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A PDZ-like carboxylate-binding motif exists in kindlin-2 F3. Mutations in this segment phenocopy the DFC and laterality organ defects of kindlin-2 and V/1b morphants. A, multiple PDZ carboxylate-binding loop sequence alignments used to identify a putative motif in the kindlin-2 F3 subdomain . Interspecies sequence alignments of the putative kindlin-2 F3 carboxylate-binding loop indicate a complete conservation from mammalian to insect lineages. B, list of kindlin-2 F3 mutants and their respective sequence alignments used in this study. Point mutations in WT sequence are underlined in red. For clarity, human sequences are used for sequence numbering on their zebrafish orthologs. C–E, direct binding of purified zebrafish kindlin-2 F3 mutants (C–E) and talin-1 F2,3 (E) to wild-type recombinant 1b tail assessed by enzyme-linked immunosorbent assay. Kindlin-2 F3 mutants: WT, black filled circle; K581E, red filled triangle; K582E, red circle; K581E/K582E, green filled triangle; G587L, cyan square; I586G/I588G, black filled triangle; R592E, green filled circle; R595E, cyan filled circle; R608E, red filled circle; W615A, black circle; and IIb, gray cross. Talin-1 F2,3 mutants: WT, magenta filled circle; W359A, magenta circle. F, summary of mutant kindlin-2 F3 direct binding affinities to wild-type integrin 1b tail. G, schematic of bicistronic mCherry-T2A-kindlin-2 vector that was used to co-express mCherry and kindlin-2 proteins. H and I, confocal images of Tg(sox17:GFP)-expressing embryos (green) injected with mRNAs that code for WT, F3, K581E, and R592E. H and I, dorsal views of 80% E embryos (H), and 6 – 8 somite stage (SS) embryos (I) are shown in all panels; anterior is to the top. H, multiple focal planes at the center of the DFC cluster. Embryos were immunolabeled with anti-cherry antibody (red) and nucleus-stained (blue); channels are merged. I, multiple focal planes at the center of KV immunolabeled with anti-acetylated tubulin antibody (white) and with anti-cherry antibody (red); channels are merged. J, bar graphs showing effects of kindlin-2 mRNA injections on DFC clustering. Number of embryos used in this study: WT, 312; F3, 353; K581E, 267; R592E, 221. K, bar graphs showing effects of kindlin-2 mRNA injections on KV lumen formation. Number of embryos used in this study: WT, 108; F3, 123; K581E, 152; R592E, 122. N.B., no binding. Analyses of binding isotherms were similar to Fig. 4. Scale bars 50 m. Error bars indicate mean S.E.
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Protein-protein interactions are driving forces in cellular processes. As a prime example, transmembrane integrins link extracellular
matrix and intracellular proteins, resulting in bidirectional signaling that regulates cell migration, proliferation, differentiation,
and survival. Here we provide the first evidence that interaction between the int...
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During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate and the stresses that...
Citations
... The three kindlin isoforms display tissue-specific expression patterns, favor binding of different integrin β subunits and even might have partially non-redundant functions (Bandyopadhyay et al., 2012;Bledzka et al., 2012;Fitzpatrick et al., 2014). While kindlin-1 is mainly present in epithelial cells, kindlin-2 is ubiquitously expressed and kindlin-3 is predominantly abundant in hematopoietic cells, but was discovered recently also in low amounts in endothelial tissues Ussar et al., 2006). ...
... Noteworthy, in our study we applied the ubiquitously expressed kindlin-2 isoform, which has been reported to bind integrin β1 with high affinity (Fitzpatrick et al., 2014). Accumulating evidence in the integrin field yet indicate specific and controversial functions for individual kindlin isoforms (Gao et al., 2019;Huet-Calderwood et al., 2014;Kadry et al., 2020) next to their tissue specificity Ussar et al., 2006). ...
... Expression of kindlin-2-QW, which displayed intermediate membrane recruitment, partially rescued spreading (Fig. 7b). This suggests that kindlin-2-QW mutant presents residual binding to integrins, in accordance with studies showing that kindlin residues other than Q614 and W615 are involved in the interaction with integrins 17,19,20,34,45,46 . The most dramatic effect was observed for kindlin-2-ΔPH, which displayed almost no full spreading, a large decrease in the fraction of partially spread cells (Fig. 7a,b), and reduced adhesion formation (Fig. 7e), supporting the idea that membrane recruitment and/or free diffusion are critical for kindlin-2 functions. ...
A bstract
Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we linked the molecular behavior and tridimensional nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displayed free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation during cell adhesion and spreading. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.
... They are encoded by three different genes, Kindlin-1 by Fermt1, Kindlin-2 by Fermt2, and Kindlin-3 by Fermt3 14,15,20,21 . Each Kindlin protein contains a FERM (F for 4.1 protein, E for ezrin, R for radixin, and M for moesin) domain that is responsible for interacting with βintegrin cytoplasmic tails 14,16,[22][23][24][25][26][27] . Bledzka et al. reported that Kindlin-2 binds actin and regulates integrin outside-in signaling 28 . ...
β-Cell dysfunction and reduction in β-cell mass are hallmark events of diabetes mellitus. Here we show that β-cells express abundant Kindlin-2 and deleting its expression causes severe diabetes-like phenotypes without markedly causing peripheral insulin resistance. Kindlin-2, through its C-terminal region, binds to and stabilizes MafA, which activates insulin expression. Kindlin-2 loss impairs insulin secretion in primary human and mouse islets in vitro and in mice by reducing, at least in part, Ca2+ release in β-cells. Kindlin-2 loss activates GSK-3β and downregulates β-catenin, leading to reduced β-cell proliferation and mass. Kindlin-2 loss reduces the percentage of β-cells and concomitantly increases that of α-cells during early pancreatic development. Genetic activation of β-catenin in β-cells restores the diabetes-like phenotypes induced by Kindlin-2 loss. Finally, the inducible deletion of β-cell Kindlin-2 causes diabetic phenotypes in adult mice. Collectively, our results establish an important function of Kindlin-2 and provide a potential therapeutic target for diabetes. Beta cell dysfunction and reduction in beta cell mass are hallmark events in the pathogenesis of diabetes mellitus. We identify focal adhesion protein Kindlin-2 as a key factor that controls insulin synthesis and secretion and beta cell mass by modulating MafA and beta-catenin proteins in pancreatic beta cells.
... However, despite plating these cells on FN-coated coverslips, these integrins showed poor recruitment into peripheral focal adhesions and central fibrillar adhesions as compared to endogenous β1 integrin labeled with monoclonal antibody clone 9EG7, detecting the extended forms of β1 integrins (Bazzoni et al., 1995;Lenter et al., 1993;Su et al., 2016) (Fig. 1A). The reduced recruitment of the C-terminally tagged β1A integrins into adhesion structures was not due to the length of the linker domain, consisting of 5 or 30 amino acids in Fig. 1B and Fig. 1D, respectively, but potentially because the free carboxylic acid is required for interaction with kindlins (Fitzpatrick et al., 2014). To avoid a non-specific interference by the cytoplasmic GFP, we decided to incorporate GFP into the extracellular domain of the β1A integrin, similar to a strategy successfully used for Drosophila β integrin. ...
... This revealed independence of the distal NPXY motif from β1D integrin recruitment and explained focal adhesion recruitment of C-terminally tagged β1D integrin (Fig. 1). The C-terminus of β1A integrin displayed a tolerance for deletion of the last amino acid, but a requirement for Y795 and a free C-terminus for full activity was observed as previously suggested (Fitzpatrick et al., 2014). ...
Heterodimeric integrin receptors control cell adhesion, migration and extracellular matrix assembly. While the α integrin subunit determines extracellular ligand specificity, the β integrin chain binds to an acidic residue of the ligand, and cytoplasmic adapter protein families such as talins, kindlins and paxillin, to form mechanosensing cell matrix adhesions. Alternative splicing of the β1 integrin cytoplasmic tail creates ubiquitouslyexpressed β1A, and the heart and skeletalmusclespecific β1D form. To study the physiological difference between these forms, we developed fluorescent β1 integrins and analyzed their dynamics, localization, and cytoplasmic adapter recruitment and effects on cell proliferation. On fibronectin, GFP-tagged β1A integrin showed dynamic exchange in peripheral focal adhesions, and long, central fibrillar adhesions. In contrast, GFP-β1D integrins exchanged slowly, forming immobile and short central adhesions.While adhesion recruitment of GFP-β1A integrin was sensitive to C-terminal tail mutagenesis, GFP-β1D integrin was recruited independently of the distal NPXY motif. In addition, a P786A mutation in the proximal, talin-binding NPXY783 motif switched β1D to a highly dynamic integrin. In contrast, the inverse A786P mutation in β1A integrin interfered with paxillin recruitment and proliferation. Thus, differential β1 integrin splicing controls integrin-dependent adhesion signaling, to adapt to the specific physiological needs of differentiated muscle cells.
... Kindlins consist of a FERM domain and a pleckstrin homology (PH) domain that is inserted into the F2 module. Biochemical interaction studies revealed that the F3 modules of kindlins bind to the membrane-distal NxxY motif; the adjacent threonine/serine residue; and, at least in integrin β1, the C-terminal carboxylate moiety (13). ...
... In addition, although the very last three nonconserved residues ( 796 EGK 798 ) in the β1-tail were not well assigned (SI Appendix, Fig. S5), deletion of the EGK sequence abolishes the binding of integrins to kindlins in both our and others' observations ( Fig. 2E) (36,37). Because the amide of E796 β1 forms a main-chain/ main-chain H-bond with the β4 F3 /β5 F3 loop in kindlin2 (Fig. 2C), deletion of EGK eliminates the H-bond, thereby disrupting the interaction, which aligns with previous findings that the binding of the β1-tail to kindlin2 requires at least one residue to the C terminus of Y795 β1 in a sequence-independent manner (36). ...
... In addition, although the very last three nonconserved residues ( 796 EGK 798 ) in the β1-tail were not well assigned (SI Appendix, Fig. S5), deletion of the EGK sequence abolishes the binding of integrins to kindlins in both our and others' observations ( Fig. 2E) (36,37). Because the amide of E796 β1 forms a main-chain/ main-chain H-bond with the β4 F3 /β5 F3 loop in kindlin2 (Fig. 2C), deletion of EGK eliminates the H-bond, thereby disrupting the interaction, which aligns with previous findings that the binding of the β1-tail to kindlin2 requires at least one residue to the C terminus of Y795 β1 in a sequence-independent manner (36). ...
Significance
Kindlin proteins play crucial roles in the integrin-signaling pathway by directly interacting with and activating integrins, which mediate the cell–extracellular matrix adhesion and signaling. Mutations of kindlins lead to diseases, such as Kindler syndrome, associated with skin blistering and atrophy; leukocyte adhesion deficiency; and cancers. However, the molecular basis underlying kindlin-mediated integrin activation remains to be determined. Here, we report the structural basis of the specific interaction between kindlins and integrins. Furthermore, we demonstrate that kindlins synergize integrin activation by forming a dimer, providing a model for understanding integrin signaling. Finally, we interpret disease-causing mutations found in kindlins at the atomic level, which can be useful for understanding and treating these diseases.
... Talin is known to regulate inside-out signaling [5] and kindlin is regarded as a coactivator of talin [8,9], mutations of which also impair the talin-mediated integrin activation [47]. The mutation of the putative binding sites in β3, such as the Y to A substitution at residue 759, causes decreased association with kindlin [18,48] and damaged integrin activation [8]. Our pull-down assays showed that GST-β3-ΔRGT bound talin and kindlin, as did GST-β3, consistent with the intact inside-out signaling in β3-ΔRGT platelets (Figs 6 and 7). ...
Previous studies in Chinese hamster ovary cells showed that truncational mutations of β3 at sites of F⁷⁵⁴ and Y⁷⁵⁹ mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F⁷⁵⁴ to C-terminus and the conservative N⁷⁵⁶ITY⁷⁵⁹ motif in platelet function have yet to be elaborated. Mice expressing β3 with F⁷⁵⁴ and Y⁷⁵⁹ truncations, or NITY deletion (β3-ΔTNITYRGT, β3-ΔRGT, or β3-ΔNITY) were established through transplanting the homozygous β3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different β3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the β3-ΔTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the β3-ΔNITY platelets. Behaviors of the β3-ΔRGT platelets were consistent with the previous observations in the β3-ΔRGT knock-in platelets. The adhesion impairment of platelets with the β3 mutants under flow was in different orders of magnitude shown as: β3-ΔTNITYRGT>β3-ΔRGT>β3-ΔNITY to fibrinogen-coated surface, and β3-ΔTNITYRGT>β3-ΔNITY>β3-ΔRGT to collagen-coated surface. To evaluate the interaction of the β3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that β3-ΔRGT interacted with kindlin but not c-Src, β3-ΔNITY interacted with c-Src but not kindlin, while β3-ΔTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin β3, i.e. T⁷⁵⁵NITYRGT⁷⁶², R⁷⁶⁰GT⁷⁶², and N⁷⁵⁶ITY⁷⁵⁹ participate in bidirectional, outside-in, and inside-out signaling, respectively and the association of c-Src or kindlin with β3 integrin may regulate these processes.
... Integrin β1and Integrin β3-mediated signaling. A hint towards this direction is provided by the observation that Kindlin-2 preferably associates with Integrin β1 tails compared to Integrin β3 (Fitzpatrick et al., 2014;Rognoni et al., 2016). In line with that, also in our OPTIC experiments we can see a preference of Kindlin-2 for Integrin β1 whereas Paxillin recruitment to Integrin β3 tails is stronger (Suppl. ...
... Two main defects have been identified in the KS mouse model that contribute to the hyperproliferation of Box 1. Integrin-binding specificity of kindlins By using pull-down assays with β-integrin-tail peptides, kindlin-1 has been shown to bind β1, β3 and β6 integrins (Bandyopadhyay et al., 2012;Rognoni et al., 2014;Harburger et al., 2009), and kindlin-2 and -3 to bind β1-, β2and β3-integrin tails (Böttcher et al., 2012;Bledzka et al., 2012;Harburger et al., 2009;Montanez et al., 2008;Moser et al., 2009aMoser et al., , 2008Ma et al., 2008) at their distal NxxY motifs and adjacent threonine and/or serine residues. Mutational analysis in the kindlin F3 subdomain revealed that a conserved (Q)W motif is essential for binding integrin tails (Fitzpatrick et al., 2014;Harburger et al., 2009;Moser et al., 2009aMoser et al., , 2008Rognoni et al., 2014). It is believed that phosphorylation of the tyrosine residue of the NxxY motif inhibits binding of kindlin . ...
... In addition to the (Q)W motif, a carboxylate-binding motif (h-G-h) in the kindlin-F3 subdomain binds the C-terminus of β1-integrin tails. Furthermore, this kindlin-F3 binding site in β1 tail might be responsible for the high affinity of kindlin-2 to the β1 tail and its lower affinities to β3 and β2 tails (Fitzpatrick et al., 2014). It has been shown that the distance between the threonine/serine residues, the NxxY motif and the carboxylate-binding site also influence the affinity of kindlins to β-integrin tails . ...
The kindlin (or fermitin) family of proteins comprises three members (kindlin-1,-2 and -3) of evolutionarily conserved focal adhesion (FA) proteins, whose best-known task is to increase integrin affinity for a ligand (also referred as integrin activation) through binding of beta-integrin tails. The consequence of kindlin-mediated integrin activation and integrin-ligand binding is cell adhesion, spreading and migration, assembly of the extracellular matrix (ECM), cell survival, proliferation and differentiation. Another hallmark of kindlins is their involvement in disease. Mutations in the KINDLIN-1 (also known as FERMT1) gene cause Kindler syndrome (KS) - in which mainly skin and intestine are affected, whereas mutations in the KINDLIN-3 (also known as FERMT3) gene cause leukocyte adhesion deficiency type III (LAD III), which is characterized by impaired extravasation of blood effector cells and severe, spontaneous bleedings. Also, aberrant expression of kindlins in various forms of cancer and in tissue fibrosis has been reported. Although the malfunctioning of integrins represent a major cause leading to kindlin-associated diseases, increasing evidence also point to integrin-independent functions of kindlins that play an important role in the pathogenesis of certain disease aspects. Furthermore, isoform-specific kindlin functions have been discovered, explaining, for example, why loss of kindlins differentially affects tissue stem cell homeostasis or tumor development. This Commentary focuses on new and isoform-specific kindlin functions in different tissues and discusses their potential role in disease development and progression.
... They are also important for the conformational stability of proteins [2]. These terminal residues serve as a binding site to interact with other proteins to form protein-protein interactions to perform a specific function [3][4][5][6][7]. Another well known function of terminal residues is their ability to serve as a cellular targeting signal [8]. ...
For the past few years, the numbers of transmembrane protein structures in Protein Data Bank have been increased substantially. It is of interest to analyze the terminal residues of transmembrane proteins by using computational approaches. Also, up to our knowledge, no analysis was reported in the literature on the study of terminal residues in transmembrane proteins. While the N-terminal position of alpha and beta transmembrane proteins are composed of signal peptides, in the present work, a careful, in-depth, computational analysis such as residue preference, stability upon mutation, solvent accessibility, hydrogen bonding and carboxy terminal pentapeptide pattern search respectively has been done on C-terminal residues. Alanine in alpha transmembrane proteins and phenylalanine in beta transmembrane proteins are highly preferred. Glutamic acid and glycine residues can be substituted at the terminal sites of alpha and beta transmembrane proteins without affecting the protein's overall stability. Hydrogen bonding of terminal residues is studied in detail. Pattern search of carboxy pentapeptides shows that identical pentapeptides with reference to the position can adopt a different secondary structure. The results discussed in this paper may help to understand the role of carboxy terminal residues in alpha and beta transmembrane proteins. From our analysis, we insist that the preferences and structural analysis of carboxy terminal residues in alpha and beta transmembrane proteins, can help to model and design novel transmembrane proteins.
