Active enzymes generate significant mechanical fluctuations while catalyzing chemical reactions, which can influence the dynamics of their surroundings. This phenomenon opens new avenues for controlling mass transport in complex and dynamically inhomogeneous environments through localized chemical reactions. Herein, we studied the uptake of transferrin molecules in retinal pigment epithelial cells via clathrin-mediated endocytosis. In the presence of enzyme catalysis in the extracellular environment, we observed a significant enhancement in the transport of fluorophore-tagged transferrin inside the cells. Total internal reflection fluorescence microscopy and fluorescence correlation spectroscopy measurements showed a substantial increase in transferrin diffusion in the presence of active fluctuations. This study sheds light on the possibility that enzyme-substrate reactions within the extracellular environment may induce long-range mechanical influences, facilitating targeted material delivery within the intracellular milieu more efficiently than passive diffusion. These insights could contribute to the development of better therapeutic strategies by overcoming limitations imposed by slow molecular diffusion under complex environments.

Tumor necrosis factor receptor 1 (TNFR1) signaling regulates cell fate in inflammation, immune responses, and tumorigenesis. While TNF-α-mediated TNFR1 pathways are well known, the role of receptor clustering remains unclear. Utilizing homo-FRET using fluorescence anisotropy, we show that intra-cluster receptor density (IRD) governs TNFR1 signaling outcomes. Soluble TNF-α (sTNF-α) increases IRD at cluster cores but decreases it at rims via receptor reorganization. Reducing IRD through membrane tension, zafirlukast, actin depolymerization, or cholesterol depletion suppresses sTNF-α signaling, whereas increasing IRD by lowering membrane tension or exposing cells in a 3D gel-like microenvironment triggers ligand-independent activation. These findings reveal IRD as a key regulator of receptor signaling, with potential relevance across related receptor families and innovative strategies in modulating TNFR1 signaling.

Ionic imbalance in the tumor microenvironment alters the function of tumor-infiltrating T lymphocytes. High extracellular K+ suppresses T cell function by negatively regulating T cell receptor (TCR) signaling. The mechanism of how monovalent cations regulate T lymphocyte function is unknown. Here, we present a mechanism that explains how cellular potassium dynamics regulate TCR function. At rest, high intracellular K+ uncouples allosteric recruitment of ZAP-70, a key signaling module, to the TCR complex. Elevated K+ concentration imparts a higher thermodynamic penalty on the binding of the ZAP-70 regulatory module to the phosphotyrosine residues in the ITAM motifs of the CD3 chain. Our data suggest that K+ functions as a key allosteric modulator, stabilizing the autoinhibited conformation of ZAP-70. Thus, it prevents spontaneous TCR activation in the resting state. Formation of the antigen-TCR complex induces K+ efflux, leading to spontaneous recruitment of ZAP-70 to the TCR. Increasing extracellular K+ concentration perturbs K+ efflux and slows ZAP-70 recruitment to the TCR complex, even upon antigen binding. Impaired ZAP-70 activation partially dampens TCR signaling, thereby altering downstream signaling. In contrast, the regulatory module in the paralogous kinase Syk, which is expressed in B cells, is insensitive to potassium concentration. At elevated K+ concentration, the interaction between the Syk regulatory module and phosphorylated ITAM motifs remains unaltered. We conclude that K+ dynamics are integral to T cell ligand discrimination and fundamental to turning off the signaling during T cell quiescence.

Plasmacytoid dendritic cells (pDCs) are innate immune cells that produce type I interferons (IFNs) upon sensing nucleic acids via Toll-like receptor 9 (TLR9). Synthetic oligodeoxynucleotides CpGA and CpGB are widely used TLR9 agonists, yet only CpGA robustly induces IFN-α in pDCs. In contrast, CpGB drives much less IFN production. The mechanism underlying this ligand-specific response is not known. Here, we identify PIEZO1, a mechanosensory ion channel, as a regulator for this ligand-specific response. We show that CpGA, unlike CpGB, self-associates into large aggregates that generate membrane tension during cellular uptake, activating PIEZO1. This triggers calcium influx and localized F-actin assembly, retaining CpGA in early endosomes to sustain IRF7 activation and IFN production. PIEZO1 deficiency or inhibition abolishes CpGA-induced IFN responses, while PIEZO1 activation enhances IFN production by CpGB. Our findings reveal a hitherto unknown biophysical checkpoint in TLR9 signaling, where PIEZO1 translates membrane tension into spatially controlled TLR9 signaling. This study uncovers a novel role for mechanosensing in nucleic acid immunity, with implications for modulating IFN responses in infections and autoimmunity.

Slow equilibration of local mechanical perturbations on the cell membrane could ideally create inhomogeneities in the mechanical state of the cell membrane, affecting its function. However, very few reports measure mechanical heterogeneities or connect them to cellular functional state. In this work, we employ optical-trap-based dual-tension measurements (per cell) to first reveal an actin-dependent tension gradient with low apical tensions. In the same cells, interference reflection microscopy reveals heterogeneous tension distribution at the base. Super-resolution imaging and fluorescence-lifetime (Flipper-TR) measurements reveal the midplane to be more contractile and tensed while confirming an actomyosin-contractility-dependent apical tension gradient and ruling out a direct contribution of actin-membrane linkages. Cargo-loaded clathrin-coated pits are bigger and denser at the tensed midplane. Inhibiting their formation suppresses the rise in midplane tension but accentuates the apical tension gradient. Thus, the tension profile is created by the actomyosin contractility profile but further tuned by its crosstalk with clathrin-mediated endocytosis.

Ezrin plays a crucial role in linking the cortical actin cytoskeleton to the plasma membrane in animal cells. Inactivated by small molecule ezrin inhibitors (EzrInh), ezrin is known to regulate cellular functions like adhesion and motility. However, whether ezrin plays any significant role in protecting cells against mechanical stresses is less explored. We use hypo-osmotic shock (HS) to challenge cells and study the mechano-protective role of ezrin. Inhibition of ezrin phosphorylation led to increased cell rupture rates on hypo-shock, with a greater impact at later time points (∼ 10 min). The time scales match with that of enhanced tension contrast at edges at the basal plane. We also observed a lowered relative retraction rate at protrusions created by hypo-shock and a less contractile mid-plane cortex – both indicate the role of a weakening of the cortex by EzrInh in compromising membrane integrity on hypo-shock. Concomitantly, EzrInh also increased stress fibres (SFs), traction forces and cell spreading clearly indicative of additional cellular pre-stress. Utilizing formin inhibitor (SMIFH2), we showed that formin-mediated actin polymerization was critical for the SF response to EzrInh while it also prevented the subsequent EzrInh-treatment from enhancing cell rupture rates. Together EzrInh reduced mechano-protection in cells by tilting of balance of actin’s organization from the cortex to SF enhancing formin-mediated SF assembly. Highlights ### Competing Interest Statement The authors have declared no competing interest.

Plasmacytoid dendritic cells (pDCs) are specialized innate immune cells which play a pivotal role in antiviral immunity by producing large quantities of type I interferons (IFNs) upon sensing nucleic acids via Toll-like receptor 9 (TLR9). Synthetic oligodeoxynucleotides (ODNs) such as CpGA and CpGB, both containing unmethylated CpG motifs, are commonly used experimental TLR9 agonists. Interestingly, CpGA and CpGB elicit markedly different responses in pDCs – CpGA induces robust IFN-α production, whereas CpGB does not. The mechanistic basis underlying this ligand-specific functional divergence has remained unclear. Here, we identify PIEZO1, a mechanosensory ion channel, as a critical determinant of ligand-specific IFN responses in human primary pDCs. We demonstrate that unlike CpGB, CpGA self-associates into larger aggregates that generate membrane tension during cellular uptake, leading to activation of PIEZO1. This activation triggers localized calcium influx and cytoskeletal remodeling, resulting in the formation of local F-actin structures that retain CpGA within early endosomes enabling sustained IRF7 nuclear translocation and robust type I IFN production. Disruption of PIEZO1 or actin polymerization abrogates CpGA-induced IFN production, while pharmacological activation of Piezo1 enhances IFN production in response to CpGB. Thus, these findings uncover a previously unrecognized biophysical checkpoint in nucleic acid sensing, wherein membrane tension is transduced via PIEZO1 into spatially controlled TLR9 signaling. Overall, our study establishes PIEZO1 mechanosensing at the plasma membrane as a key regulatory event in nucleic acid-induced immunity, with different functional outcomes based on the cargo structure, opening potential new avenues for modulating type I IFN responses in infection and autoimmunity. ### Competing Interest Statement The authors have declared no competing interest. Council of Scientific and Industrial Research, https://ror.org/021wm7p51, FBR MLP140 to D.G. Department of Science and Technology, https://ror.org/0101xrq71, Swarnajayanti Fellowship (DST/SJF/LSA-03/2016-17) to D.G. Wellcome Trust/DBT India Alliance, https://ror.org/04reqzt68, India Alliance Internediate Fellowship (IA/I/13/1/500885) to B.S. Science and Engineering Research Board, SERB\_CRG\_2336 to B.S.

The functions of the plasma membrane of a cell are coupled to its mechanical state. To understand the players that contribute to the tight regulation of membrane mechanics with its local heterogeneities, the tension of the cell membrane requires to be mapped out from the basal to the apical membrane. In this work, optical-trap-based tension measurements performed at two axial (z) planes per cell reveal an apico-basal gradient, that is understood better on comparison with basal membrane mechanics and membrane compaction measurements by a combination of high resolution microscopy (IRM) and fluorescence lifetime (Flipper-TR) measurements, respectively. While the apico-basal gradient in the apparent tension (low to high) was found to depend on an intact actin cytoskeleton, analysing the colocalization of the cortical actin with the motor protein myosin and the actin-membrane linker ezrin suggested that cytoskeleton contractility is a major determinant of the patterned tension. Tension-sensitive lifetime of Flipper-TR, validated the gradient but failed to catch the dependence on actin due to the dual and opposite effects of tension on lifetime. Finally, measurements utilizing fluorescently tagged transferrin (Tf) demonstrated that the functional state of the membrane also showed similar height dependence. Thus, planes where tension maximized showed a peak in accumulation of Tf colocalizing with clathrin indicative of the presence of more pits. This enabled us to demonstrate that cells exist in tightly regulated patterned tension states that is coupled to their cytoskeleton as well membrane functions like endocytosis. ### Competing Interest Statement The authors have declared no competing interest.

We describe a noncanonical, membrane receptor-like regulation of the human copper transporter-1 (CTR1) in response to copper stimuli. CTR1 is the sole high-affinity trimeric plasma-membrane copper-importing channel that self-regulates by undergoing endocytosis to limit copper uptake. We observed that preceding copper-induced endocytosis, CTR1 forms clusters on the plasma membrane, a phenomenon that is typically observed in membrane receptors. We deciphered the mechanism of CTR1 clustering and studied its ramifications on the physical properties of plasma membranes harboring these clusters that could favor endocytosis. Membrane tension and fluctuation are fundamental regulators of pre- and post-endocytic events. Using coarse-grain molecular dynamics (MD)-simulations and coupled interference reflection microscopy-total internal reflection fluorescence microscopy (TIRF) we demonstrated that CTR1 clusters induce positive membrane curvature, an increase in local membrane tension, and a decrease in local membrane fluctuation; alterations that favor the formation of endocytic pits. Clustering is facilitated by copper-sequestering methionine-rich extracellular amino-terminus of CTR1. MD-simulations and IRM-TIRF imaging revealed that CTR1 clustering is facilitated by membrane cholesterol, depletion of which delays CTR1 endocytosis. CTR1 clustering promotes clathrin-coated pit formation that engages recruitment of adaptor protein AP-2. To summarize, we report a hitherto unknown “pre-endocytic” “receptor-like” phenomenon of ligand-induced clustering of a metal channel, which in turn regulates self-endocytosis by modulating membrane properties.

Tumor Necrosis Factor Receptor 1 (TNFR1) signaling determines cell fate during inflammation, immunopathogenesis, and tumorigenesis. TNFR1 proteins homo-oligomerize into clusters on the plasma membrane. The potential impact of TNFR1 clustering on downstream signaling remains unexplored. Homo-FRET measurements elucidate that alterations in intra-cluster receptor density (IRD) dictate the outcomes of downstream TNFR1 signaling. Soluble TNF-α (sTNF-α) elevates IRD within the TNFR1 clusters core while diminishing it in the rim, through intra-cluster dynamic reorganization of TNFR1. Decreasing TNFR1 IRD through increasing membrane tension, administering TNFR1 antagonist zafirlukast, actin depolymerization, or depleting cholesterol impedes sTNF-α-mediated stimulation. Conversely, increasing IRD by reducing membrane tension or exposing cells to 3D gel-like microenvironment induces ligand-independent TNFR1 signaling. These findings suggest a broader applicability of IRD in modulating signaling pathways across other receptor families, offering insights for innovative strategies in TNFR1 signaling modulation. ### Competing Interest Statement The authors have declared no competing interest.

The excessive cosolute densities in the intracellular fluid create a physicochemical condition called macromolecular crowding (MMC). Intracellular MMC entropically maintains the biochemical thermodynamic equilibria by favoring associative reactions while hindering transport processes. Rapid cell volume shrinkage during extracellular hypertonicity elevates the MMC and disrupts the equilibria, potentially ushering cell death. Consequently, cells actively counter the hypertonic stress through regulatory volume increase (RVI) and restore the MMC homeostasis. Here, we establish fluorescence anisotropy of EGFP as a reliable tool for studying cellular MMC and explore the spatiotemporal dynamics of MMC during cell volume instabilities under multiple conditions. Our studies reveal that the actin cytoskeleton enforces spatially varying MMC levels inside adhered cells. Within cell populations, MMC is uncorrelated with nuclear DNA content but anti-correlated with the cell spread area. Although different cell lines have statistically similar MMC distributions, their responses to extracellular hypertonicity vary. The intensity of the extracellular hypertonicity determines a cell's ability for RVI, which correlates with nuclear factor kappa beta (NFkB) activation. Pharmacological inhibition and knockdown experiments reveal that tumor necrosis factor receptor 1 (TNFR1) initiates the hypertonicity-induced NFkB signaling and RVI. At severe hypertonicities, the elevated MMC amplifies cytoplasmic microviscosity and hinders receptor interacting protein kinase 1 (RIPK1) recruitment at the TNFR1 complex, incapacitating the TNFR1-NFkB signaling and consequently, RVI. Together, our studies unveil the involvement of TNFR1-NFkB signaling in modulating RVI and demonstrate the pivotal role of MMC in determining cellular osmoadaptability.

Biological cells sample their surrounding microenvironments using nanoscale force sensors on the cell surfaces. These surface-based force and stress sensors generate physical and chemical responses inside the cell. The inherently well-connected cytoskeleton and its physical contacts with the force elements on the nuclear membrane lead these physicochemical responses to cascade all the way inside the cell nucleus, physically altering the nuclear state. These physical alterations of the cell nucleus, through yet-unknown complex steps elicit physical and functional response from the chromatin in the form of altered gene expression profiles. This mechanism of force/stress sensing by the cell and then its nuclear response has been shown to play a vital role in maintaining robust cellular homeostasis, controlling gene expression profiles during developmental phases as well as cell differentiation. Over the last few years, there has been appreciable progress toward identification of the molecular players responsible for force sensing. However, the actual sensing mechanism of cell surface bound force sensors and more importantly cascading of the signals, both physical (via cytosolic force sensing elements such as microtubule and actin framework) and chemical (cascade of biochemical signaling from cell surface to nuclear surface and further to the chromatin), inside the cell is poorly understood. In this chapter, we present a review of the currently known molecular players in cellular as well as nuclear force sensing repertoire and their possible mechanistic aspects. We also introduce various biophysical concepts that are used to describe the force/stress sensing and response of a cell. We hope this will help asking clearer questions and designing pointed experiments for better understanding of the force-dependent design principles of the cell surface, nuclear surface, and gene expression.

Cells inhabit a mechanical microenvironment that they continuously sense and adapt to. The plasma membrane (PM), serving as the boundary of the cell, plays a pivotal role in this process of adaptation. In this Review, we begin by examining well-studied processes where mechanoregulation proves significant. Specifically, we highlight examples from the immune system and stem cells, besides discussing processes involving fibroblasts and other cell types. Subsequently, we discuss the common molecular players that facilitate the sensing of the mechanical signal and transform it into a chemical response covering integrins YAP/TAZ and Piezo. We then review how this understanding of molecular elements is leveraged in drug discovery and tissue engineering alongside a discussion of the methodologies used to measure mechanical properties. Focusing on the processes of endocytosis, we discuss how cells may respond to altered membrane mechanics using endo- and exocytosis. Through the process of depleting/adding the membrane area, these could also impact membrane mechanics. We compare pathways from studies illustrating the involvement of endocytosis in mechanoregulation, including clathrin-mediated endocytosis (CME) and the CLIC/GEEC (CG) pathway as central examples. Lastly, we review studies on cell-cell fusion during myogenesis, the mechanical integrity of muscle fibers, and the reported and anticipated roles of various molecular players and processes like endocytosis, thereby emphasizing the significance of mechanoregulation at the PM.

We use the short-time inference scheme [Manikandan , ], obtained within the framework of stochastic thermodynamics, to infer a lower bound to entropy production rate from flickering data generated by interference reflection microscopy of HeLa cells. We can clearly distinguish active cell membranes from their adenosine-triphosphate-depleted selves and even spatiotemporally resolve activity down to the scale of about 1 µm. Our estimate of activity is . Published by the American Physical Society 2024

Copper, an essential micronutrient, plays crucial role in major physiological processes. However, excess copper is cytotoxic. We explored the mechanism of copper import by the mammalian high-affinity Copper Transporter CTR1. A bona fide ion channel CTR1, interestingly, behaves as a plasma membrane receptor as it endocytoses upon binding copper. In mammalian cells, CTR1 primarily localizes on plasma membrane(PM) and forms functional homotrimeric channel to mobilize copper. At higher extracellular copper, CTR1 is endocytosed as a self-regulatory mechanism to limit copper uptake.

Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms, including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis are poorly understood. To investigate this, we studied the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes, as well as transferrin-labelled pits close to the basal plasma membrane, also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked, but knocking down AP2-dependent pit formation stopped the tension recovery. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested by depleting both. The data strongly supports Clathrin and AP2-dependent pit-formation to be central to the reduction in fluctuations confirmed by super-resolution microscopy. Furthermore, we propose that cholesterol-dependent pits spontaneously regulate tension under ATP-depleted conditions.

T cells are crucial for efficient antigen-specific immune responses and thus their migration within the body, to inflamed tissues from circulating blood or to secondary lymphoid organs, plays a very critical role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such mechanosensing in cell migration are not established. We explored if the professional mechanosensor Piezo1 plays any role during integrin-dependent chemotaxis of human T cells. We found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on and follows focal adhesion formation at the leading edge and local increase in membrane tension upon chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and calpain activation, in turn, are crucial for the integrin LFA1 (CD11a/CD18) recruitment at the leading edge of the chemotactic human T cells. Thus, we find that Piezo1 activation in response to local mechanical cues constitutes a membrane-intrinsic component of the 'outside-in' signaling in human T cells, migrating in response to chemokines, that mediates integrin recruitment to the leading edge.

Cell migration is vital for multiple physiological functions and is involved in the metastatic dissemination of tumour cells in various cancers. For effective directional migration, cells often reorient their Golgi apparatus and, therefore, the secretory traffic towards the leading edge. However, not much is understood about the regulation of Golgi's reorientation. Herein, we address the role of gap junction protein Connexin 43 (Cx43), which connects cells, allowing the direct exchange of molecules. We utilized HeLa WT cells lacking Cx43 and HeLa 43 cells, stably expressing Cx43, and found that functional Cx43 channels affected Golgi morphology and reduced the reorientation of Golgi during cell migration. Although the migration velocity of the front was reduced in HeLa 43, the front displayed enhanced coherence in movement, implying an augmented collective nature of migration. On BFA treatment, Golgi was dispersed and the high heterogeneity in inter-regional front velocity of HeLa WT cells was reduced to resemble the HeLa 43. HeLa 43 had higher vimentin expression and stronger basal F-actin. Furthermore, non-invasive measurement of basal membrane height fluctuations revealed a lower membrane tension. We, therefore, propose that reorientation of Golgi is not the major determinant of migration in the presence of Cx43, which induces collective-like coherent migration in cells.

20 T cells are crucial for efficient antigen-specific immune responses and thus their migration within the 21 body, to inflamed tissues from circulating blood or to secondary lymphoid organs, play a very critical 22 role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between 23 endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse 24 external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such 25 mechanosensing in cell migration are not established. We explored if the professional 26 mechanosensor Piezo1 play any role during integrin-dependent chemotaxis of human T cells. We 27 found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility 28 on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on 29 and follows focal adhesion formation at the leading edge and local increase in membrane tension on 30 chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and 31 calpain activation, in turn are crucial for the integrin LFA-1 recruitment at the leading edge of the 32 chemotactic human T cells. Thus we find that Piezo1 activation in response to local mechanical cues 33 constitutes a membrane-intrinsic component of the ‘outside-in’ signaling in human T cells, migrating 34 in response to chemokines, that mediates integrin recruitment to the leading edge. 35