Mechanosensitive channels of large conductance (MscL) protect bacteria from hypo-osmotic stress by opening a large pore in response to membrane tension. The transmembrane TM1 and TM2 helices together with the N-terminal amphipathic helix function as the primary force-sensing structural domains, whereas the role of the periplasmic loop in the channel gating remains poorly understood. Using experimental and computational approaches, our study demonstrates that mutating the loop residues (A64, Q65, G66, D67) or inserting a four-glycine flexible hinge at D67 site modified the properties of the channel recorded in giant E. coli spheroplasts and liposomes composed of azolectin or negatively charged lipids. Extending the periplasmic loop disrupted mechanical force transmission, reducing channel sensitivity to applied tension. Q65R channel mutant exhibited increased sensitivity in azolectin liposomes that was reduced in negatively charged liposomes and spheroplast membranes. The Q65E mutant showed decreased sensitivity across all preparations tested and exhibited channel flickering in negatively charged liposomes. Molecular dynamics simulations revealed that Q65E produced larger but more structurally restricted conformational changes, whereas wild-type MscL and Q65R exhibited rapid and extensive pore opening over a shorter timeframe. Our findings establish the periplasmic loop as a structural domain fine-tuning MscL gating through electrostatic interactions with surrounding lipids.
The mechanisms underlying mechanical differences among cancer cell lines with varying metastatic potential remain poorly understood, despite their clear therapeutic relevance. While comparisons between malignant and benign cell mechanics reveal systematic differences, these are difficult to attribute to specific molecular drivers, complicating mechanistic interpretation. Here, we investigate whether controlled expression of a single oncogene is sufficient to induce measurable morphological, mechanical, and migratory changes. Using atomic force microscopy-based microrheology on live-cell nuclei, we show that inducible expression of the oncogene p95HER2 in breast cancer cells leads to a reduced viscoelastic modulus and a lower transition frequency, consistent with nuclear fluidization. Fluorescence staining of the actin cytoskeleton reveals no detectable reorganization of actin at the nuclear region upon p95HER2 expression, suggesting that the mechanical changes originate primarily from alterations in the nucleus rather than from cytoskeletal remodeling. Consistent with this interpretation, western blotting of isolated nuclei demonstrates reduced lamin A/C expression in p95HER2-expressing cells, and measurements of isolated nuclei confirm that they are both taller and softer than control nuclei. Among Jeffrey’s model viscoelastic parameters, only the outer dashpot viscosity η2 shows a marked reduction, suggesting it is specifically sensitive to lamin A/C levels. Cells expressing p95HER2 exhibit distinct morphological alterations and increased migratory capacity in a pore migration assay, quantified using deep-learning-based segmentation. Enhanced nuclear fluidity, driven at least in part by reduced lamin A/C, may facilitate migration by promoting efficient passage through endothelial barriers.
Network models offer a powerful framework for linking filament-scale properties to cell and tissue mechanobiology. Lattice-based, off-lattice, and active network approaches explain emergent phenomena including nonlinear elasticity, rigidity transitions, and force transmission in cytoskeletal and extracellular matrices. This Review synthesizes key theoretical advances and experimental insights, and outlines challenges in extending network models to living tissues, including mechanochemical coupling, multiscale integration, and structural heterogeneity.
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.
In addition to actin assembly at the growth cone, neuronal axons elongate via interactions between microtubules, dynein motor proteins and cross-linking proteins. Dynein translocates microtubules toward the growth cone and exerts extensile forces for axonal outgrowth. During this process, microtubules likely experience compressive loads, which can result in bending deformation called buckling. Such buckled microtubules are impaired in their ability to bear compressive forces and may not contribute significantly to axonal outgrowth. If microtubules are interconnected by cross-linking proteins, they are less likely to be buckled and thus can resist larger compressive loads. Despite the importance of microtubule buckling and connectivity, their effects on axonal outgrowth have not been investigated to date. In this study, using an agent-based computational model, we created a microtubule bundle to simulate the microtubule system in axons. Motor activity elongated the bundle against a mechanical load. We found that intermediate cross-linking density maximized bundle elongation, because microtubules were easily buckled at low cross-linking density, whereas their displacements were inhibited at high cross-linking density. When microtubules were stiffer, the bundle elongated longer even with lower cross-linking density, since stiff microtubules experience less buckling by compressive loads. Our study provides new insights into the mechanisms driving axonal outgrowth.
Regulatory T cells (Tregs) suppress immune responses through multiple mechanisms, including contact-dependent removal of pMHCII and costimulatory ligands from antigen-presenting cells via trogocytosis and trans-endocytosis. These pathways require membrane deformation, receptor engagement, and vesicular trafficking. We propose that Treg efficiency in these processes is shaped by cellular mechanics—particularly membrane tension and actin organization—yielding a distinct biophysical signature for antigen-specific suppression.
Laser optical tweezers (LOT) is a single-molecule force spectroscopy (SMFS) method that can provide direct, real-time assessments of forces acting on individual molecules at sub-piconewton resolution. Furthermore, LOT allows for the direct manipulation of single molecules under controlled conditions, thereby enabling detailed examination of processes including chromatin compaction, templated protein misfolding, DNA packaging by the bacteriophage T4 motor, function of transcription and translation machinery, and the roles of SNARE protein complexes in membrane fusion. The main advantage of LOT is its ability to connect the mechanical properties of individual biomolecules at the molecular level with larger (sub)cellular processes, and, hence, it creates a mechanistic link between the biophysics of single molecules and the complex nature of cells. This is a crucial step in advancing mechanobiology and improving our understanding of how biomolecules function in their biological context, particularly in post-reductionist research.
Mechanosensitive (MS) channels are membrane proteins that respond to mechanical stimuli and are essential across prokaryotic and eukaryotic organisms. The E. coli mechanosensitive channel of large conductance (MscL) provides a powerful model for dissecting protein-lipid interactions underlying mechanotransduction. Here, we investigated the contribution of four periplasmic loop residues (A64, Q65, G66, D67) to MscL gating. Using site-directed spin labelling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy, we show that these residues interact directly with the lipid bilayer during the channel opening through membrane tension sensitivity of the channel. We further examined how mutations at these periplasmic loop residues affect biophysical properties of MscL in giant E. coli spheroplasts and liposomes composed of azolectin or negatively charged lipids (70% phosphatidylcholine and 30% phosphatidylglycerol) using patch clamp electrophysiology, patch fluorometry and molecular dynamics simulations. Substitution of Q65 residue with arginine (Q65R) increased channel sensitivity to membrane tension, whereas replacement with glutamic acid (Q65E) decreased the sensitivity across all systems tested. Molecular dynamics simulations revealed that under in-plane radial tension Q65E exhibited larger conformational changes under tension, whereas the wild-type (WT) and Q65R mutant channels showed rapid and extensive opening at later stages within a shorter time frame. Insertion of a four-glycine hinge at D67 also reduced tension sensitivity, consistent with impaired force transmission. Substitution of A64 and G66 to either glutamic acid (A64E, G66E) or arginine (A64R) decreased tension sensitivity of the channel only in liposomes of negatively charged lipids. Together, these findings deepen our understanding of protein-lipid interactions governing MscL opening kinetics and highlights the contribution of the periplasmic loop in regulating mechanosensitivity.
AAA+ ATPases are essential ATP-driven molecular machines with diverse cellular functions, including protein unfolding, active transport, and chromatin remodeling. Despite their broad importance, the precise mechanisms by which energy transduction drives protein unfolding in AAA-ATPase motors remain unclear. Here, we present a probabilistic model that simulates nonequilibrium chemomechanical transduction of ring-like AAA-ATPase motors during substrate unfolding in the 26S proteasome. By capturing the sequential cycling of ATP hydrolysis around the ATPase ring, our model explores a wider range of coordinated conformational transitions than previously observed experimentally. Our simulations reveal multiple high-probability pathways for state transition during hand-over-hand translocation of substrate, elucidating the nonequilibrium dynamics of around-the-ring energy transduction in AAA-ATPase motors. These findings, extensively examined by experiments, provide quantitative insights into chemomechanical coupling that are likely conserved across the AAA+ protease or unfoldase superfamily. This work offers a theoretical framework for understanding ring-like AAA+ translocation motors in general.
Mitochondria are central hubs of cellular bioenergetics, converting chemical free energy into ATP while inevitably releasing heat during respiration. Fluorescence-based thermometry has been interpreted to show intracellular "hot spots" more than 10 °C above the bulk physiological temperature, implying that mitochondria might operate far outside conventional thermal bounds. Such claims, however, appear inconsistent with basic biophysics: the small size of mitochondria, their aqueous and highly conductive environment, and their limited power output all argue against large steady-state temperature gradients. This discrepancy has prompted renewed scrutiny of both the physical limits of intracellular heat transfer and the biological interpretation of nanoscale thermal measurements. A key open question is whether nonequilibrium biochemical processes, such as respiration-driven proton pumping, could act as nanoscale heat pumps that maintain higher local temperatures than allowed by passive diffusion alone. Here, we develop a model-independent thermodynamic analysis based solely on the Second Law of Thermodynamics to bound the maximal temperature difference that any biochemically driven mechanism can sustain across the inner mitochondrial membrane and show that even under idealized conditions the achievable temperature rise is restricted to a small fraction of a degree, effectively closing this loophole.
A major requirement for humans is a breathable atmosphere for adequate respiratory CO2/O2 gas exchange. In microgravity, despite environmental life support systems regulating air exchange, astronauts complain about air quality, with elevated CO2-levels resulting in detrimental health and performance effects. Using high-fidelity computational fluid dynamics, we create a model of human respiratory ventilation to show how gravity biophysically shapes and drives respiratory exchange on Earth and in microgravity. On Earth, gravity influences gas exchange through buoyancy and biothermal convection, generating a ‘human thermal body plume’ that drives airflow around the human body, and so facilitates effective gas exchange. We show that the absence of biothermal convection in microgravity reduces this airflow around the human body. This impairs gas exchange by creating an environmental breathing deadspace immediately in front of the face, leading to significant CO2-rebreathing, with direct implications for astronaut health and countermeasures. This model was also used to estimate engineering requirements for directed external airflow equivalent to that generated by the human thermal body plume to alleviate this problem. Our model further shows that in Earth-normal 1 g, increasing ambient air temperature can also reduce efficient respiratory exchange, resulting in breathing conditions equivalent to those in microgravity, with implications for respiratory health on Earth.
While the formation of single-stranded regions in DNA plays important roles in processes such as replication, repair, and transcription, the underlying mechanics and kinetics of these structures are not well understood. Here, we combine single-molecule Convex Lens-induced Confinement (CLiC) microscopy with stemless molecular beacons to detect and study correlations in the formation of multiple single-stranded regions within plasmids in suspended solution conditions. We demonstrate that negative supercoiling not only increases denaturation of these sites, but also augments oligo-plasmid binding interactions. The observed denaturation disagrees with theoretical predictions stemming from equilibrium thermodynamics, suggesting the structures may form metastable states. To our knowledge, this is the first single-molecule study of plasmids in suspended solution conditions to make measurements correlating the formation of multiple single-stranded regions within the same plasmid. Beyond DNA mechanics, these methods have the potential to enable insights in a broad range of fields, such as informing the development of therapeutics sensitive to DNA and RNA structure.
The mechanical environment surrounding cells influences their behavior. Multiple methods have been developed to measure local mechanics with mechanical perturbation, which is invasive. For sheets of confluent cells, the geometry of the boundaries of the cells can be used to estimate the local membrane tension and internal cell pressure through stress inference (SI), which is image-based and therefore non-invasive. SI is sensitive to noise and has not been validated in a physical system using a direct pressure measurement. We developed a large-scale model of confluent cells using densely-packed balloons with interior pressure sensors to allow for simultaneous measurement of pressure and geometry. We also developed a new SI method which resulted in a correlation coefficient >0.8 with the balloon model, less noise sensitivity in computational models, and measured pressure change in red onion epithelial cells. These results demonstrate the validity and utility of SI in multiscale physical and computational applications.
Macrophages are innate immune cells contributing to tissue homeostasis and various pathologies. Signals from their environment can lead macrophages to adapt distinct functional phenotypes, a process called polarization. Because macrophages have been previously shown to degrade the nuclear envelope proteins lamin A/C upon pro-inflammatory polarization, and lamins are considered key determinants of nuclear deformability, we aimed to address the effect of pro-inflammatory stimulation on nuclear mechanics. We present the surprising finding that polarized bone marrow-derived macrophages have less deformable nuclei than unpolarized macrophages, despite their reduced lamin A/C levels. Furthermore, pro-inflammatory macrophages exhibited altered chromatin dynamics relative to unpolarized macrophages, including redistribution of trimethylated histone H3K9 (H3K9me3) from the nuclear periphery to the interior and increased chromatin compaction. Our findings suggest a model in which pro-inflammatory stimulation of macrophages induces chromatin changes that drive nuclear stiffening, and that in these cells, chromatin, rather than the nuclear lamina, is the major driver for resisting nuclear deformation. These findings may have functional relevance for the physiological function of polarized macrophages, as the mechanical properties of the nucleus can influence how these cells adapt and respond to their environments in the context of cell migration or inflammatory disease pathologies.
The incorporation of a functional perfusable microvascular network (MVN) is a common requirement for most organ on-chip-models. Long-term perfusion of MVNs is often required for the maturation of organ phenotypes and disease pathologies and to model the transport of cells and drugs entering organs. Here, we use a microfluidic pump to apply continuous, recirculating physiological flow through self-assembler microvascular networks. In our microphysiological system (MPS), we observe that flow can recover perfusion in regressed MVNs and maintain perfusable MVNs for at least 51 days. Throughout the 51 days, however, the MVNs are continuously remodeling to align with the direction of bulk flow and only appear to attain morphological homeostasis with the use of maintenance medium without growth factors. We observed that the flow resistance of the MVNs decreases over time, and using a computational model, we show that stable vessels have higher flow rates and velocities compared to regressing vessels. Cytokine analysis suggests that static conditions generate an inflammatory state, and that continuous flow reduces inflammation over an extended period. Finally, through bulk RNA sequencing we identify that both the endothelial and fibroblast cells are actively engaged in flow-induced vascular and matrix remodeling and that these effects persist for at least 2 weeks. This MPS can be applied to study hemodynamically driven processes, such as metastatic dissemination or drug distribution, or to model long-term diseases previously not captured by MPS, such as chronic inflammation or aging-associated diseases.
A long-wavelength approximation of Fluid-Structure-Interaction (FSI) waves in vascular networks is developed, the relevance of which is first discussed. Long-wavelength FSI-waves continuity condition within networks are derived. The resulting coupled wave system is solved within any network generalizing a recently proposed quantum-graph eigen-mode decomposition approach. The spectral condition associated with the secular matrix evaluation is derived, providing the intrinsic pulsating modes of arterial pulse waves. The theory is applied to the circle of Willis arterial network for which the spectrum is explicitly computed and compared with clinical observations related to the intra-cranial pressure dynamics. Retrieving the cardiac frequency modes from the observed pulse signal produce consistent results between the new modes predictions and observations. This leads to a new understanding of intra-cranial pressure frequency content, related to pressure pulse propagation within the arterial network, containing relevant clinical information about the arterial’s physiological and mechanical response.
While migratory cells can quickly change their mode of migration in complex three-dimensional environments, it is not clear why. Understanding the dynamic and reciprocal relationship migrating cells have with their microenvironments may help reveal why migratory plasticity, or mode-switching, is a common feature of eukaryotic cell motility. In this review, we discuss the physical and mechanical properties of cells and the environments they move through, and how those properties can influence each other. Given the dual role of the cytoskeleton in cell migration and cellular mechanics, we suggest that migratory plasticity derives from the necessity for the cell to maintain mechanical homeostasis in diverse physical environments.
The eye presents a very dynamic biomechanical environment, and thus ocular cells must be highly mechanosensitive and mechanoresponsive. Moreover, defects in mechanobiological pathways contribute to a number of sight-threatening ocular diseases, highlighting the importance of ocular mechanobiology. We here give a concise overview of the mechanobiology of ocular cells in the lens and cornea (and how mechanobiology plays a role in associated pathologies in these tissues), before providing a detailed review of the mechanobiology of the common blinding disease, glaucoma. Mechanical stimuli are intimately linked with the pathology of glaucoma, both in terms of altered homeostasis of the eye's internal pressure control system and in the response of neural cells to elevated pressure in the eye. A complex array of mechanosensory elements (stretch-activated ion channels, integrins, G protein-coupled receptors) work together with intersecting networks of mechanotransducing pathways in cells of both the posterior and anterior eye in glaucoma. Despite intense research efforts over the past decades, much remains unknown about the mechanobiology of glaucoma. Continued investigation of glaucomatous mechanobiology is important, as it may reveal novel targets for treating this challenging disease.
Soil compaction and escalating global drought increase soil strength and stiffness. It remains unclear which plant root biomechanical mechanisms/traits enable growth in these harsh conditions. Here, we combine synchrotron X-ray computed tomography with spatially resolved X-ray diffraction to characterize the biomechanics of a replica root-soil system. We map the strain field around the root tip analog, finding strong agreement with finite element simulations, thereby demonstrating a promising new in vivo measurement protocol.
Mild traumatic brain injury (mTBI) is an acute injury with immediate and medium-term symptom presentation. However, our mechanistic understanding of mTBI and how mechanical loading of soft cellular tissues leads to injury is limited. The aim of this review is to introduce this interdisciplinary field to non-experts and provide an overview of our current understanding of how mechanical trauma contributes to cellular injury. Here, we compare the significance of various measures of mechanical loading including strain magnitude, strain rate, loading mode, and frequency, and their relative significance for cell and tissue injury in in vitro and ex vivo experimental models reported in the literature. Interestingly, while it is difficult to define a precise injury threshold value based on strain magnitude alone, cellular injury is commonly observed at strain rates of >0.1 s-1, higher than rates observed in many normal cell functions (< 0.01 s-1). We explore the role of the plasma membrane, cytoskeleton, and specialized structures in maintaining cell integrity during traumatic injury.
