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David R Critchley - One of the best experts on this subject based on the ideXlab platform.
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structural studies on full length Talin1 reveal a compact auto inhibited dimer implications for Talin activation
Journal of Structural Biology, 2013Co-Authors: Benjamin T. Goult, Neil Bate, Igor L. Barsukov, Alexandre R Gingras, David R Critchley, Bipin Patel, Mark F Swift, Petra M Kopp, Niels Volkmann, Dorit HaneinAbstract:Talin is a large adaptor protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain (the head) linked to a flexible rod comprised of 13 amphipathic helical bundles (R1–R13) that terminate in a C-terminal helix (DD) that forms an anti-parallel dimer. We derived a three-dimensional structural model of full-length Talin at a resolution of approximately 2.5 nm using EM reconstruction of full-length Talin and the known shapes of the individual domains and inter-domain angles as derived from small angle X-ray scattering. Talin adopts a compact conformation consistent with a dimer in which the two Talin rods form a donut-shaped structure, with the two Talin heads packed side by side occupying the hole at the center of this donut. In this configuration, the integrin binding site in the head domain and the actin-binding site at the carboxy-terminus of the rod are masked, implying that Talin must unravel before it can support integrin activation and engage the actin cytoskeleton.
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Talin contains a c terminal calpain2 cleavage site important in focal adhesion dynamics
PLOS ONE, 2012Co-Authors: Neil Bate, Alexandre R Gingras, Bipin Patel, Benjamin T. Goult, Alexia I Bachir, Rick Horwitz, David R CritchleyAbstract:Talin is a large (∼2540 residues) dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs), where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of Talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the Talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of Talin1 in vivo. Expression of a GFP-tagged Talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the Talin head and rod domains. Moreover, incorporation of both mutations into a single Talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of Talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the Talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The Talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the Talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the Talin rod may provide a mechanism both for terminating the signalling function of the Talin rod and indeed for inactivating full-length Talin thereby promoting FA turnover at the rear of the cell.
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Subcellular localization of Talin is regulated by inter-domain interactions.
The Journal of biological chemistry, 2012Co-Authors: Asoka Banno, Neil Bate, David R Critchley, Benjamin T. Goult, Ho-sup Lee, Mark H GinsbergAbstract:Talin, which is composed of head (THD) and rod domains, plays an important role in cell adhesion events in diverse species including most metazoans and Dictyostelium discoideum. Talin is abundant in the cytosol; however, it mediates adhesion by associating with integrins in the plasma membrane where it forms a primary link between integrins and the actin cytoskeleton. Cells modulate the partitioning of Talin between the plasma membrane and the cytosol to control cell adhesion. Here, we combine nuclear magnetic resonance spectroscopy (NMR) with subcellular fractionation to characterize two distinct THD-rod domain interactions that control the interaction of Talin with the actin cytoskeleton or its localization to the plasma membrane. An interaction between a discrete vinculin-binding region of the rod (VBS1/2a; Tln1(482–787)), and the THD restrains Talin from interacting with the plasma membrane. Furthermore, we show that vinculin binding to VBS1/2a results in Talin recruitment to the plasma membrane. Thus, we have structurally defined specific inter-domain interactions between THD and the Talin rod domain that regulate the subcellular localization of Talin.
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Talin dependent integrin activation is required for fibrin clot retraction by platelets
Blood, 2011Co-Authors: Jacob R Haling, Susan J Monkley, David R Critchley, Brian G PetrichAbstract:Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of Talin, we show for the first time that the capacity of Talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which Talin functions are required for this process, we tested clot retraction in platelets expressing a Talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between Talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and Talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in Talin1(L325R) platelets, but not Talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that Talin1(L325R) can form functional links to the actin cytoskeleton.
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Talin 1 and 2 are required for myoblast fusion sarcomere assembly and the maintenance of myotendinous junctions
Development, 2009Co-Authors: Francesco J Conti, Susan J Monkley, David R Critchley, Malcolm R Wood, Ulrich MullerAbstract:Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, Talin 1 regulates the stability of myotendinous junctions (MTJs), but the function of Talin 2 in skeletal muscle is not known. Here we show that MTJ integrity is affected in Talin 2-deficient mice. Concomitant ablation of Talin 1 and 2 leads to defects in myoblast fusion and sarcomere assembly, resembling defects in muscle lacking beta1 integrins. Talin 1/2-deficient myoblasts express functionally active beta1 integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather by disruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesion complexes is perturbed in the remaining muscle fibers of Talin 1/2-deficient mice. We conclude that Talin 1 and 2 are crucial for skeletal muscle development, where they regulate myoblast fusion, sarcomere assembly and the maintenance of MTJs.
Mark H Ginsberg - One of the best experts on this subject based on the ideXlab platform.
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optogenetic based localization of Talin to the plasma membrane promotes activation of β3 integrins
Journal of Biological Chemistry, 2021Co-Authors: Zhongji Liao, Alexandre R Gingras, Mark H Ginsberg, Frederic Lagarrigue, Sanford J ShattilAbstract:Interaction of Talin with the cytoplasmic tails of integrin β triggers integrin activation, leading to an increase of integrin affinity/avidity for extracellular ligands. In Talin knockout mice, loss of Talin interaction with platelet integrin αIIbβ3 causes a severe hemostatic defect, and loss of Talin interaction with endothelial cell integrin αVβ3 affects angiogenesis. In normal cells, Talin is auto-inhibited and localized in the cytoplasm. Here we employed an optogenetic platform to assess whether recruitment of full-length Talin to the plasma membrane was sufficient to induce integrin activation. A dimerization module (CRY2 fused to the N-terminus of Talin; CIBN-CAAX) responsive to 450 nm (blue) light was inserted into CHO cells and endothelial cells also expressing αIIbβ3 or αVβ3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of CRY2-Talin to the CIBN-CAAX-decorated plasma membrane. This resulted in β3 integrin activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of Talin was not sufficient for integrin activation, as membrane-associated Rap1-GTP was also required. Moreover, Talin mutations that interfered with its direct binding to Rap1 abrogated β3 integrin activation. Altogether, these results define a role for the plasma membrane recruitment of Talin in β3 integrin activation, and they suggest a nuanced sequence of events thereafter involving Rap1-GTP.
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Kindlins, Integrin Activation and the Regulation of Talin Recruitment to αIIbβ3
PloS one, 2012Co-Authors: Bryan N. Kahner, Mark H Ginsberg, Asoka Banno, Hisashi Kato, Sanford J ShattilAbstract:Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the Talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since Talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate Talin recruitment to plasma membranes and to αIIbβ3, full-length Talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and Talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect Talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected Talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, Talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified Talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial Talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment.
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Subcellular localization of Talin is regulated by inter-domain interactions.
The Journal of biological chemistry, 2012Co-Authors: Asoka Banno, Neil Bate, David R Critchley, Benjamin T. Goult, Ho-sup Lee, Mark H GinsbergAbstract:Talin, which is composed of head (THD) and rod domains, plays an important role in cell adhesion events in diverse species including most metazoans and Dictyostelium discoideum. Talin is abundant in the cytosol; however, it mediates adhesion by associating with integrins in the plasma membrane where it forms a primary link between integrins and the actin cytoskeleton. Cells modulate the partitioning of Talin between the plasma membrane and the cytosol to control cell adhesion. Here, we combine nuclear magnetic resonance spectroscopy (NMR) with subcellular fractionation to characterize two distinct THD-rod domain interactions that control the interaction of Talin with the actin cytoskeleton or its localization to the plasma membrane. An interaction between a discrete vinculin-binding region of the rod (VBS1/2a; Tln1(482–787)), and the THD restrains Talin from interacting with the plasma membrane. Furthermore, we show that vinculin binding to VBS1/2a results in Talin recruitment to the plasma membrane. Thus, we have structurally defined specific inter-domain interactions between THD and the Talin rod domain that regulate the subcellular localization of Talin.
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Talin phosphorylation by cdk5 regulates smurf1 mediated Talin head ubiquitylation and cell migration
Nature Cell Biology, 2009Co-Authors: Cai Huang, Zenon Rajfur, Nima Yousefi, Zaozao Chen, Kenneth A Jacobson, Mark H GinsbergAbstract:Cell migration is a dynamic process that requires temporal and spatial regulation of integrin activation and focal adhesion assembly/disassembly. Talin, an actin and beta-integrin tail-binding protein, is essential for integrin activation and focal adhesion formation. Calpain-mediated cleavage of Talin has a key role in focal adhesion turnover; however, the Talin head domain, one of the two cleavage products, stimulates integrin activation, localizes to focal adhesions and maintains cell edge protrusions, suggesting that other steps, downstream of Talin proteolysis, are required for focal adhesion disassembly. Here we show that Talin head binds Smurf1, an E3 ubiquitin ligase involved in cell polarity and migration, more tightly than full-length Talin does and that this interaction leads to Talin head ubiquitylation and degradation. We found that Talin head is a substrate for Cdk5, a cyclin-dependent protein kinase that is essential for cell migration, synaptic transmission and cancer metastasis. Cdk5 phosphorylated Talin head at Ser 425, inhibiting its binding to Smurf1, thus preventing Talin head ubiquitylation and degradation. Expression of the mutant tal(S425A), which resists Cdk5 phosphorylation thereby increasing its susceptibility to Smurf1-mediated ubiqitylation, resulted in extensive focal adhesion turnover and inhibited cell migration. Thus, Talin head produced by calpain-induced cleavage of Talin is degraded through Smurf1-mediated ubiquitylation; moreover, phosphorylation by Cdk5 regulates the binding of Smurf1 to Talin head, controlling Talin head turnover, adhesion stability and ultimately, cell migration.
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riam activates integrins by linking Talin to ras gtpase membrane targeting sequences
Journal of Biological Chemistry, 2009Co-Authors: Wilma Puzonmclaughlin, Sanford J Shattil, Mark H GinsbergAbstract:Abstract Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule (RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to promote Talin-dependent integrin activation. Here, we show that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to Talin, thereby recruiting Talin to the plasma membrane and activating integrins. The MRL proteins bound directly to Talin via short, N-terminal sequences predicted to form amphipathic helices. RIAM-induced integrin activation required both its capacity to bind to Rap1 and to Talin. Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the Talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A. This minimized Rap-RIAM module was sufficient to target Talin to the plasma membrane and to mediate integrin activation, even in the absence of Rap1 activity. We identified a short Talin binding sequence in Lamellipodin (Lpd), another MRL protein; Talin binding Lpd sequence joined to a Rap1 membrane-targeting sequence is sufficient to recruit Talin and activate integrins. These data establish the mechanism whereby MRL proteins interact with both Talin and Ras GTPases to activate integrins.
Benjamin T. Goult - One of the best experts on this subject based on the ideXlab platform.
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Direct binding of Talin to Rap1 is required for cell-ECM adhesion in Drosophila
Journal of cell science, 2018Co-Authors: Darius Camp, Amanda Haage, Veronika Solianova, William M. Castle, Emily Lostchuck, Benjamin T. Goult, Guy TanentzapfAbstract:Attachment of cells to the extracellular matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin (encoded by rhea in flies) is necessary for linking integrins to the cytoskeleton, and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. Here, we show that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies that seen with null alleles of Talin. Moreover, we show that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion.
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The mechanical response of Talin
Nature communications, 2016Co-Authors: Mingxi Yao, Benjamin T. Goult, Michael P Sheetz, Benjamin Klapholz, Christopher P. Toseland, Yingjian Guo, Peiwen Cong, Jie YanAbstract:Talin, a force-bearing cytoplasmic adapter essential for integrin-mediated cell adhesion, links the actin cytoskeleton to integrin-based cell–extracellular matrix adhesions at the plasma membrane. Its C-terminal rod domain, which contains 13 helical bundles, plays important roles in mechanosensing during cell adhesion and spreading. However, how the structural stability and transition kinetics of the 13 helical bundles of Talin are utilized in the diverse Talin-dependent mechanosensing processes remains poorly understood. Here we report the force-dependent unfolding and refolding kinetics of all Talin rod domains. Using experimentally determined kinetics parameters, we determined the dynamics of force fluctuation during stretching of Talin under physiologically relevant pulling speeds and experimentally measured extension fluctuation trajectories. Our results reveal that force-dependent stochastic unfolding and refolding of Talin rod domains make Talin a very effective force buffer that sets a physiological force range of only a few pNs in the Talin-mediated force transmission pathway.
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Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity
Journal of Cell Biology, 2016Co-Authors: Abhishek Kumar, Benjamin T. Goult, Mingxing Ouyang, Koen Van Den Dries, Ewan J Mcghee, Keiichiro Tanaka, Marie D Anderson, Alex Groisman, Kurt I Anderson, Martin A SchwartzAbstract:Integrin-dependent adhesions are mechanosensitive structures in which Talin mediates a linkage to actin filaments either directly or indirectly by recruiting vinculin. Here, we report the development and validation of a Talin tension sensor. We find that Talin in focal adhesions is under tension, which is higher in peripheral than central adhesions. Tension on Talin is increased by vinculin and depends mainly on actin-binding site 2 (ABS2) within the middle of the rod domain, rather than ABS3 at the far C terminus. Unlike vinculin, Talin is under lower tension on soft substrates. The difference between central and peripheral adhesions requires ABS3 but not vinculin or ABS2. However, differential stiffness sensing by Talin requires ABS2 but not vinculin or ABS3. These results indicate that central versus peripheral adhesions must be organized and regulated differently, and that ABS2 and ABS3 have distinct functions in spatial variations and stiffness sensing. Overall, these results shed new light on Talin function and constrain models for cellular mechanosensing.
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The Mechanical Properties of Talin Rod Domain
Biophysical Journal, 2016Co-Authors: Mingxi Yao, Benjamin T. Goult, Michael P Sheetz, Jie YanAbstract:Focal adhesion protein Talin has emerged as a key protein the mechanotransduction pathway linking integrin based cell adhesion to actin cytoskeleton. The force-dependent structural transitions of the c-terminal rod domain, consisting of 13 alpha-helical bundles, are critical for Talin's mechanosensing function during cell adhesion and spreading but remain poorly understood. We systematically determined the force-dependent unfolding/refolding kinetics of all Talin rod domains and obtained the dynamics of force fluctuation of Talin rod under experimentally measured extension fluctuation time trajectories. The results revealed Talin rod's role as a viscoelastic spring and tension buffer that maintains at a level < 10 pN along Talin mediated force-transmission pathway. These results provide the first quantitative understanding of how Talin serves as a mechanical clutch during cell spreading.
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structural studies on full length Talin1 reveal a compact auto inhibited dimer implications for Talin activation
Journal of Structural Biology, 2013Co-Authors: Benjamin T. Goult, Neil Bate, Igor L. Barsukov, Alexandre R Gingras, David R Critchley, Bipin Patel, Mark F Swift, Petra M Kopp, Niels Volkmann, Dorit HaneinAbstract:Talin is a large adaptor protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain (the head) linked to a flexible rod comprised of 13 amphipathic helical bundles (R1–R13) that terminate in a C-terminal helix (DD) that forms an anti-parallel dimer. We derived a three-dimensional structural model of full-length Talin at a resolution of approximately 2.5 nm using EM reconstruction of full-length Talin and the known shapes of the individual domains and inter-domain angles as derived from small angle X-ray scattering. Talin adopts a compact conformation consistent with a dimer in which the two Talin rods form a donut-shaped structure, with the two Talin heads packed side by side occupying the hole at the center of this donut. In this configuration, the integrin binding site in the head domain and the actin-binding site at the carboxy-terminus of the rod are masked, implying that Talin must unravel before it can support integrin activation and engage the actin cytoskeleton.
G. Isenberg - One of the best experts on this subject based on the ideXlab platform.
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The effect of intact Talin and Talin tail fragment on actin filament dynamics and structure depends on pH and ionic strength.
European journal of biochemistry, 2001Co-Authors: Wolfgang H. Goldmann, Daniel Hess, G. IsenbergAbstract:We employed quasi-elastic light scattering and electron microscopy to investigate the influence of intact Talin and Talin tail fragment on actin filament dynamics and network structure. Using these methods, we confirm previous reports that intact Talin induces cross-linking as well as filament shortening on actin networks. We now show that the effect of intact Talin as well as Talin tail fragment on actin networks is controlled by pH and ionic strength. At pH 7.5, actin filament dynamics in the presence of intact Talin and Talin tail fragment are characterized by a rapid decay of the dynamic structure factor and by a square root power law for the stretched exponential decay which is in contrast with the theory for pure actin solutions. At pH 6 and low ionic strength, intact Talin cross-links actin filaments more tightly than Talin tail fragment. Talin head fragment showed no effect on actin networks, indicating that the actin binding sites reside probably exclusively within the tail domain.
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Examining F-actin interaction with intact Talin and Talin head and tail fragment using static and dynamic light scattering
European journal of biochemistry, 1997Co-Authors: Wolfgang H. Goldmann, Daniel Hess, Zeno Guttenberg, Stefan H. E. Kaufmann, Robert M. Ezzell, G. IsenbergAbstract:We examined the binding kinetics of intact Talin and Talin head and tail fragment with F-actin at pH 7.0 and at low ionic strength. We observed by a transient kinetic method a fast followed by a slower binding process for intact Talin and Talin tail fragment with filamentous actin. The latter can be attributed to F-actin cross-linking and/or bundling, which was observed in cosedimentation assays as well as by low shear viscometry and electron microscopy [Zhang, J., Robson, R. M., Schmidt, J. M. & Stromer, M. H. (1996) Biochem. Biophys. Res. Commun. 218, 530–537]. This finding is supported by dynamic light scattering measurements, indicating changes in internal actin filament dynamics due to cross-linking/bundling events with intact Talin and Talin and Talin tail fragment. No binding of the Talin head fragment with F-actin was detected by either method.
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Probing phosphatidylinositolphosphates and adenosinenucleotides on Talin nucleated actin polymerization.
FEBS letters, 1996Co-Authors: G. Isenberg, V Niggli, U Pieper, S Kaufmann, W H GoldmannAbstract:We have investigated the binding of PI, PIP and PIP2 to Talin and the effect of phosphoinositides and adenosinenucleotides on Talin-induced actin polymerization. At physiological salt concentrations, Talin coprecipitates with liposomes when containing phosphoinositides but not when containing PI. The nucleating effect of Talin as reflected by a twofold increase of fluorescence during the polymerization of actin labelled with NBD is not inhibited by phosphoinositides. The polymerization of ADP-actin versus ATP-actin was investigated in the presence and absence of Talin by NBD fluorescence. ADP-actin nucleation induced by Talin is comparably efficient as with ATP-actin. These experimental findings in summary have implications when evaluating the role of Talin during cell activation.
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Probing actin and liposome interaction of Talin and Talin-vinculin complexes: a kinetic, thermodynamic and lipid labeling study.
Biochemistry, 1992Co-Authors: W H Goldmann, Stefan H. E. Kaufmann, Verena Niggli, G. IsenbergAbstract:Talin purified from human platelets and chicken gizzard smooth muscle is an actin and lipid binding protein. Here, we have investigated the effect of vinculin on (a) Talin-nucleated actin polymerization and (b) insertion of Talin into lipid bilayers. Calorimetric data show ternary complex formation between Talin, vinculin, and actin. Actin-Talin, actin-vinculin and actin-(Talin-vinculin) binding and rate constants as well as actin polymerization rates for all three protein species have been determined by steady state titration, stopped-flow, and fluorescence assay. In contrast to an increase of the polymerization rate by a factor of less than 2 for actin-Talin and actin-(Talin-vinculin) when lowering the temperature, we measured a decrease in rates for actin alone and actin-vinculin. The overall equilibrium constants (Keq) in the van't Hoff plot proved linear and were of one-step reactions. Thermodynamic data exhibited signs of van der Waal's binding forces. Using the photoactivatable lipid analogue [3H]PTPC/11, which selectively labels membrane-embedded hydrophobic domains of proteins, we also show that Talin partially inserts into the hydrophobic bilayer of liposomes. This insertion occurs in a similar manner irrespective of preincubation with vinculin.
Bipin Patel - One of the best experts on this subject based on the ideXlab platform.
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structural studies on full length Talin1 reveal a compact auto inhibited dimer implications for Talin activation
Journal of Structural Biology, 2013Co-Authors: Benjamin T. Goult, Neil Bate, Igor L. Barsukov, Alexandre R Gingras, David R Critchley, Bipin Patel, Mark F Swift, Petra M Kopp, Niels Volkmann, Dorit HaneinAbstract:Talin is a large adaptor protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain (the head) linked to a flexible rod comprised of 13 amphipathic helical bundles (R1–R13) that terminate in a C-terminal helix (DD) that forms an anti-parallel dimer. We derived a three-dimensional structural model of full-length Talin at a resolution of approximately 2.5 nm using EM reconstruction of full-length Talin and the known shapes of the individual domains and inter-domain angles as derived from small angle X-ray scattering. Talin adopts a compact conformation consistent with a dimer in which the two Talin rods form a donut-shaped structure, with the two Talin heads packed side by side occupying the hole at the center of this donut. In this configuration, the integrin binding site in the head domain and the actin-binding site at the carboxy-terminus of the rod are masked, implying that Talin must unravel before it can support integrin activation and engage the actin cytoskeleton.
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Talin contains a c terminal calpain2 cleavage site important in focal adhesion dynamics
PLOS ONE, 2012Co-Authors: Neil Bate, Alexandre R Gingras, Bipin Patel, Benjamin T. Goult, Alexia I Bachir, Rick Horwitz, David R CritchleyAbstract:Talin is a large (∼2540 residues) dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs), where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of Talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the Talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of Talin1 in vivo. Expression of a GFP-tagged Talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the Talin head and rod domains. Moreover, incorporation of both mutations into a single Talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of Talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the Talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The Talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the Talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the Talin rod may provide a mechanism both for terminating the signalling function of the Talin rod and indeed for inactivating full-length Talin thereby promoting FA turnover at the rear of the cell.
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the structure of the n terminus of kindlin 1 a domain important for alphaiibbeta3 integrin activation
Journal of Molecular Biology, 2009Co-Authors: Benjamin T. Goult, Neil Bate, Igor L. Barsukov, Bipin Patel, Iain D. Campbell, David A Calderwood, Mohamed Bouaouina, David S Harburger, Nicholas J Anthis, Gordon C K RobertsAbstract:The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the Talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the β-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for Talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the Talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the Talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the Talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the Talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in Talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in Talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the Talin F0 and F1 domains. Comparison of the kindlin-1 and Talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support Talin-induced αIIbβ3 integrin activation and for the localization of kindlin-1 to focal adhesions.
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The Activity of the Vinculin Binding Sites in Talin Is Influenced by the Stability of the Helical Bundles That Make Up The Talin Rod
The Journal of biological chemistry, 2006Co-Authors: Bipin Patel, Gordon C K Roberts, Alexandre R Gingras, Jonas Emsley, Audrey A. Bobkov, L. Miya Fujimoto, Man Zhang, Robert C. Liddington, Daniela Mazzeo, Igor L. BarsukovAbstract:Abstract The Talin rod contains ∼11 vinculin binding sites (VBSs), each defined by hydrophobic residues in a series of amphipathic helices that are normally buried within the helical bundles that make up the rod. Consistent with this, Talin failed to compete for binding of the vinculin Vd1 domain to an immobilized Talin polypeptide containing a constitutively active VBS. However, Talin did bind to GST-Vd1 in pull-down assays, and isothermal titration calorimetry measurements indicate a Kd of ∼9 μm. Interestingly, Vd1 binding exposed a trypsin cleavage site in the Talin rod between residues 898 and 899, indicating that there are one or more active VBSs in the N-terminal part of the Talin rod. This region comprises a five helix bundle (residues 482-655) followed by a seven-helix bundle (656-889) and contains five VBSs (helices 4, 6, 9, 11, and 12). The single VBS within 482-655 is cryptic at room temperature. In contrast, Talin 482-889 binds Vd1 with high affinity (Kd ∼ 0.14 μm), indicating that one or more of the four VBSs within 656-889 are active, and this likely represents the vinculin binding region in intact Talin. In support of this, hemagglutinin-tagged Talin 482-889 localized efficiently to focal adhesions, whereas 482-655 did not. Differential scanning calorimetry showed a strong negative correlation between Vd1 binding and helical bundle stability, and a 755-889 mutant with a more stable fold bound Vd1 much less well than wild type. We conclude that the stability of the helical bundles that make up the Talin rod is an important factor determining the activity of the individual VBSs.
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activation of a vinculin binding site in the Talin rod involves rearrangement of a five helix bundle
The EMBO Journal, 2004Co-Authors: E Papagrigoriou, Neil Bate, Ian J Fillingham, Wolfgang H Ziegler, Gordon C K Roberts, Igor L. Barsukov, Alexandre R Gingras, Ronald Frank, Bipin Patel, David R CritchleyAbstract:The interaction between the cytoskeletal proteins Talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the Talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin-binding site (VBS), folds into a five-helix bundle whereas Talin 656–789 is a four-helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the Talin 482–655 fold. We demonstrate that the Talin 482–655 five-helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of Talin. Activation of the VBS in Talin and the recruitment of vinculin may support the maturation of small integrin/Talin complexes into more stable adhesions.
