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Systemic capillary leak syndrome (SCLS) is a rare life-threatening disorder due to profound vascular leak. The trigger and the cause of the disease is currently unknown and there is no specific treatment. Here, we identified a rare heterozygous splice-site variant in theTLN1gene in a familial SCLS case, suggestive of autosomal dominant inheritance with incomplete penetrance. Talin1 has a key role in cell adhesions by activating and linking integrins to the actin cytoskeleton. This variant causes in-frame skipping of exon 54 and is predicted to affect talin’s c-terminal actin binding site (ABS3). Modelling the SCLS-TLN1variant by mimicking the actin-binding disruption inTLN1heterozygous endothelial cells resulted in disorganized endothelial adherens junctions. Mechanistically, we established that disruption of talin’s ABS3 sequestrates talin’s interacting partner, vinculin, at cell-extracellular matrix adhesions, leading to destabilization of the endothelial barrier. We propose that pathogenic variant inTLN1underlie SCLS, providing insight into the molecular mechanism of the disease which can be explored for future therapeutic interventions.SUMMARYSystemic capillary leak syndrome (SCLS) is a rare potentially lethal disease with unknown etiology and non-specific treatment. Here, we established a heterozygous splice variant of talin1, a key cell adhesion protein, as a genetic link to a familial SCLS case.

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. In this study we analysed mutations in talin-1 reported in the Catalogue of Somatic Mutations in Cancer. A total of 11 talin mutants were selected and expressed in talin-deficient fibroblasts and their functional and structural effects were characterised in detail. An I392N point mutation in the F3 domain caused a three-fold increase in invasion, and enhanced migration compared to wildtype talin. Mutations R1368W and L1539P in the R7 and R8 domains caused increased invasion and proliferation and affected talin-vinculin complexation, but were not linked to changes in their binding affinities with known substrates KANK1 and RIAM measured for isolated talin domains. Lastly, L2509P, a mutation in the dimerisation domain of talin, prevented talin dimer formation, actin recruitment and FAKpTyr397 activation leading to anisotropic cell spreading and loss of random migration. Altogether, this study suggests that cancer derived point mutations in talin-1 can drastically affect cell behaviour and so may contribute to cancer progression.

More than 250 proteins are associated with the formation of integrin adhesion complexes involving a vast number of complex interactions between them. These interactions enable adhesions to serve as dynamic and diverse mechanosignaling centers. Our laboratory focuses on the biochemical and structural study of these interactions to help unpick this complex network. Here, we describe the general pipeline of biochem- ical assays and methods we use. The chapter is split into two sections: (1) protein production and characterization and (2) biochemical assays for the characterization of binding between full-length proteins and/or specific regions of proteins with other proteins, peptides, and phospholipids. The suite of assays we use routinely includes circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy for sample quality assessment, prior to biochemical analysis using NMR, fluorescence polarization (FP), microscale thermophoresis (MST), size-exclusion chromatography multiangle light scattering (SEC-MALS), and pulldown/cosedimentation-based approaches. The results of our analysis feed into in vivo studies that allow for the elucidation of the biological role of each interaction.

More than 250 proteins are associated with the formation of integrin adhesion complexes involving a vast number of complex interactions between them. These interactions enable adhesions to serve as dynamic and diverse mechanosignaling centers. Our laboratory focuses on the biochemical and structural study of these interactions to help unpick this complex network. Here, we describe the general pipeline of biochemical assays and methods we use. The chapter is split into two sections: (1) protein production and characterization and (2) biochemical assays for the characterization of binding between full-length proteins and/or specific regions of proteins with other proteins, peptides, and phospholipids. The suite of assays we use routinely includes circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy for sample quality assessment, prior to biochemical analysis using NMR, fluorescence polarization (FP), microscale thermophoresis (MST), size-exclusion chromatography multiangle light scattering (SEC-MALS), and pulldown/cosedimentation-based approaches. The results of our analysis feed into in vivo studies that allow for the elucidation of the biological role of each interaction.

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 α-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.

Focal adhesions are complex multi-molecular structures that link the actin cytoskeleton to the extracellular matrix through integrin adhesion receptors and play a key role in regulation of many cellular functions. LAR (also known as PTPRF) is a receptor protein tyrosine phosphatase that regulates PDGF signalling and localises to focal adhesions. We have observed that loss of LAR phosphatase activity in mouse embryonic fibroblasts results in reduced numbers of focal adhesions and decreased adhesion to fibronectin. To understand how LAR regulates cell adhesion we used phosphoproteomic data, comparing global phosphorylation events in wild-type and LAR phosphatase-deficient cells, to analyse differential kinase activity. Kinase prediction analysis of LAR-regulated phosphosites identified a node of cytoskeleton- and adhesion-related proteins centred on cyclin-dependent kinase-1 (CDK1). We found that loss of LAR activity resulted in reduced activity of CDK1, and that CDK1 activity was required for LAR-mediated focal adhesion complex formation. We also established that LAR regulates CDK1 activity through c-Abl and Akt family proteins. In summary, we have identified a new role for a receptor protein tyrosine phosphatase in regulating CDK1 activity and hence cell adhesion to the extracellular matrix.