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 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

Force-dependent regulation of KANK1-talin complex at focal adhesions

KANK proteins mediate cross-talk between dynamic microtubules and integrin- based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin−KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin−KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions

Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors

The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets

Talin rod domain-containing protein 1 (TLNRD1) is a novel actin-bundling protein which promotes filopodia formation

Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain-containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif-containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly

Talin rod domain–containing protein 1 (TLNRD1) is a novel actin-bundling protein which promotes filopodia formation

Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain–containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif–containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly

Linking cell-matrix adhesions and microtubules: the role of KANK proteins

In this thesis, we characterized the KANK family proteins and their role as mediators of the crosstalk between the microtubule (MT) cytoskeleton and integrin-mediated cell-matrix adhesions. We tried to better understand how KANKs are recruited to FAs, how they interact with talin and what other factors contribute to their function and subcellular localization. In Chapter 2, we establish KANK family proteins as novel binding partners of the FA protein talin. Thereby KANKs form the link between FAs and the CMSC. Further, we could show that the talin-KANK interaction is required for proper formation of the CMSC at the cortex and that perturbation of this connection leads to MT overgrowth. In Chapter 3, we describe the interaction between talin and KANK1 in more detail. We found that the interaction between talin rod domain R7 and the KANK1 KN domain is force-regulated and that it can withstand forces in the piconewton range under certain force geometry. In Chapter 4, we focus on the subcellular localization of KANK1 and explain how the clustering of KANKs around FAs is mediated. We were able to demonstrate that this localization is not due to the coiled coil nor the ankyrin repeat domains but due to the linker region L2 of KANK1. In Chapter 5, we describe the differences between KANK1 and KANK2 with a particular focus on their different subcellular localizations and their role in cell migration. KANK1 is predominantly localized at the cell periphery whereas KANK2 is more concentrated in the central region. We were able to show that the localization of KANK2 is mediated by its KN domain and binding to talin. Further, have seen that depletion of both KANK1 and 2 does not affect cell migration of HeLa cells. On the other hand, loss of KANK2 significantly hampered cell migration of SUM159PT breast cancer cells. In Chapter 6, we discuss how KANK family proteins regulate the crosstalk between MTs and FAs. We combine our work on KANKs as linkers between FAs and CMSC (Chapter 2), the force-regulated interaction between talin and KANKs (Chapter 3), the role of KANK1’s linker region L2 in localizing KANK1 around FAs (Chapter 4), and the role of KANK family proteins in cell migration (Chapter 5) and put them into broader perspective. Further, we discuss some of the partially contradictory findings on KANK family proteins from us and others. We also present some preliminary data on KANK family proteins in endothelial cells and discuss an involvement of KANKs in ECM reorganization (fibronectin fibrillogenesis) and angiogenesis

Linking cell-matrix adhesions and microtubules: the role of KANK proteins

In this thesis, we characterized the KANK family proteins and their role as mediators of the crosstalk between the microtubule (MT) cytoskeleton and integrin-mediated cell-matrix adhesions. We tried to better understand how KANKs are recruited to FAs, how they interact with talin and what other factors contribute to their function and subcellular localization. In Chapter 2, we establish KANK family proteins as novel binding partners of the FA protein talin. Thereby KANKs form the link between FAs and the CMSC. Further, we could show that the talin-KANK interaction is required for proper formation of the CMSC at the cortex and that perturbation of this connection leads to MT overgrowth. In Chapter 3, we describe the interaction between talin and KANK1 in more detail. We found that the interaction between talin rod domain R7 and the KANK1 KN domain is force-regulated and that it can withstand forces in the piconewton range under certain force geometry. In Chapter 4, we focus on the subcellular localization of KANK1 and explain how the clustering of KANKs around FAs is mediated. We were able to demonstrate that this localization is not due to the coiled coil nor the ankyrin repeat domains but due to the linker region L2 of KANK1. In Chapter 5, we describe the differences between KANK1 and KANK2 with a particular focus on their different subcellular localizations and their role in cell migration. KANK1 is predominantly localized at the cell periphery whereas KANK2 is more concentrated in the central region. We were able to show that the localization of KANK2 is mediated by its KN domain and binding to talin. Further, have seen that depletion of both KANK1 and 2 does not affect cell migration of HeLa cells. On the other hand, loss of KANK2 significantly hampered cell migration of SUM159PT breast cancer cells. In Chapter 6, we discuss how KANK family proteins regulate the crosstalk between MTs and FAs. We combine our work on KANKs as linkers between FAs and CMSC (Chapter 2), the force-regulated interaction between talin and KANKs (Chapter 3), the role of KANK1’s linker region L2 in localizing KANK1 around FAs (Chapter 4), and the role of KANK family proteins in cell migration (Chapter 5) and put them into broader perspective. Further, we discuss some of the partially contradictory findings on KANK family proteins from us and others. We also present some preliminary data on KANK family proteins in endothelial cells and discuss an involvement of KANKs in ECM reorganization (fibronectin fibrillogenesis) and angiogenesis

Force-Dependent Regulation of Talin-KANK1 Complex at Focal Adhesions

KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin-KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions

Force-Dependent Regulation of Talin-KANK1 Complex at Focal Adhesions

KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin-KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions