Congying Wu

Cells in highly crowded environments are exposed to fluctuating mechanical forces. While cells can activate the cortical migration machinery to escape from undesirable compressive stress, the consequence to less motile cells and of prolonged extensive confinement is yet to be uncovered. Here, we demonstrate that nuclear deformation generated by axial confinement triggers a specific form of regulated cell death – ferroptosis. We show that axial confinement is sensed by the nucleus and results in Drp1-dependent mitochondrial fragmentation and mitochondrial ROS accumulation. Meanwhile, we detect cPLA2 translocation to mitochondria. These mitochondrial ROS accumulation and arachidonic acid production concertedly lead to lipid peroxidation and evoke ferroptosis. Interestingly, we find in osteoarthritis, a disease intimately associated with mechanical overloading and inflammation, characteristics of confinement-induced ferroptosis including mitochondrial localization of cPLA2 and high ROS. Together, our findings unveil a pivotal role of cell nucleus and mitochondria in linking mechanical confinement with cell death, highlighting the orchestration of Drp1 and cPLA2 in confinement-induced ferroptosis.

We reveal that bleb membranes are enriched in PI(3,4,5)P₃ but depleted in PI(4,5)P₂ compared with non-bleb regions. We find that the Septin-SH3KBP1-PI3K axis establishes differential phosphoinositide distribution, regulates phosphoinositide diffusion, and confers resistance to anoikis in detached tumor cells, providing novel targets to counteract anoikis resistance in cancer.

Cells interact dynamically with the extracellular matrix (ECM), which provides both structural support and biochemical signals that regulate various cellular processes. Among these, the mechanical properties of the ECM, particularly stiffness, play a crucial role in governing cell differentiation, migration, and survival. Recent studies have highlighted the intricate relationship between ECM stiffness and cellular metabolism, influencing key pathways such as glucose, lipid, and amino acid metabolism. This review explores how ECM stiffness modulates these metabolic processes, emphasizing the underlying mechanotransduction mechanisms. In addition, we discuss emerging techniques that enable the investigation of ECM-mediated force sensing and response, providing new insights into the mechanoregulation of metabolism and its implications in disease and therapy.

Genes regulating the finger-like cellular protrusions—filopodia have long been implicated in cancer metastasis. However, depleting the flat lamellipodia but retaining filopodia drastically hampers cell migration on spread surface, obscuring the role of filopodia in cell motility. It has been noticed recently that cells under confinement may employ distinct migratory machineries. However, the regulating factors have mainly been focused on cell blebbing, nuclear deformation and cell rear contractility, without much emphasis on cell protrusions and even less on filopodia. Here, by micropore-based screening, we identified espin as an active regulator for confined migration and that its overexpression was associated with metastasis. In comparison to fascin, espin showed stronger actin bundling in vitro and induced shorter and thicker filopodia in cells. Combining the imaging-compatible microchannels and DNA-based tension probes, we uncovered that espin overexpression induced excessive filopodia at the leading edge and along the sides, exerting force for confined migration. Our results demonstrate an important role for filopodia and the regulating protein—espin in confined cell migration and shed new light on cytoskeletal mechanisms underlying metastasis.

Cell migration towards stiff substrates has been coined as durotaxis and implicated in development, wound healing, and cancer, where complex interplays between immune and non-immune cells are present. Compared to the emerging mechanisms underlying the strongly adhesive mesenchymal durotaxis, little is known about whether immune cells - migrating in amoeboid mode - could follow mechanical cues. Here, we develop an imaging-based confined migration device with a stiffness gradient. By tracking live cell trajectory and analyzing the directionality of T cells and neutrophils, we observe that amoeboid cells can durotax. We further delineate the underlying mechanism to involve non-muscle myosin IIA (NMIIA) polarization towards the soft-matrix-side but may not require differential actin flow up- or down-stiffness gradient. Using the protista Dictyostelium, we demonstrate the evolutionary conservation of amoeboid durotaxis. Finally, these experimental phenomena are theoretically captured by an active gel model capable of mechanosensing. Collectively, these results may shed new lights on immune surveillance and recently identified confined migration of cancer cells, within the mechanically inhomogeneous tumor microenvironment or the inflamed fibrotic tissues.

Actin homeostasis is fundamental for cell structure and consumes a large portion of cellular ATP. It has been documented in the literature that certain glycolytic enzymes can interact with actin, indicating an intricate interplay between the cytoskeleton and cellular metabolism. Here we report that hyperosmotic stress triggers actin severing and subsequent phase separation of the actin-binding protein tropomyosin 4 (TPM4). TPM4 condensates recruit glycolytic enzymes such as HK2, PFKM, and PKM2, while wetting actin filaments. Notably, the condensates of TPM4 and glycolytic enzymes are enriched of NADH and ATP, suggestive of their functional importance in cell metabolism. At cellular level, actin filament assembly is enhanced upon hyperosmotic stress and TPM4 condensation, while depletion of TPM4 impairs osmolarity-induced actin reorganization. At tissue level, colocalized condensates of TPM4 and glycolytic enzymes are observed in renal tissues subjected to hyperosmotic stress. Together, our findings suggest that stress-induced actin perturbation may act on TPM4 to organize glycolytic hubs that tether energy production to cytoskeletal reorganization.

Cell migration towards stiff substrates has been coined as durotaxis and implicated in development, wound healing and cancer, where complex interplays between immune and non-immune cells are present. Compared to the emerging mechanisms underlying the strongly adhesive mesenchymal durotaxis, little is known about whether immune cells - migrating in amoeboid mode - could follow mechanical cues. Here we develop an imaging-based confined migration device with stiffness gradient. By tracking live cell trajectory and analyzing the directionality of T cells and neutrophils, we observe that amoeboid cells can durotax. We further delineate the underlying mechanism to involve non-muscle myosin IIA (NMIIA) polarization towards the soft-matrix-side but may not require differential actin flow up- or down-stiffness gradient. Using the protista Dictyostelium, we demonstrate the evolutionary conservation of amoeboid durotaxis. Finally, these experimental phenomena are theoretically captured by an active gel model capable of mechanosensing. Collectively, these results may shed new lights on immune surveillance and recently identified confined migration of cancer cells, within the mechanically inhomogeneous tumor microenvironment or the inflamed fibrotic tissues.

Actin homeostasis is fundamental for cell structure and consumes a large portion of cellular ATP. It has been documented in the literature that certain glycolytic enzymes can interact with actin, indicating an intricate interplay between the cytoskeleton and cellular metabolism. Here we report that hyperosmotic stress triggers actin severing and subsequent phase separation of the actin-binding protein TPM4. TPM4 condensates glycolytic enzymes such as HK2, PFKM, and PKM2, and adhere to and wrap around actin filaments. Notably, the condensates of TPM4 and glycolytic enzymes are enriched of NADH and ATP, suggestive of their functional importance in cell metabolism. At cellular level, actin filaments assembly is enhanced upon hyperosmotic stress and TPM4 condensation, while depletion of TPM4 impaired osmolarity-induced actin reorganization. At tissue level, co-localized condensates of TPM4 and glycolytic enzymes are observed in renal tissues subjected to hyperosmotic stress. Together, our findings suggest that stress-induced actin perturbation may act on TPM4 to organize glycolytic hubs that tether energy production to cytoskeletal reorganization. Graphical

Cells rely on substrate adhesion to activate diverse signaling pathways essential for survival. In the absence of proper adhesion, cells undergo anoikis, a form of programmed cell death. Poorly attached cells often exhibit rounded morphologies and form small round protrusions called blebs. While the role of blebs in amoeboid migration is well-documented, recent studies have highlighted their function in anoikis resistance with the help of curvature-recognized proteins. However, little is known about whether the most abundant membrane components-phospholipids function in blebs-facilitated cell survival. Here, we found enriched PI(3,4,5)P3 and diluted PI(4,5)P2 at the bleb membrane, compared to non-bleb plasma membrane regions. Our experimental results showed that both phosphoinositides (PIs) have restricted diffusion pattern between the bleb and non-bleb membranes. Subsequently, we reveal SH3KBP1 to play a crucial role in recruiting PI3K and organizing the Septin-SH3KBP1-PI3K complex at the bleb necks. This process then contributes to differential PIs distribution and anoikis resistance. These novel insights into PIs dynamics elucidate their role in cell blebbing, cancer cell behavior and metastasis, presenting potential targets for new therapeutic interventions.