Biology subjects

Goult, B. T.

Publications and source records attributed to Goult, B. T..

26 records · Page 2Linked to original sources

bioRxiv2022

A subpopulation of Talin 1 resides in the nucleus and regulates gene expression

Talin 1 (TLN1) is best known for its role at focal adhesions, where it activates {beta}-integrin receptors and transmits mechanical stimuli to the actin cytoskeleton. Interestingly, the localization of TLN1 is not restricted to the focal adhesions, but its function in other cellular compartments remains poorly understood. By utilizing both biochemical and confocal microscopy analyses, we show that TLN1 localizes to the nucleus and that it strongly interacts with the chromatin. Importantly, depletion of endogenous TLN1 results in extensive changes in the gene expression profile of human breast epithelial cells. To determine the impact of nuclear TLN1 on gene regulation, we expressed a TLN1 fusion protein containing a nuclear localization signal. Our results revealed that nuclear TLN1 regulates a specific subset of the TLN1-dependent genes. Taken together, we show that apart from localizing at the plasma membrane and cytoplasm, TLN1 also resides in the nucleus where it functions in the regulation of gene expression.

molecular biology↗

bioRxiv2021

Force-dependent interactions between talin and full-length vinculin

Talin and vinculin are part of a multi-component system involved in mechanosensing in cell-matrix adhesions. Both exist in auto-inhibited forms, and activation of vinculin requires binding to mechanically activated talin, yet how forces affect talins interaction with vinculin has not been investigated. Here by quantifying the force-dependent talin-vinculin interactions and kinetics using single-molecule analysis, we show that mechanical exposure of a single vinculin binding site (VBS) in talin is sufficient to relieve the autoinhibition of vinculin resulting in high-affinity binding. We provide evidence that the vinculin undergoes dynamic fluctuations between an auto-inhibited closed conformation and an open conformation that is stabilized upon binding to the VBS. Furthermore, we discover an additional level of regulation in which the mechanically exposed VBS binds vinculin significantly more tightly than the isolated VBS alone. Molecular dynamics simulations reveal the basis of this new regulatory mechanism, identifying a sensitive force-dependent change in the conformation of an exposed VBS that modulates binding. Together, these results provide a comprehensive understanding of how the interplay between force and autoinhibition provides exquisite complexity within this major mechanosensing axis.

biophysics↗

bioRxiv2021

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1

Talin is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behaviour of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronised phosphorylation of a large number of protein targets. CDK1 activity also maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesiveness that are required for successful completion of mitosis. Using a combination of biochemical, structural and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8 through the use of recombinant fragments. Talin also contains a consensus CDK1 phosphorylation motif centred on S1589; a site that was phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of KANK and weakened the mechanical response of the region, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, therefore provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division in multicellular organisms. SummaryThe direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division.

cell biology↗

bioRxiv2020

Talin Rod Domain Containing Protein 1 (TLNRD1) is a novel actin- bundling protein which promotes filopodia formation

Talin is a mechanosensitive adapter protein which couples integrins to the cytoskeleton and regulates integrin-mediated adhesion. Talin rod domain-containing protein-1 (TLNRD1) shares 22% homology with the R7R8 domains of talin, and is highly conserved throughout vertebrate evolution, however little is known about its function. Here we show that TLNRD1 is an -helical protein which shares the same atypical topology as talin R7R8, but forms a novel antiparallel dimer arrangement. Actin co-sedimentation assays and electron microscopy reveal TLNRD1 is an actin-bundling protein that forms tight actin bundles. In addition, TLNRD1 binds to the same LD-motif containing proteins, RIAM and KANK, as talin, and thus may act in competition with talin. Filopodia are cell protrusions supported by tightly bundled actin filaments and TLNRD1 localises to filopodia tips, increases filopodia number and promotes cell migration in 2D. Together our results suggest that TLNRD1 has similar functionality to talin R7R8, serving as a nexus between the actin and microtubule cytoskeletons independent of adhesion complexes.

cell biology↗

bioRxiv2020

Myosin-X is required for integrin activation at filopodia tips

Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin localization and regulation in filopodia are poorly defined. Here, we observed that active integrins accumulate at the tip of myosin-X(MYO10)-positive filopodia while inactive integrins are uniformly distributed. RNAi depletion of 10 integrin activity modulators identified talin as the principal integrin activator in filopodia. Deletion of the MYO10-FERM domain, or mutation of the {beta}1-integrin-binding residues within, revealed MYO10 as facilitating integrin activation but not transport in filopodia. However, MYO10-FERM alone could not activate integrins, potentially due to dual binding to both a- and {beta}-integrin tails. As swapping MYO10-FERM with talin-FERM enabled integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation.

cell biology↗

bioRxiv2020

Cancer associated talin point mutations disorganise cell adhesion and migration

Talin-1 is a key component of the multiprotein adhesion complexes which mediate cell migration, adhesion and integrin signalling and has been linked to cancer in several studies. We analysed talin-1 mutations reported in the COSMIC (Catalogue of Somatic Mutations in Cancer) database and developed a bioinformatics pipeline to predict the severity of each mutation. These predictions were then assessed using biochemistry and cell biology experiments. With this approach we were able to identify several talin-1 mutations affecting integrin activity, actin recruitment and Deleted in Liver Cancer 1 localization. We explored potential changes in talin-1 signalling responses by assessing impact on migration, invasion and proliferation. Altogether, this study describes a pipeline approach of experiments for crude characterization of talin-1 mutants in order to evaluate their functional effects and potential pathogenicity. Our findings suggest that cancer related point mutations in talin-1 can affect cell behaviour and so may contribute to cancer progression.

cancer biology↗

bioRxiv2019

Formation of talin-vinculin pre-complexes dictates maturation of nascent adhesions by accelerated force transmission and vinculin recruitment

Talin, vinculin, and paxillin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). Using machine learning, traction microscopy, single-particle-tracking, and fluorescence fluctuation analysis, we find that talin, vinculin, and paxillin are recruited in near-synchrony to NAs maturing to focal adhesions. After initial recruitment of all three proteins under minimal load, vinculin accumulates in these NAs at a ~5 fold higher rate than in non-maturing NAs and with faster growth in traction. We identify a domain in talin, R8, which exposes a vinculin-binding-site (VBS) without requiring load. Stabilizing this domain via mutation lowers load-free vinculin binding to talin, impairs maturation of NAs, and reduces the rate of additional vinculin recruitment. Taken together, our data show that talins concurrent localization with vinculin, before engagement with integrins, is essential for NA maturation, which entails traction-mediated unfolding of talin and exposure of additional VBSs triggering further vinculin binding.

cell biology↗

bioRxiv2017

ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes

Integrin transmembrane heterodimeric receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. A diverse set of methods have been used to investigate cytoplasmic interactions between integrins and intracellular proteins. These predominantly consist of peptide-based pull-downs and biochemical immuno-isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe a technique called ProLIF (Protein-Liposome Iinenteractions by Flow cytometry) to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments on liposomes as individual or {beta} subunits or as {beta} heterodimers and, using flow cytometry, to rapidly and quantitatively measure protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. By combining integrins with membrane lipids to generate proteoliposomes, the effects of membrane composition such as PI(4,5)P2 presence on protein recruitment to the integrin CTs can be analyzed. ProLIF requires no specific instrumentation, apart from a standard flow cytometer and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.

molecular biology↗