Proteomic profiling identifies a direct interaction between heat shock transcription factor 2 and the focal adhesion adapter talin-1.

Heat shock factor 2 (HSF2) is a versatile transcription factor that regulates gene expression under stress conditions, during development, and in disease. Despite recent advances in characterizing HSF2-dependent target genes, little is known about the protein networks associated with this transcription factor. In this study, we performed co-immunoprecipitation coupled with mass spectrometry analysis to identify the HSF2 interactome in mouse testes, where HSF2 is required for normal sperm development. Endogenous HSF2 was discovered to form a complex with several adhesion-associated proteins, a finding substantiated by mass spectrometry analysis conducted in human prostate carcinoma PC-3 cells. Notably, this group of proteins included the focal adhesion adapter protein talin-1 (TLN1). Through co-immunoprecipitation and proximity ligation assays, we demonstrate the conservation of the HSF2-TLN1 interaction from mouse to human. Additionally, employing sequence alignment analyses, we uncovered a TLN1-binding motif in the HSF2 C terminus that binds directly to multiple regions of TLN1 in vitro. We provide evidence that the 25 C-terminal amino acids of HSF2, fused to EGFP, are sufficient to establish a protein complex with TLN1 and modify cell-cell adhesion in human cells. Importantly, this TLN1-binding motif is absent in the C-terminus of a closely related HSF family member, HSF1, which does not form a complex with TLN1. These results highlight the unique molecular characteristics of HSF2 in comparison to HSF1. Taken together, our data unveil the protein partners associated with HSF2 in a physiologically relevant context and identifies TLN1 as the first adhesion-related HSF2-interacting partner.

Talin-1 variants associated with spontaneous coronary artery dissection (SCAD) highlight how even subtle changes in multi-functional scaffold proteins can manifest in disease.

Variants of talin-1 (TLN1) have recently been linked with spontaneous coronary artery dissection (SCAD) a condition where a tear can form in the wall of a heart artery necessitating immediate medical care. One talin-1 variant, A2013T, has an extensive familial pedigree of SCAD, which led to the screening for, and identification of, further talin-1 variants in SCAD patients. Here we evaluated these variants with commonly used pathogenicity prediction tools and found it challenging to reliably classify SCAD-associated variants, even A2013T where the evidence of a causal role is strong. Using biochemical and cell biological methods, we show that SCAD-associated variants in talin-1, which would typically be classified as non-pathogenic, still cause a measurable impact on protein structure and cell behaviour, including cell movement and wound healing capacity. Together, this indicates that even subtle variants in central mechanosensitive adapter proteins, can give rise to significant health impacts at the individual level, suggesting the need for a possible re-evaluation of the scoring criteria for pathogenicity prediction for talin variants.

Cellular stiffness sensing through talin 1 in tissue mechanical homeostasis.

Tissue mechanical properties are determined mainly by the extracellular matrix (ECM) and actively maintained by resident cells. Despite its broad importance to biology and medicine, tissue mechanical homeostasis remains poorly understood. To explore cell-mediated control of tissue stiffness, we developed mutations in the mechanosensitive protein talin 1 to alter cellular sensing of ECM. Mutation of a mechanosensitive site between talin 1 rod-domain helix bundles R1 and R2 increased cell spreading and tension exertion on compliant substrates. These mutations promote binding of the ARP2/3 complex subunit ARPC5L, which mediates the change in substrate stiffness sensing. Ascending aortas from mice bearing these mutations showed less fibrillar collagen, reduced axial stiffness, and lower rupture pressure. Together, these results demonstrate that cellular stiffness sensing contributes to ECM mechanics, directly supporting the mechanical homeostasis hypothesis and identifying a mechanosensitive interaction within talin that contributes to this mechanism.

Molecular dynamics simulations reveal how vinculin refolds partially unfolded talin rod helices to stabilize them against mechanical force.

Vinculin binds to specific sites of mechanically unfolded talin rod domains to reinforce the coupling of the cell's exterior to its force generation machinery. Force-dependent vinculin-talin complexation and dissociation was previously observed as contraction or extension of the unfolded talin domains respectively using magnetic tweezers. However, the structural mechanism underlying vinculin recognition of unfolded vinculin binding sites (VBSs) in talin remains unknown. Using molecular dynamics simulations, we demonstrate that a VBS dynamically refolds under force, and that vinculin can recognize and bind to partially unfolded VBS states. Vinculin binding enables refolding of the mechanically strained VBS and stabilizes its folded α-helical conformation, providing resistance against mechanical stress. Together, these results provide an understanding of a recognition mechanism of proteins unfolded by force and insight into the initial moments of how vinculin binds unfolded talin rod domains during the assembly of this mechanosensing meshwork.

TLNRD1 is a CCM complex component and regulates endothelial barrier integrity.

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity.

Alternative molecular mechanisms for force transmission at adherens junctions via ß-catenin-vinculin interaction.

Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing. Here, we demonstrate that, in absence of α-catenin, ß-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that ß-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and ß-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving ß-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin.

Mechanically operated signalling scaffolds.

Cellular signalling is a complex process and involves cascades of enzymes that, in response to a specific signal, give rise to exact cellular responses. Signalling scaffold proteins organise components of these signalling pathways in space and time to co-ordinate signalling outputs. In this review we introduce a new class of mechanically operated signalling scaffolds that are built into the cytoskeletal architecture of the cell. These proteins contain force-dependent binary switch domains that integrate chemical and mechanical signals to introduce quantised positional changes to ligands and persistent alterations in cytoskeletal architecture providing mechanomemory capabilities. We focus on the concept of spatial organisation, and how the cell organises signalling molecules at the plasma membrane in response to specific signals to create order and distinct signalling outputs. The dynamic positioning of molecules using binary switches adds an additional layer of complexity to the idea of scaffolding. The switches can spatiotemporally organise enzymes and substrates dynamically, with the introduction of ∼50 nm quantised steps in distance between them as the switch patterns change. Together these different types of signalling scaffolds and the proteins engaging them, provide a way for an ordering of molecules that extends beyond current views of the cell.

The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion.

Cell adhesion requires linkage of transmembrane receptors to the cytoskeleton through intermediary linker proteins. Integrin-based adhesion to the extracellular matrix (ECM) involves large adhesion complexes that contain multiple cytoskeletal adapters that connect to the actin cytoskeleton. Many of these adapters, including the essential cytoskeletal linker Talin, have been shown to contain multiple actin-binding sites (ABSs) within a single protein. To investigate the possible role of having such a variety of ways of linking integrins to the cytoskeleton, we generated mutations in multiple actin binding sites in Drosophila talin. Using this approach, we have been able to show that different actin-binding sites in talin have both unique and complementary roles in integrin-mediated adhesion. Specifically, mutations in either the C-terminal ABS3 or the centrally located ABS2 result in lethality showing that they have unique and non-redundant function in some contexts. On the other hand, flies simultaneously expressing both the ABS2 and ABS3 mutants exhibit a milder phenotype than either mutant by itself, suggesting overlap in function in other contexts. Detailed phenotypic analysis of ABS mutants elucidated the unique roles of the talin ABSs during embryonic development as well as provided support for the hypothesis that talin acts as a dimer in in vivo contexts. Overall, our work highlights how the ability of adhesion complexes to link to the cytoskeleton in multiple ways provides redundancy, and consequently robustness, but also allows a capacity for functional specialization.

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.

Mechanotransduction through protein stretching.

Cells sense and respond to subtle changes in their physicality, and via a myriad of different mechanosensitive processes, convert these physical cues into chemical and biochemical signals. This process, called mechanotransduction, is possible due to a highly sophisticated machinery within cells. One mechanism by which this can occur is via the stretching of mechanosensitive proteins. Stretching proteins that contain force-dependent regions results in altered geometry and dimensions of the connections, as well as differential spatial organization of signals bound to the stretched protein. The purpose of this mini-review is to discuss some of the intense recent activity in this area of mechanobiology that strives to understand how protein stretching can influence signaling outputs and cellular responses.

Mechanosensing through talin 1 contributes to tissue mechanical homeostasis.

It is widely believed that tissue mechanical properties, determined mainly by the extracellular matrix (ECM), are actively maintained. However, despite its broad importance to biology and medicine, tissue mechanical homeostasis is poorly understood. To explore this hypothesis, we developed mutations in the mechanosensitive protein talin1 that alter cellular sensing of ECM stiffness. Mutation of a novel mechanosensitive site between talin1 rod domain helix bundles 1 and 2 (R1 and R2) shifted cellular stiffness sensing curves, enabling cells to spread and exert tension on compliant substrates. Opening of the R1-R2 interface promotes binding of the ARP2/3 complex subunit ARPC5L, which mediates the altered stiffness sensing. Ascending aortas from mice bearing these mutations show increased compliance, less fibrillar collagen, and rupture at lower pressure. Together, these results demonstrate that cellular stiffness sensing regulates ECM mechanical properties. These data thus directly support the mechanical homeostasis hypothesis and identify a novel mechanosensitive interaction within talin that contributes to this mechanism.

3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.

Cell invasion is a multi-step process, initiated by the acquisition of a migratory phenotype and the ability to move through complex 3D extracellular environments. We determine the composition of cell-matrix adhesion complexes of invasive breast cancer cells in 3D matrices and identify an interaction complex required for invasive migration. ßPix and myosin18A (Myo18A) drive polarized recruitment of non-muscle myosin 2A (NM2A) to adhesion complexes at the tips of protrusions. Actomyosin force engagement then displaces the Git1-ßPix complex from paxillin, establishing a feedback loop for adhesion maturation. We observe active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. The recruitment of NM2A to adhesions creates a non-muscle myosin isoform gradient, which extends from the protrusion to the nucleus. We postulate that this gradient facilitates coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

Force-dependent focal adhesion assembly and disassembly: A computational study.

Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.

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

The cAMP-dependent protein kinase (Protein Kinase A; PKA) is a ubiquitous, promiscuous kinase whose activity is focused and 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 the extracellular matrix (ECM). Recent observations demonstrate that PKA is an active component of focal adhesions (FA), intracellular complexes coupling ECM-bound integrins to the actin cytoskeleton, suggesting the existence of one or more FA AKAPs. Using a combination of 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-R13. Direct binding assays and nuclear magnetic resonance 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. Finally, single-molecule experiments with talin1 and PKA, and experiments in cells manipulated to alter actomyosin contractility demonstrate that the PKA-talin interaction is regulated by mechanical force across the talin molecule. These observations identify the first mechanically-gated anchoring protein for PKA, a new force-dependent binding partner for talin1, and thus a new mechanism for coupling cellular tension and signal transduction.

Next-generation protein-based materials capture and preserve projectiles from supersonic impacts.

Extreme energy-dissipating materials are essential for a range of applications. The military and police force require ballistic armour to ensure the safety of their personnel, while the aerospace industry requires materials that enable the capture, preservation and study of hypervelocity projectiles. However, current industry standards display at least one inherent limitation, such as weight, breathability, stiffness, durability and failure to preserve captured projectiles. To resolve these limitations, we have turned to nature, using proteins that have evolved over millennia to enable effective energy dissipation. Specifically, a recombinant form of the mechanosensitive protein talin was incorporated into a monomeric unit and crosslinked, resulting in a talin shock-absorbing material (TSAM). When subjected to 1.5 km s-1 supersonic shots, TSAMs were shown to absorb the impact and capture and preserve the projectile.

Clutching at Guidance Cues: The Integrin-FAK Axis Steers Axon Outgrowth.

Integrin receptors are essential contributors to neurite outgrowth and axon elongation. Activated integrins engage components of the extracellular matrix, enabling the growth cone to form point contacts, which connect the extracellular substrate to dynamic intracellular protein complexes. These adhesion complexes facilitate efficient growth cone migration and neurite extension. Major signalling pathways mediated by the adhesion complex are instigated by focal adhesion kinase (FAK), whilst axonal guidance molecules present in vivo promote growth cone turning or retraction by local modulation of FAK activity. Activation of FAK is marked by phosphorylation following integrin engagement, and this activity is tightly regulated during neurite outgrowth. FAK inhibition slows neurite outgrowth by reducing point contact turnover; however, mutant FAK constructs with enhanced activity stimulate aberrant outgrowth. Importantly, FAK is a major structural component of maturing adhesion sites, which provide the platform for actin polymerisation to drive leading edge advance. In this review, we discuss the coordinated signalling of integrin receptors and FAK, as well as their role in regulating neurite outgrowth and axon elongation. We also discuss the importance of the integrin-FAK axis in vivo, as integrin expression and activation are key determinants of successful axon regeneration following injury.

Myosin-X recruits lamellipodin to filopodia tips.

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

The MeshCODE to scale-visualising synaptic binary information.

The Mercator projection map of the world provides a useful, but distorted, view of the relative scale of countries. Current cellular models suffer from a similar distortion. Here, we undertook an in-depth structural analysis of the molecular dimensions in the cell's computational machinery, the MeshCODE, that is assembled from a meshwork of binary switches in the scaffolding proteins talin and vinculin. Talin contains a series of force-dependent binary switches and each domain switching state introduces quantised step-changes in talin length on a micrometre scale. The average dendritic spine is 1 µm in diameter so this analysis identifies a plausible Gearbox-like mechanism for dynamic regulation of synaptic function, whereby the positioning of enzymes and substrates relative to each other, mechanically-encoded by the MeshCODE switch patterns, might control synaptic transmission. Based on biophysical rules and experimentally derived distances, this analysis yields a novel perspective on biological digital information.

The mechanical cell - the role of force dependencies in synchronising protein interaction networks.

The role of mechanical signals in the proper functioning of organisms is increasingly recognised, and every cell senses physical forces and responds to them. These forces are generated both from outside the cell or via the sophisticated force-generation machinery of the cell, the cytoskeleton. All regions of the cell are connected via mechanical linkages, enabling the whole cell to function as a mechanical system. In this Review, we define some of the key concepts of how this machinery functions, highlighting the critical requirement for mechanosensory proteins, and conceptualise the coupling of mechanical linkages to mechanochemical switches that enables forces to be converted into biological signals. These mechanical couplings provide a mechanism for how mechanical crosstalk might coordinate the entire cell, its neighbours, extending into whole collections of cells, in tissues and in organs, and ultimately in the coordination and operation of entire organisms. Consequently, many diseases manifest through defects in this machinery, which we map onto schematics of the mechanical linkages within a cell. This mapping approach paves the way for the identification of additional linkages between mechanosignalling pathways and so might identify treatments for diseases, where mechanical connections are affected by mutations or where individual force-regulated components are defective.