner resulting in significant inter-cell heterogeneity. While several different methods have been developed to interrogate the mechanical properties of single cells, throughput to capture this heterogeneity remains an issue. While new high-throughput techniques are slowly emerging, they are primarily aimed at characterizing cells in suspension, whereas high-throughput measurements of adherent cells have proven to be more challenging. Here, we demonstrate single-cell, high-throughput characterization of adherent cells using acoustic force spectroscopy. We demonstrate that cells undergo marked changes in viscoelasticity as a function of temperature, the measurements of which are facilitated by a closed microfluidic culturing environment that can rapidly change temperature between 21 {degrees}C and 37 {degrees}C. In addition, we show quantitative differences in cells exposed to different pharmacological treatments specifically targeting the membrane-cytoskeleton interface. Further, we utilize the high-throughput format of the AFS to rapidly probe, in excess of 1000 cells, three different cell-lines expressing different levels of a mechanosensitive protein, Piezo1, demonstrating the ability to differentiate between cells based on protein expression levels.

bioengineering↗

bioRxiv2020

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk

BackgroundKCNMA1 encodes the -subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited and KCNMA1 has not been investigated as an AF candidate gene. Methods and ResultsKCNMA1 sequencing in 118 patients with familial AF identified a novel complex variant in one kindred. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced cardiac arrhythmias. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. ConclusionsOur data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish and fly and suggest that KCa1.1 loss of function may predispose to AF.

genetics↗

bioRxiv2020

Force-dependent Piezo1 recruitment to focal adhesions regulates adhesion maturation and turnover specifically in non-transformed cells

Mechanosensing is an integral part of many physiological processes including stem cell differentiation, fibrosis, and cancer progression. Two major mechanosensing systems - focal adhesions and mechanosensitive ion channels, can convert mechanical features of the microenvironment into biochemical signals. We report here surprisingly that the mechanosensitive Ca2+-channel Piezo1, previously perceived to be diffusive on plasma membranes, binds to matrix adhesions in a force-dependent manner, promoting adhesion maturation and cell spreading in normal but not in tumor cells. In the absence of Piezo1, matrix adhesions are smaller in normal cells mimicking transformed cells where adhesions do not change with or without Piezo1. A novel adhesion-targeted calcium sensor shows robust Piezo1-dependent, calcium influx at adhesions in normal cells; but not in transformed cells. A linker domain in Piezo1 is needed for binding to adhesions and overexpression of the domain blocks Piezo1 binding to adhesions decreasing adhesion size and cell spread area. Thus, we suggest that Piezo1 is a novel component of focal adhesions in non-transformed cells that catalyzes adhesion maturation and growth through force-dependent calcium signaling, but this function is absent in most cancer cells.

cell biology↗

bioRxiv2019

Membrane stiffness is a key determinant of E coli MscS channel mechanosensitivity

Prokaryotic mechanosensitive (MS) channels have an intimate relationship with membrane lipids. Membrane lipids may influence channel activity by directly interacting with bacterial MS channels or by influencing the global properties of the membrane such as area stretch and bending moduli. Previous work has implicated membrane stiffness as a key determinant of the mechanosensitivity of E. coli (Ec)MscS. Here we systematically tested this hypothesis using patch fluorometry of azolectin liposomes doped with lipids of increasing area stretch moduli. Increasing DOPE content of azolectin liposomes causes a rightward shift in the tension response curve of EcMscS. These rightward shifts are further magnified by the addition of stiffer forms of PE such as the branched chain lipid DPhPE and the fully saturated lipid DSPE. Furthermore, a comparison of the branched chain lipid DPhPC to the stiffer DPhPE showed a rightward shift in the tension response curve in the presence of the stiffer DPhPE. We show that these changes are not due to changes in membrane bending rigidity as the tension threshold of EcMscS in membranes doped with PC18:1 and PC18:3 are the same, despite a two-fold difference in their bending rigidity. We also show that after prolonged pressure application sudden removal of force in softer membranes causes a rebound reactivation of EcMscS and we discuss the relevance of this phenomenon to bacterial osmoregulation. Collectively, our data demonstrate that membrane stiffness is a key determinant of the mechanosensitivity of EcMscS.

biophysics↗

bioRxiv2019

Acquisition of Temporal Order Involves a Reverberating Network in Hippocampal Field CA3

Here we report that hippocampal field CA3 maintains self-sustained activity for remarkable periods following a brief input and that this effect is extremely sensitive to minor perturbations. Using novel behavioral tests, that as with human episodic learning do not involve training or explicit rewards, we show that partial silencing of the network in mice blocks acquisition of temporal order, but not the identity or location, of odors. These results suggest a solution to the question of how hippocampus adds time to episodic memories.

neuroscience↗

bioRxiv2019

Disruption of membrane cholesterol organization impairs the concerted activity of PIEZO1 channel clusters

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-{beta}-Cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFPs response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure-response curve, increased channel latency in response to mechanical stimuli and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM super-resolution imaging revealed that, at the nano-scale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of poly-unsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane mechanical properties and lateral organization of membrane cholesterol domains which coordinates the concerted activity of PIEZO1 channels.\n\nSUMMARYThe essential mammalian mechanosensitive channel PIEZO1 organizes in the plasma membrane into nanometric clusters which depend on the integrity of cholesterol domains to rapidly detect applied force and especially inactivate syncronously, the most commonly altered feature of PIEZO1 in pathology.

biophysics↗