Machine learning interpretable models of cell mechanics from protein images.

Cellular form and function emerge from complex mechanochemical systems within the cytoplasm. Currently, no systematic strategy exists to infer large-scale physical properties of a cell from its molecular components. This is an obstacle to understanding processes such as cell adhesion and migration. Here, we develop a data-driven modeling pipeline to learn the mechanical behavior of adherent cells. We first train neural networks to predict cellular forces from images of cytoskeletal proteins. Strikingly, experimental images of a single focal adhesion (FA) protein, such as zyxin, are sufficient to predict forces and can generalize to unseen biological regimes. Using this observation, we develop two approaches-one constrained by physics and the other agnostic-to construct data-driven continuum models of cellular forces. Both reveal how cellular forces are encoded by two distinct length scales. Beyond adherent cell mechanics, our work serves as a case study for integrating neural networks into predictive models for cell biology.

DNA-based ForceChrono probes for deciphering single-molecule force dynamics in living cells.

Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.

Organization, dynamics and mechanoregulation of integrin-mediated cell-ECM adhesions.

The ability of animal cells to sense, adhere to and remodel their local extracellular matrix (ECM) is central to control of cell shape, mechanical responsiveness, motility and signalling, and hence to development, tissue formation, wound healing and the immune response. Cell-ECM interactions occur at various specialized, multi-protein adhesion complexes that serve to physically link the ECM to the cytoskeleton and the intracellular signalling apparatus. This occurs predominantly via clustered transmembrane receptors of the integrin family. Here we review how the interplay of mechanical forces, biochemical signalling and molecular self-organization determines the composition, organization, mechanosensitivity and dynamics of these adhesions. Progress in the identification of core multi-protein modules within the adhesions and characterization of rearrangements of their components in response to force, together with advanced imaging approaches, has improved understanding of adhesion maturation and turnover and the relationships between adhesion structures and functions. Perturbations of adhesion contribute to a broad range of diseases and to age-related dysfunction, thus an improved understanding of their molecular nature may facilitate therapeutic intervention in these conditions.

The Architecture of Talin1 Reveals an Autoinhibition Mechanism.

Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.

Focal Adhesion-Independent Cell Migration.

Cell migration is central to a multitude of physiological processes, including embryonic development, immune surveillance, and wound healing, and deregulated migration is key to cancer dissemination. Decades of investigations have uncovered many of the molecular and physical mechanisms underlying cell migration. Together with protrusion extension and cell body retraction, adhesion to the substrate via specific focal adhesion points has long been considered an essential step in cell migration. Although this is true for cells moving on two-dimensional substrates, recent studies have demonstrated that focal adhesions are not required for cells moving in three dimensions, in which confinement is sufficient to maintain a cell in contact with its substrate. Here, we review the investigations that have led to challenging the requirement of specific adhesions for migration, discuss the physical mechanisms proposed for cell body translocation during focal adhesion-independent migration, and highlight the remaining open questions for the future.

Focal adhesions are controlled by microtubules through local contractility regulation.

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

The focal adhesion protein talin is a mechanically gated A-kinase anchoring protein.

Protein kinase A (PKA) is a ubiquitous, promiscuous kinase whose activity is specified through subcellular localization mediated by A-kinase anchoring proteins (AKAPs). PKA has complex roles as both an effector and a regulator of integrin-mediated cell adhesion to extracellular matrix (ECM). Recent observations demonstrate that PKA is an active component of focal adhesions (FA), suggesting the existence of one or more FA AKAPs. Using a promiscuous biotin ligase fused to PKA type-IIα regulatory (RIIα) subunits and subcellular fractionation, we identify the archetypal FA protein talin1 as an AKAP. Talin is a large, mechanosensitive scaffold that directly links integrins to actin filaments and promotes FA assembly by recruiting additional components in a force-dependent manner. The rod region of talin1 consists of 62 α-helices bundled into 13 rod domains, R1 to R13. Direct binding assays and NMR spectroscopy identify helix41 in the R9 subdomain of talin as the PKA binding site. PKA binding to helix41 requires unfolding of the R9 domain, which requires the linker region between R9 and R10. Experiments with single molecules and in cells manipulated to alter actomyosin contractility demonstrate that the PKA-talin interaction is regulated by mechanical force across the talin molecule. Finally, talin mutations that disrupt PKA binding also decrease levels of total and phosphorylated PKA RII subunits as well as phosphorylation of VASP, a known PKA substrate, within FA. These observations identify a mechanically gated anchoring protein for PKA, a force-dependent binding partner for talin1, and a potential pathway for adhesion-associated mechanotransduction.

S100A11 promotes focal adhesion disassembly via myosin II-driven contractility and Piezo1-mediated Ca2+ entry.

S100A11 is a small Ca2+-activatable protein known to localize along stress fibers (SFs). Analyzing S100A11 localization in HeLa and U2OS cells further revealed S100A11 enrichment at focal adhesions (FAs). Strikingly, S100A11 levels at FAs increased sharply, yet transiently, just before FA disassembly. Elevating intracellular Ca2+ levels with ionomycin stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NMII) inhibitor blebbistatin or with an inhibitor of the stretch-activatable Ca2+ channel Piezo1 suppressed S100A11 recruitment, implicating S100A11 in an actomyosin-driven FA recruitment mechanism involving Piezo1-dependent Ca2+ influx. Applying external forces on peripheral FAs likewise recruited S100A11 to FAs even if NMII activity was inhibited, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function were indispensable, indicating that NMII contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. S100A11-knockout cells display enlarged FAs and had delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel function for S100A11 in promoting actomyosin contractility-driven FA disassembly.

Caskin2 is a novel talin- and Abi1-binding protein that promotes cell motility.

Talin (herein referring collectively to talin 1 and 2) couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE regulatory complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2-Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in cortical microtubule stabilization complexes. Taken together, our results identify Caskin2 as a novel talin-binding protein that might not only connect integrin-mediated adhesion to actin polymerization but could also play a role in crosstalk between integrins and microtubules.

DLC1 promotes mechanotransductive feedback for YAP via RhoGAP-mediated focal adhesion turnover.

Angiogenesis is a tightly controlled dynamic process demanding a delicate equilibrium between pro-angiogenic signals and factors that promote vascular stability. The spatiotemporal activation of the transcriptional co-factors YAP (herein referring to YAP1) and TAZ (also known WWTR1), collectively denoted YAP/TAZ, is crucial to allow for efficient collective endothelial migration in angiogenesis. The focal adhesion protein deleted-in-liver-cancer-1 (DLC1) was recently described as a transcriptional downstream target of YAP/TAZ in endothelial cells. In this study, we uncover a negative feedback loop between DLC1 expression and YAP activity during collective migration and sprouting angiogenesis. In particular, our study demonstrates that signaling via the RhoGAP domain of DLC1 reduces nuclear localization of YAP and its transcriptional activity. Moreover, the RhoGAP activity of DLC1 is essential for YAP-mediated cellular processes, including the regulation of focal adhesion turnover, traction forces, and sprouting angiogenesis. We show that DLC1 restricts intracellular cytoskeletal tension by inhibiting Rho signaling at the basal adhesion plane, consequently reducing nuclear YAP localization. Collectively, these findings underscore the significance of DLC1 expression levels and its function in mitigating intracellular tension as a pivotal mechanotransductive feedback mechanism that finely tunes YAP activity throughout the process of sprouting angiogenesis.

c-Src-induced vascular malformations require localised matrix degradation at focal adhesions.

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

Phase transition of tensin-1 during the focal adhesion disassembly and cell division.

Biomolecular condensates are nonmembranous structures that are mainly formed through liquid-liquid phase separation. Tensins are focal adhesion (FA) proteins linking the actin cytoskeleton to integrin receptors. Here, we report that GFP-tagged tensin-1 (TNS1) proteins phase-separate to form biomolecular condensates in cells. Live-cell imaging showed that new TNS1 condensates are budding from the disassembling ends of FAs, and the presence of these condensates is cell cycle dependent. TNS1 condensates dissolve immediately prior to mitosis and rapidly reappear while postmitotic daughter cells establish new FAs. TNS1 condensates contain selected FA proteins and signaling molecules such as pT308Akt but not pS473Akt, suggesting previously unknown roles of TNS1 condensates in disassembling FAs, as the storage of core FA components and the signaling intermediates.

Testis-specific serine kinase 6 (TSSK6) is abnormally expressed in colorectal cancer and promotes oncogenic behaviors.

Cancer testis antigens (CTAs) are a collection of proteins whose expression is normally restricted to the gamete but abnormally activated in a wide variety of tumors. The CTA, Testis-specific serine kinase 6 (TSSK6), is essential for male fertility in mice. The functional relevance of TSSK6 to cancer, if any, has not previously been investigated. Here we find that TSSK6 is frequently anomalously expressed in colorectal cancer and patients with elevated TSSK6 expression have reduced relapse-free survival. Depletion of TSSK6 from colorectal cancer cells attenuates anchorage-independent growth, invasion, and growth in vivo. Conversely, overexpression of TSSK6 enhances anchorage independence and invasion in vitro as well as in vivo tumor growth. Notably, ectopic expression of TSSK6 in semi-transformed human colonic epithelial cells is sufficient to confer anchorage independence and enhance invasion. In somatic cells, TSSK6 co-localizes with and enhances the formation of paxillin and tensin-positive foci at the cell periphery, suggesting a function in focal adhesion formation. Importantly, TSSK6 kinase activity is essential to induce these tumorigenic behaviors. Our findings establish that TSSK6 exhibits oncogenic activity when abnormally expressed in colorectal cancer cells. Thus, TSSK6 is a previously unrecognized intervention target for therapy, which could exhibit an exceptionally broad therapeutic window.

The plasma membrane of focal adhesions has a high content of cholesterol and phosphatidylcholine with saturated acyl chains.

Cellular functions, such as differentiation and migration, are regulated by the extracellular microenvironment, including the extracellular matrix (ECM). Cells adhere to ECM through focal adhesions (FAs) and sense the surrounding microenvironments. Although FA proteins have been actively investigated, little is known about the lipids in the plasma membrane at FAs. In this study, we examine the lipid composition at FAs with imaging and biochemical approaches. Using the cholesterol-specific probe D4 with total internal reflection fluorescence microscopy and super-resolution microscopy, we show an enrichment of cholesterol at FAs simultaneously with FA assembly. Furthermore, we establish a method to isolate the lipid from FA-rich fractions, and biochemical quantification of the lipids reveals that there is a higher content of cholesterol and phosphatidylcholine with saturated fatty acid chains in the lipids of the FA-rich fraction than in either the plasma membrane fraction or the whole-cell membrane. These results demonstrate that plasma membrane at FAs has a locally distinct lipid composition compared to the bulk plasma membrane.

Vinculin Y822 is an important determinant of ligand binding.

Vinculin is an actin-binding protein present at cell-matrix and cell-cell adhesions, which plays a critical role in bearing force experienced by cells and dissipating it onto the cytoskeleton. Recently, we identified a key tyrosine residue, Y822, whose phosphorylation plays a critical role in force transmission at cell-cell adhesions. The role of Y822 in human cancer remains unknown, even though Y822 is mutated to Y822C in uterine cancers. Here, we investigated the effect of this amino acid substitution and that of a phosphodeficient Y822F vinculin in cancer cells. We observed that the presence of the Y822C mutation led to cells that proliferate and migrate more rapidly and contained smaller focal adhesions when compared to cells with wild-type vinculin. In contrast, the presence of the Y822F mutation led to highly spread cells with larger focal adhesions and increased contractility. Furthermore, we provide evidence that Y822C vinculin forms a disulfide bond with paxillin, accounting for some of the elevated phosphorylated paxillin recruitment. Taken together, these data suggest that vinculin Y822 modulates the recruitment of ligands.

Focal adhesion-mediated cell anchoring and migration: from in vitro to in vivo.

Cell-extracellular matrix interactions have been studied extensively using cells cultured in vitro. These studies indicate that focal adhesion (FA)-based cell-extracellular matrix interactions are essential for cell anchoring and cell migration. Whether FAs play a similarly important role in vivo is less clear. Here, we summarize the formation and function of FAs in cultured cells and review how FAs transmit and sense force in vitro. Using examples from animal studies, we also describe the role of FAs in cell anchoring during morphogenetic movements and cell migration in vivo. Finally, we conclude by discussing similarities and differences in how FAs function in vitro and in vivo.

Dynamically regulated focal adhesions coordinate endothelial cell remodelling in developing vasculature.

The assembly of a mature vascular network involves coordinated endothelial cell (EC) shape changes, including the process of EC elongation. How EC elongation is dynamically regulated in vivo is not fully understood. Here, we have generated a zebrafish mutant that is deficient for the integrin adaptor protein Talin 1 (Tln1). Using a new focal adhesion (FA) marker line expressing endothelial Vinculinb-eGFP, we demonstrate that EC FAs function dynamically and are lost in our tln1 mutants, allowing us to uncouple the primary roles of FAs in EC morphogenesis from the secondary effects that occur due to systemic vessel failure or loss of blood flow. Tln1 loss led to compromised F-actin rearrangements, perturbed EC elongation and disrupted cell-cell junction linearisation in vessel remodelling. Finally, chemical induction of actin polymerisation restored actin dynamics and EC elongation during vascular morphogenesis. Together, we identify that FAs are essential for EC elongation and junction linearisation in flow-pressured vessels and that they influence actin polymerisation in cellular morphogenesis. These observations can explain the severely compromised vessel beds and vascular leakage observed in mutant models that lack integrin signalling. This article has an associated 'The people behind the papers' interview.

Polarized focal adhesion kinase activity within a focal adhesion during cell migration.

Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.

Wnt5a-Vangl1/2 signaling regulates the position and direction of lung branching through the cytoskeleton and focal adhesions.

Lung branching morphogenesis requires reciprocal interactions between the epithelium and mesenchyme. How the lung branches are generated at a defined location and projected toward a specific direction remains a major unresolved issue. In this study, we investigated the function of Wnt signaling in lung branching in mice. We discovered that Wnt5a in both the epithelium and the mesenchyme plays an essential role in controlling the position and direction of lung branching. The Wnt5a signal is mediated by Vangl1/2 to trigger a cascade of noncanonical or planar cell polarity (PCP) signaling. In response to noncanonical Wnt signaling, lung cells undergo cytoskeletal reorganization and change focal adhesions. Perturbed focal adhesions in lung explants are associated with defective branching. Moreover, we observed changes in the shape and orientation of the epithelial sheet and the underlying mesenchymal layer in regions of defective branching in the mutant lungs. Thus, PCP signaling helps define the position and orientation of the lung branches. We propose that mechanical force induced by noncanonical Wnt signaling mediates a coordinated alteration in the shape and orientation of a group of epithelial and mesenchymal cells. These results provide a new framework for understanding the molecular mechanisms by which a stereotypic branching pattern is generated.

Acidity-induced ITGB6 promote migration and invasion of lung cancer cells by epithelial-mesenchymal transition and focal adhesion.

Non-small cell lung cancer (NSCLC) is a prevalent tumor and acidic tumor microenvironment provides an energy source driving tumor progression. We previously demonstrated significantly upregulated Integrin ß6 (ITGB6) in NSCLC cells. This study was designed to investigate the role of ITGB6 in NSCLC metastasis and explore the potential mechanisms. The expression of ITGB6 was evaluated in patients with NSCLC. Migration and invasion assays were utilized to investigate the role of ITGB6, and ChIP-qPCR and dual-luciferase reporter experiments preliminarily analyzed the relationship between ETS proto-oncogene 1 (ETS1) and ITGB6. Bioinformatics analysis and rescue models were performed to explore the underlying mechanisms. The results demonstrated that ITGB6 was upregulated in NSCLC patients and the difference was even more pronounced in patients with poor prognosis. Functionally, acidity-induced ITGB6 promoted migration and invasion of NSCLC cells in vitro, and epithelial-mesenchymal transition (EMT) and focal adhesion were the important mechanisms responsible for ITGB6-involved metastasis. Mechanistically, we revealed ETS1 enriched in the ITGB6 promoter region and promoted transcription to triggered the activation of subsequent signaling pathways. Moreover, ChIP-qPCR and dual-luciferase reporter experiments demonstrated that ETS1 played an important role in directly mediating ITGB6 expression. Furthermore, we found ITGB6 was responsible for the acidic microenvironment-mediated migration and invasion processes in NSCLC by performing rescue experiments with ITGB6 knockdown. Our findings indicated acidic microenvironment directly induced ETS1 to regulate the expression of ITGB6, and then the highly expressed ITGB6 further mediate EMT and activates the downstream focal adhesion pathways, eventually promotes the invasion and migration in NSCLC progression and metastasis.