Transmembrane signaling receptors, such as integrins, organize as nanoclusters that provide several advantages, including increasing avidity, sensitivity (increasing the signal-to-noise ratio), and robustness (signaling threshold) of the signal in contrast to signaling by single receptors. Furthermore, compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, whether nanoclusters function as signaling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at subdiffraction limited resolution of ∼100 nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in an immobile population of integrin β3 organized as nanoclusters, which (ii) in turn serve to organize nanoclusters of associated key adhesome proteins-vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signaling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct protein dynamics, which is closely correlated to their function in signaling. (iv) Long-lived nanoclusters function as signaling hubs─wherein immobile integrin nanoclusters organize phosphorylated FAK to form stable nanoclusters in close proximity to them, which are disassembled in response to inactivation signal by removal of force and in turn activation of phosphatase PTPN12. (v) Signaling takes place in response to external signals such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. We term these functional nanoclusters as integrin signaling transit and relay nodes (STARnodes). Taken together, these results demonstrate that integrin STARnodes seed signaling downstream of the integrin receptors by organizing hubs of signaling proteins (FAK, paxillin, vinculin) to relay the incoming signal intracellularly and bring about robust function.

Recent studies have shown that mechanical properties such as extracellular matrix stiffness, fluid flow, weight loading, compression, and stretching can affect cellular functions. Some examples of cell responses to mechanical properties could be the migration of cancer cells from rigid to soft surfaces or the differentiation of fibroblasts into myofibroblasts. Cellular responses to mechanical changes can modify the insertion of proteins in the extracellular matrix (ECM), causing an increase in tissue stiffness with functional consequences. In general, mechanical and physical factors can affect any kind of cell phenotype in culture conditions and in vivo tissues. Cells sense mechanical stimuli by applying force and restructuring their shape and functions in response to the resistance of the stimuli. Furthermore, mechanical triggers can develop a “memory” for altering cellular plasticity and adaptation. This phenomenon is called cellular mechanical memory (CMM), a singular feature of mesenchymal stem cells (MSCs). Controlled targeting of CMM may resolve the scarcity of viable cells needed for cell based therapy (CBT) and implement studies concerning cancer research, fibrosis, and senescence. This review focusses on cells from the mesodermal lineage, such as MSCs, fibroblasts and chondrocytes, and the role of CMM as a potential target for CBT.

The risk for developing insulin resistance and type II diabetes increases with age. Although lifestyle factors contribute to age-related insulin resistance, aging itself independently reduces insulin sensitivity, partially via an increase in inflammation and cellular senescence. Low-frequency ultrasound (LFU) has been shown to rejuvenate senescent cells and to reduce the proinflammatory senescence-associated secretory phenotype. Because diabetes is more common in aged individuals, there is an increased need to develop effective therapeutics for aged individuals with this condition. This study investigated the effects of LFU treatment on muscle function, blood glucose control, and skeletal muscle gene expression in aged, insulin-resistant, and diabetic mice. Insulin resistance was induced via a high-fat, high-sucrose (HFHS) diet, and diabetes was induced via an HFHS diet plus a low dose of streptozotocin. Insulin-resistant and diabetic mice exhibited impaired glucose metabolism and physical function, as well as an altered transcriptomic profile in skeletal muscle, indicating an increase in inflammation and an immune response. LFU treatment reversed much of the transcriptomic changes that occurred with insulin resistance and diabetes but had no effect on blood glucose control or physical function. LFU demonstrates potential as a noninvasive therapy for reducing inflammation and altering immune cell function in skeletal muscle in insulin-resistant and diabetic populations.NEW & NOTEWORTHY This study introduces low-frequency ultrasound (LFU) as a novel, noninvasive therapy that attenuates insulin resistance- and diabetes-induced transcriptional changes in aged skeletal muscle. LFU primarily reduced inflammatory and immune-related gene expression, potentially by promoting a shift toward an anti-inflammatory (M2) macrophage profile. These findings suggest that LFU may target underlying inflammatory mechanisms of insulin resistance and diabetes in aging muscle.

The presence of senescent cells causes age-related pathologies since their removal by genetic or pharmacological means, as well as possibly by exercise, improves outcomes in animal models. An alternative to depleting such cells would be to rejuvenate them to promote their return to a replicative state. Here we report that treatment of non-growing senescent cells with low-frequency ultrasound (LFU) rejuvenates the cells for growth. Notably, there are 15 characteristics of senescent cells that are reversed by LFU, including senescence-associated secretory phenotype (SASP) plus decreased cell and organelle motility. There is also inhibition of β-galactosidase, p21, and p16 expression, telomere length is increased, while nuclear 5mC, H3K9me3, γH2AX, nuclear p53, ROS, and mitoSox levels are all restored to normal levels. Mechanistically, LFU causes Ca2+ entry and increased actin dynamics that precede dramatic increases in autophagy and an inhibition of mTORC1 signaling plus movement of Sirtuin1 from the nucleus to the cytoplasm. Repeated LFU treatments enable the expansion of primary cells and stem cells beyond normal replicative limits without altering phenotype. The rejuvenation process is enhanced by co-treatment with cytochalasin D, rapamycin, or Rho kinase inhibition but is inhibited by blocking Sirtuin1 or Piezo1 activity. Optimized LFU treatment parameters increased mouse lifespan and healthspan. These results indicate that mechanically induced pressure waves alone can reverse senescence and aging effects at the cellular and organismal level, providing a non-pharmacological way to treat the effects of aging.

Cancer cells undergo rigidity independent growth due to low expression of mechanosensory cytoskeletal protein known as tropomyosin 2.1, acting as rigidity sensing module. This enables their survival on soft matrices by inciting distinct response to the mechanical cues, in contrast to non-transformed cells. We previously found that treatment of cancer cells with low-frequency ultrasound results in calcium influx through activation of piezo1 channels, microtubule depolymerization through calpain proteases, activation of RhoA-ROCK pathway and increased myosin IIA-mediated actomyosin contractility, ultimately causing cell death in calcium-induced calcium release manner. However, the underlying processes connecting the initiation of apoptosis upon mechanical stimuli remain inadequately understood. Our research focus involves ER-mitochondrial stress pathway comprising of mitochondria-associated ER membranes (MAMs), that might be involved in causing apoptosis in cancer cells exhibiting variations in their mechanoresponse via differential cell killing, while leaving the normal cells unaffected. Recently, we found lowering of mitochondrial membrane potential in cancer cells post-ultrasound treatment. Mitochondrial distribution of normal and cancer cells after ultrasound treatment varied dramatically, leading to increased punctate forms in cancer cells. Inhibition of mitochondrial fission showed significant decrease in apoptosis of cancer cells after ultrasound treatment. We conclude crucial role of mitochondrial fission, ROS and electron transport chain in low-frequency ultrasound-mediated apoptosis in cancer cells along with the cytoskeletal proteins. Tuning of desired parameters involving frequency, pressure and pulse duration could aid in the development of non-invasive cancer therapeutics. Aditi Singh, Felix Margadant, Michael Sheetz. Mechanistic insights into low-frequency ultrasound-induced cancer cell apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1623.

Rigidity sensing enables cells to detect and respond to extracellular matrix stiffness, directing survival and apoptotic decisions. Tropomyosin 2.1 (Tpm2.1), a key actin-binding protein, is essential for this process and is frequently downregulated in cancer. While Tpm2.1 overexpression restores rigidity sensing and suppresses malignant growth, the downstream pathways governing cell fate remain poorly defined. Here, we show that Tpm2.1-mediated rigidity sensing represses anchorage-independent growth by downregulating oncogenic networks, including PI3K–Akt and EphA2 signaling. Unexpectedly, a majority of Tpm2.1-overexpressing cells remained viable in suspension, revealing a previously unrecognized decoupling between rigidity sensing and anoikis. Transcriptomic profiling of apoptotic and non-apoptotic cells in suspension identified compensatory survival programs marked by PI3K–Akt activation and induction of cell–cell adhesion genes. These findings uncover a dominant anoikis-resistant state that persists despite cytoskeletal normalization, suggesting that rigidity sensing alone is insufficient to reestablish anchorage dependence in cancer cells. ### Competing Interest Statement The authors have declared no competing interest. Cancer Prevention and Research Institute of Texas, RR210018 The University of Texas Medical Branch at Galveston, https://ror.org/016tfm930 Council for Higher Education of Israel and Bar-Ilan University

Increased susceptibility of elderly populations to infection is a major concern, as recently experienced by the coronavirus pandemic. Therefore, the treatment of infected elderly individuals is of immediate interest. Here, we report the use of ultrasound treatment to reduce Salmonella infection in elderly individuals. Salmonella enterica serovar Typhimurium enters the human body through the ingestion of contaminated food or water and causes gastroenteritis. The Centers for Disease Control (CDC) reports 420 deaths per year in the United States caused by the Salmonella pathogen, especially in the elderly population. In this study, we investigated the susceptibility to Salmonella infection in young and old mice by enumerating the number of viable bacteria in the liver 3 days post-infection. We then explored the effect of ultrasound treatment (low-frequency ultrasound [LFU]) prior to infecting aged mice with Salmonella. We observed a higher colonization of Salmonella infection in the livers of old mice compared to young mice. Colonization was significantly reduced when old mice were treated with LFU prior to infection, which significantly reduced colonization. Chemokine analysis revealed a significant increase in ICAM-1, SDF-1, and KC/CXCL1 in aged, treated mice. Ultrasound treatment rejuvenated the immune system in old mice, reducing their susceptibility to Salmonella infection. IMPORTANCE:Our study demonstrates the efficacy of low-frequency ultrasound (LFU) treatment in significantly rejuvenating the immune system in aged mice and reducing their susceptibility to Salmonella infection. These findings underscore the potential of LFU as a therapeutic intervention to boost immune function in elderly populations, reducing the risk of infectious diseases.

Nanoscale organization of transmembrane receptors is critical for cellular functions, enabled by the nanoscale engineering of bioligand presentation. Previously, a spatial threshold of ≤60 nm for integrin binding ligands in cell-matrix adhesion is demonstrated using monoliganded gold nanoparticles. However, the ligand geometric arrangement is limited to hexagonal arrays of monoligands, while plasmonic quenching limits further investigation by fluorescence-based high-resolution imaging. Here, these limitations are overcome with dielectric TiO2 nanopatterns, eliminating fluorescence quenching, thus enabling super-resolution fluorescence microscopy on nanopatterns. By dual-color super-resolution imaging, high precision and consistency among nanopatterns, bioligands, and integrin nanoclusters are observed, validating the high quality and integrity of both nanopattern functionalization and passivation. By screening TiO2 nanodiscs with various diameters, an increase in fibroblast cell adhesion, spreading area, and Yes-associated protein (YAP) nuclear localization on 100 nm diameter compared with smaller diameters was observed. Focal adhesion kinase is identified as the regulatory signal. These findings explore the optimal ligand presentation when the minimal requirements are sufficiently fulfilled in the heterogenous extracellular matrix network of isolated binding regions with abundant ligands. Integration of high-fidelity nano-biopatterning with super-resolution imaging allows precise quantitative studies to address early signaling events in response to receptor clustering and their nanoscale organization.

Recent studies show that tumor cells undergo apoptosis after mechanical stretching, which promotes normal cell growth. Since ultrasound can produce similar sub-cellular mechanical stresses on the nanoscale, here we test the effect of ultrasound-mediated mechanical forces on tumors and normal cell survival. Surprisingly, tumor cells undergo apoptosis through a calpain-dependent mitochondrial pathway that relies upon calcium entry through the mechanosensitive Piezo1 channels. This is a general property of all tumor cell lines tested irrespective of tissue origin, but normal cells are unaffected. In vivo, ultrasound treatment promotes tumor cell killing in a mouse model with invasive CT26 cancer cell subcutaneous tumors and in the chick chorioallantoic membrane (CAM) model with relatively minor damage to chick embryos. Further, patient-derived pancreatic tumor organoids are killed by ultrasound treatment. Because ultrasound-mediated mechanical forces cause apoptosis of tumor cells from many different tissues in different microenvironments, it may offer a safe, non-invasive approach to augment tumor treatments.

Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity (increasing the signal-to-noise ratio) and robustness (signalling above a threshold rather than activation by a single receptor) of the signal compared to signalling by single receptors. Compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, if nanoclusters function as signalling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at sub-diffraction limited resolution of ~100nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in immobile population of integrin β3 organised as nanoclusters, which (ii) in turn serve to organise nanoclusters of associated key adhesome proteins- vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signalling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct dynamics dependent on function. (iv) long-lived nanoclusters function as signalling hubs- wherein phosphorylated FAK and paxillin formed stable nanoclusters in close proximity to immobile integrin nanoclusters which are disassembled in response to inactivation signal by phosphatase PTPN12 (v) signalling takes place in response to an external signal such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. Taken together, these results demonstrate that signalling downstream of transmembrane receptors is organised as hubs of signalling proteins (FAK, paxillin, vinculin) seeded by nanoclusters of the transmembrane receptor (integrin).

Cell adhesion receptors are transmembrane proteins that bind cells to their environment. These proteins typically cluster into disk-shaped or linear structures. Here, we show that such clustering patterns spontaneously emerge when the receptor senses the membrane deformation gradient, for example, by reaching a lower-energy conformation when the membrane is tilted relative to the underlying binding substrate. Increasing the strength of the membrane gradient-sensing mechanism first yields isolated disk-shaped clusters and then long linear structures. Our theory is coherent with experimental estimates of the parameters, suggesting that a tilt-induced clustering mechanism is relevant in the context of cell adhesion.

Collective cell migration is crucial in various physiological processes, including wound healing, morphogenesis, and cancer metastasis. Adherens Junctions (AJs) play a pivotal role in regulating cell cohesion and migration dynamics during tissue remodeling. While the role and origin of the junctional mechanical tension at AJs have been extensively studied, the influence of the actin cortex structure and dynamics on junction plasticity remains incompletely understood. Moreover, the mechanisms underlying stress dissipation at junctions are not well elucidated. Here, we found that the ligand-independent phosphorylation of epithelial growth factor receptor (EGFR) downstream of de novo E-cadherin adhesion orchestrates a feedback loop, governing intercellular viscosity via the Rac pathway regulating actin dynamics. Our findings highlight how the E-cadherin-dependent EGFR activity controls the migration mode of collective cell movements independently of intercellular tension. This modulation of effective viscosity coordinates cellular movements within the expanding monolayer, inducing a transition from swirling to laminar flow patterns while maintaining a constant migration front speed. Additionally, we propose a vertex model with adjustable junctional viscosity, capable of replicating all observed cellular flow phenotypes experimentally.

Insulin resistance and type II diabetes become increasingly prevalent with advanced age. Exercise training improves blood glucose control. However, implementing exercise interventions in aged populations is challenging, making alternate treatments important. We previously found that pancreatic senescence can be reduced with low-frequency ultrasound treatment (LFU). The purpose of this study was to determine whether whole-body LFU improves blood glucose control in aged insulin-resistant mice. Twenty-one-month-old C57BL/6j mice were fed a high-fat, high-sucrose (HFHS) or a normal chow (NC) diet for 28 days (n=4-6). Following the initial 14 days of dietary intervention, LFU or sham treatments were administered (30 minutes, 3x/week). The HFHS diet increased body weight (main effect: 41.3%; p<0.0001) and elevated fasting blood glucose and insulin (main effect: 15.6% and 121.3%, respectively; p<0.01). The HFHS diet also impaired glucose tolerance (main effect: 26.6%; p<0.001) measured by blood glucose area under the curve (AUC) during an oral glucose tolerance test (OGTT). Insulin sensitivity was impaired by the HFHS diet, indicated by increased HOMA-IR and reduced QUICKI values (main effect: p<0.001). Interestingly, LFU treatment reduced fasting blood glucose (main effect: -13.2%, p<0.01). Furthermore, LFU mice exhibited increased insulin production, indicated by higher insulin AUC during the OGTT and increased HOMA-β values (main effect: p<0.05). Despite increasing insulin secretion during the OGTT, LFU did not improve blood glucose AUC. No changes occurred in muscle or liver mitochondrial respiratory capacity with HFHS diet or LFU. Overall, these preliminary results suggest that LFU lowers fasting blood glucose and increases insulin secretion. Funding for this work was provided by a Claude D. Pepper Older Americans Independence Center Pilot Project Grant (MS and BR) and a CPRIT Foundation grant (RR180025; MS).

Nano-Biopatterning In article number 2309284 by Haogang Cai, Rishita Changede, and co-workers, a dielectric nano-biopatterning technology is developed, enabling super-resolution fluorescence microscopy that is challenging for conventional plasmonic nanopatterns with quenching effects. Through both simulations and experiments, nanopattern interactions with adjacent fluorescence and surrounding lipid membranes are studied. This technology will facilitate precise quantitative cell biology studies to investigate molecular-scale signaling events in response to receptor clustering and their nanoscale organization. Image credit: Nanzhong Deng, New York University.

This study investigates differences in focal adhesion (FA) morphology and Talin cleavage levels between transformed and non-transformed cell lines. Utilizing fluorescently tagged wild-type Talin and Talin mutants with calpain cleavage site mutations, FA structures were visualized. Mutations in different Talin cleavage sites showed varying impacts on FA morphology and distribution across melanoma cell lines (Meljuso, A375P, A2058) and a non-transformed cell line (HFF). Western blot analysis, ratiometric fluorescence intensity-based measurements, and FRAP experiments revealed higher Talin cleavage levels within FAs of transformed cell lines compared to non-transformed cells. Additionally, growth assays indicated that reducing calpain cleavage levels attenuated transformed cell growth. These findings suggest that Talin cleavage level is crucial for FA morphology and assembly, with higher levels observed in transformed cells, influencing their growth dynamics.

Upon interaction with the extracellular matrix, the integrin receptors form nanoclusters as a first biochemical response to ligand binding. Here, we uncover a critical biodesign principle where these nanoclusters are spatially self-organized, facilitating effective mechanotransduction. Mouse Embryonic Fibroblasts (MEFs) with integrin β3 nanoclusters organized themselves with an intercluster distance of ∼550 nm on uniformly coated fibronectin substrates, leading to larger focal adhesions. We determined that this spatial organization was driven by cell-intrinsic factors since there was no pre-existing pattern on the substrates. Altering this spatial organization using cyclo-RGD functionalized Titanium nanodiscs (of 100 nm, corroborating to the integrin nanocluster size) spaced at intervals of 300 nm (almost half), 600 nm (normal) or 1000 nm (almost double) resulted in abrogation in mechanotransduction, indicating that a new parameter i.e., an optimal intercluster distance is necessary for downstream function. Overexpression of α-actinin, which induces a kink in the integrin tail, disrupted the establishment of the optimal intercluster distance, while simultaneous co-overexpression of talin head with α-actinin rescued it, indicating a concentration-dependent competition, and that cytoplasmic activation of integrin by talin head is required for the optimal intercluster organization. Additionally, talin head-mediated recruitment of FHOD1 that facilitates local actin polymerization at nanoclusters, and actomyosin contractility were also crucial for establishing the optimal intercluster distance and a robust mechanotransduction response. These findings demonstrate that cell-intrinsic machinery plays a vital role in organizing integrin receptor nanoclusters within focal adhesions, encoding essential information for downstream mechanotransduction signalling.

Actomyosin tension has been shown to be a ubiquitous driver of tissue morphogenesis[1][1], [2][2]. The Rho pathway, a prominent regulatory network influencing cortical tension, plays a central role in both tissue reorganisation and cell migration[3][3]–[6][4]. While viscous dissipation in the actin network is commonly regarded as a constant passive parameter in cell migration in both 2D and 3D contexts, there is limited knowledge concerning the regulation of dissipative forces arising from viscous drag between cells during collective rearrangement. Here, we found that the phosphorylation of Epithelial Growth Factor Receptor (EGFR) downstream of de novo E-cadherin adhesion[7][5], [8][6] orchestrates a feedback loop, thereby governing intercellular viscosity via the Rac pathway regulating actin dynamics. Our findings highlight how the E-cadherin-dependent EGFR activity controls the migration mode of collective cell movements independently of intercellular tension. Combining molecular cell biology, micropatterning, and in silico simulation, our work suggests the existence of a regulatory loop by which cells can tune junctional actin viscosity, with implications for the phenomenology of morphogenetic movements.### Competing Interest StatementThe authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-6 [5]: #ref-7 [6]: #ref-8

Cellular senescence is a state of irreversible cell cycle arrest and is one of the hallmarks of aging. Accumulation of senescent cells in various tissues and organs of aged individuals contributes to diseases including cancer, diabetes, cardiovascular, osteoporosis. Cancer is one of the negative outcomes of aging process. In vitro senescent and cancer cells both respond to mechanical stimuli. However, underlies molecular mechanism of onset of skin cancer in aged individual is yet unknown. Here, we address this question by studying the senescence to cancer transition on viscoelastic surfaces. We probed the senesce to cancer transition on surface of viscoelastic, elastic, and plastic surfaces. Senescence fibroblast cells on viscoelastic surface formed cluster increased growth and reduced senescent beta gal staining. We found that Senescent cells also exhibited reduced spread area compared to elastic and plastic surface. Transition of senescent cells to cancer lead by the cross talk between mtor signaling pathway and protein kinase c beta. In conclusion our study shows that change in viscoelastic property of microenvironment cause the accumulated senescent to transform to cancer phenotype.