Talin and kindlin use integrin tail allostery and direct binding to activate integrins

Integrin affinity regulation, also termed integrin activation, is essential for metazoan life. Although talin and kindlin binding to the β-integrin cytoplasmic tail is indispensable for integrin activation, it is unknown how they achieve this function. By combining NMR, biochemistry and cell biology techniques, we found that talin and kindlin binding to the β-tail can induce a conformational change that increases talin affinity and decreases kindlin affinity toward it. We also discovered that this asymmetric affinity regulation is accompanied by a direct interaction between talin and kindlin, which promotes simultaneous binding of talin and kindlin to β-tails. Disrupting allosteric communication between the β-tail-binding sites of talin and kindlin or their direct interaction in cells severely compromised integrin functions. These data show how talin and kindlin cooperate to generate a small but critical population of ternary talin–β-integrin–kindlin complexes with high talin–integrin affinity and high dynamics.

Integrins adhere cells to the extracellular matrix, probe biochemical and biophysical properties of the extracellular matrix and convert the information into cellular responses such as spreading, migration, proliferation, survival and differentiation.
Integrins are α-β heterodimers consisting of a large ectodomain, a single-span transmembrane (TM) helix and a short C-terminal cytoplasmic tail (CT).A hallmark of integrins is the ability to reversibly switch between conformations with low and high affinity for ligand.The affinity switch requires the binding of the adaptor proteins talin and kindlin to the β-integrin CT (β-CT).In mammals, there are two talin (TLN1 and TLN2) and three kindlin (KIND1, KIND2 and KIND3) isoforms.Talin and kindlin colocalize in integrin-containing focal adhesions and cooperate to enable integrin-ligand binding and cell adhesion [1][2][3] .The mechanism underlying this cooperation remains a major unresolved question in adhesion biology.
A ternary talin-β-integrin-kindlin complex has been observed for different kindlin and β-integrin isoforms by NMR spectroscopy, analytical ultracentrifugation or super-resolution microscopy [14][15][16] and is considered crucial for talin-kindlin cooperativity.However, this hypothesis has not been proven so far.Theoretically, one would expect that talin and kindlin binding to the β-CT cooperate by amplifying each other's activity 10 .In the case of the β 3 -CT, surface plasmon resonance experiments point to independent binding of TLN1 and KIND2 to the β 3 -CT 14 .On the contrary, pulldown experiments reported competition Article https://doi.org/10.1038/s41594-023-01139-9Data Fig. 1e).This global signal intensity decrease in the β 1 -CT is consistent with a molecular weight increase upon formation of a ternary complex.In addition, the 1 H- 13 C correlation spectra of β 1 -CT in the presence of both TLN1-F3 and ΔKIND2 (Fig. 1d and Extended Data Fig. 1c) reveal that methyl signals of several amino acids between the membrane-proximal and -distal NPxY motifs (A786-V791) are line broadened beyond detection, which is neither observed for all the other peaks nor is it in binary β 1 -CT-TLN1-F3 or β 1 -CT-ΔKIND2 complexes.This observation indicates changes in the binding regime (on and off kinetics) during formation of the ternary TLN1-F3-β 1 -CT-ΔKIND2 complex and possibly a conformational change of β 1 -CT upon simultaneous binding of TLN1-F3 and ΔKIND2.Because simultaneous as well as sequential addition of TLN1-F3 and ΔKIND2 to labeled β 1 -CT produces identical 1 H- 13 C correlation spectra, we conclude that the ternary complex forms at thermodynamic equilibrium (Extended Data Fig. 1d).

Talin and kindlin affinities for β-tails
To investigate whether talin and kindlin influence each other upon β 1 -CT or β 3 -CT binding, we determined their dissociation constants (K d values) for β-CTs at thermodynamic equilibrium.In the simplest case of ternary talin-β-CT-kindlin complex formation, the assumption is that either talin or kindlin binds to β-CT first and then the remaining free adaptor binds to the occupied β-CT, which can be illustrated with an 'energy square' (equation ( 1)): Talin + kindlin + β-CT Talin + kindlin-β-CT talin-β-CT-kindlin.
kindlin + talin-β-CT Measurement of the dissociation constant of talin or kindlin for the β-CT that is either free (K d ) or occupied with kindlin or talin (K d *) allows us to prove or disprove the ternary-complex model.As the total energy does not change in a closed system, microscopic reversibility demands that the product of dissociation and association constants (K a = 1/K d ) around a reaction cycle of an energy square must equal 1 (equation (2): Furthermore, a comparison of the dissociation constants of talin and kindlin for free or occupied β-CT allows us to differentiate independent (K d = K d *), competitive (K d < K d *) or mutually reinforced binding of talin and kindlin to β-CT (K d > K d *) and assign a potential function to the ternary talin-β-integrin-kindlin complex.
We determined the talin-and kindlin-binding mode to β 1 -CT by microscale thermophoresis (MST) 20 , which quantifies changes in fluorescence induced by a temperature-related intensity change as well as thermophoresis of a fluorescently labeled probe.The extent of temperature-related intensity change due to ligand binding and thermophoresis due to size, charge and solvation entropy differences were used to quantify binding affinities in titration experiments.To minimize heat effects, we measured the fluorescence changes only 1.5 s before and after turning on the infrared laser (Extended Data Fig. 1f).MST-based K d measurements of recombinant THD1, THD2 and KIND2 (Fig. 1a and Extended Data Fig. 1a) for ATTO 488-labeled (488)-β 1 -CTs were performed in the absence and the presence of KIND2, THD1 or THD2 at near-saturation binding concentrations (Extended Data Fig. 1f-h).The maximal solubilities of KIND2 and THD1, which were about 500 µM (40 mg ml −1 ) and 2 mM (100 mg ml −1 ), respectively, of recombinant KIND2 and KIND3 by TLN1 (ref.17), and molecular dynamics simulations suggested that talin and kindlin differentially influence each other during β 3 -CT binding 18 .
Here, we investigated the mechanistic basis of talin and kindlin association with β-CT.We found that ternary interactions of THD and KIND2 with β 1 -CT or β 3 -CT can induce an allosteric change of the β-CT, which increases THD and decreases KIND2 affinity.We also observed a direct talin-kindlin interaction, which likely enables rebinding and maintaining the populations of ternary talin-β-integrin-kindlin complexes at a critical threshold.The complex cooperativity between talin and kindlin results in an intrinsic cycle of assembly and disassembly of ternary talin-β-integrin-kindlin complexes that is solely governed by molecular communication between talin and kindlin.

Results β 1 -tail, TLN1 and KIND2 form a ternary complex at equilibrium
To determine whether the β 1A CT splice isoform (β 1 -CT) assembles, similar to β 3 -CT 14 , a ternary complex with TLN1 and KIND2 in vitro, we used NMR spectroscopy to characterize the interaction of isotope-labeled β 1 -CT with the unlabeled F3 domain of TLN1 (TLN1-F3) and KIND2 lacking the flexible loop in F1 and the PH domain (ΔKIND2; Fig. 1a and Extended Data Fig. 1a) to reduce molecular weight and enhance solubility and NMR spectral quality.NMR [ 1 H, 15 N and 1 H, 13 C]methyl correlation spectra (Fig. 1b-d and Extended Data Fig. 1b,c) are affected by the presence of a binding partner, and changes appear either as chemical shift perturbation (CSP), defined as shift of an NMR signal (Extended Data Fig. 1b), or line broadening leading to decreasing signal intensity (Fig. 1b and Extended Data Fig. 1e).Interestingly, ΔKIND2 binding affected the β 1 -CT G778 residue, which is located in the talin-binding site, whereas TLN1-F3 binding did not affect β 1 -CT G797 in the kindlin-binding site, pointing to a different mutual influence of kindlin and talin during β 1 -CT binding.Of note, the line width of an NMR signal is related to the molecular weight of the protein tumbling in solution.Thus, the line broadening observed during complex formation will reflect the increase in the molecular weight of the complex.In addition, the stability of the complex, that is, the binding off-rate and local conformational dynamics due to interaction with the binding partners, can provide an additional contribution to the line width.Residues in β 1 -CT undergoing the greatest line broadening shown by NMR signal are those in the talinor kindlin-binding sites.The extent of line broadening likely reflects the molecular weight of the complex, although additional contributions from binding kinetics or conformational dynamics cannot be excluded.
Superposition of 1 H- 15 N correlation spectra of β 1 -CT in the absence and the presence of TLN1-F3 or ΔKIND2 shows reduced peak intensities assigned to a region from A773 to A786 including the membrane-proximal NPxY motif for TLN1-F3 and from Y783 to the C-terminal end of the β 1 -CT including the membrane-distal NPxY motif for ΔKIND2 (Fig. 1b,c and Extended Data Fig. 1e).This finding agrees well with the reported talin-and kindlin-binding sites 4,11,12 .Furthermore, 1 H-15 N (Fig. 1c and Extended Data Fig. 1e) and 1 H-13 C (Fig. 1d and Extended Data Fig. 1c) correlation spectra show minor CSPs and small changes in signal intensities assigned to the membrane-proximal, α-helical region from H758 to K770, suggesting that, in contrast to β 3 -CT, this conserved region is not an important binding site for TLN1-F3 in β 1 -CT 12,19 .Interestingly, spectral peak intensities assigned to the β 1 -CT Y783-A786 region are reduced upon addition of TLN1-F3 as well as ΔKIND2, suggesting that talin-and kindlin-binding sites overlap in β 1 -CT (Fig. 1c).
To confirm unidirectional competition between talin and kindlin with an orthogonal assay that includes lipid-binding sites for THD1 and KIND2, we incorporated recombinant, biotinylated β 1 and β 3 TM-and CT-containing polypeptides (β 1 -TM-CT, β 3 -TM-CT; Fig. 1a) into 10% phosphatidylinositol phosphate-and 90% phosphocholine-containing nanodiscs (Extended Data Fig. 1a).The reconstituted nanodiscs were immobilized on streptavidin beads and analyzed in a flow cytometry-based reporter-displacement assay (FC-RDA) that allowed us to determine the concentration of unlabeled THD1 or KIND2 required to decrease binding of fluorescently labeled THD1 and KIND2 to 50%, respectively (IC 50 ; Fig. 1h,i).The IC 50 values of unlabeled THD1 competing with fluorescently labeled THD1 and of unlabeled KIND2 competing with fluorescently labeled KIND2 report affinities.The IC 50 values of unlabeled THD1 competing with fluorescently labeled KIND2 and of unlabeled KIND2 competing with fluorescently labeled THD1 report the capability to displace the other adaptor, which is related to the apparent dissociation constant K d,app *, measured by MST.FC-RDA measurements revealed that affinities of THD1 as well as KIND2 for β 1 -TM-CTs and β 3 -TM-CTs embedded in PIP2-or PIP3-containing nanodiscs were in the range of around 80-100 nM and, for α 5 -TM lacking the cytoplasmic domain (tailless α 5 -TM) embedded in PIP2-or PIP3-containing nanodiscs, were in the range of around 300-500 nM.As the tailless α 5 -TM interacts with neither talin nor kindlin, these findings indicate that charged lipids contribute the largest binding energy for talin and kindlin, whereas β-tails make a minor contribution (Extended Data Table 1), which is in line with reports for THD1 and β 3 -CT 22 .The ability of THD1 to compete with labeled KIND2 and of KIND2 with labeled THD1 with similar IC 50 values from tailless α 5 -TM embedded in PIP2-or PIP3-containing nanodiscs indicates that talin and kindlin compete with similar efficiency for the same lipid-binding sites.In line with the data obtained by MST (Fig. 1g), KIND2 was unable to effectively outcompete THD1 binding to β 1 -TM-CTs as well as β 3 -TM-CTs embedded in PIP3-or PIP2-containing nanodiscs, whereas THD1 readily displaced KIND2 (Fig. 1i and Extended Data Fig. 1k).MST data also showed that THD1 failed to displace KIND2 from talin-binding-impaired β 1 -CT Y783A and β 3 -CT Y772A (Extended Data Fig. 1l,m), which altogether indicates that THD1 displaced KIND2 in a β-CT-binding-dependent manner.

KIND2 directly binds TLN1 to stabilize the ternary complex
To achieve microscopic reversibility, the decrease in KIND2 affinity for β 3 -CT in the presence of THD1 (Fig. 1g) must be accompanied by the same decrease in THD1 affinity in the presence of KIND2.Because THD1 affinity, however, does not decrease in the presence of KIND2, we hypothesized that allostery in the β-CT and/or between talin and KIND2 caused by a direct talin-KIND2 interaction counteracts the decrease in THD1 affinity and may lead to unidirectional binding preference.To investigate the talin-kindlin interaction, we probed for an interaction of TLN1-F3 with KIND2 in the absence and presence of β 1 -CT using NMR.First, we examined whether TLN1-F3 and KIND2 bind in the absence of β-CTs by adding KIND2 in a 0.75-fold ratio to uniformly 15 N-labeled TLN1-F3.Analysis of 1 H- 15 N heteronuclear single quantum coherence (HSQC) NMR spectra revealed CSPs in the α 1 -helix of TLN1-F3 located between the integrin-binding site and the flexible linker segment that joins THD and the rod domain in full-length talin (Fig. 2a-c and Extended Data Fig. 2a).NMR spectral changes in the α 1 -helix of TLN1-F3 differ from those observed in NMR spectra of binary complexes between β 1 -CT and uniformly 15 N-labeled TLN1-F3 (Extended Data Fig. 2b-d), indicating that the KIND2 and β-CT-binding sites localize b, Overlay of one-dimensional traces of amide signals of residues G778 and G797 in talin-and kindlin-binding regions of β 1 -CT in 1 H- 15 N HSQC NMR spectra of 15 N-labeled β 1 -CT before (black) and after addition of increasing stoichiometries of ΔKIND2 (top, green) or TLN1-F3 (middle, blue) or both TLN1-F3 and ΔKIND2 (bottom, purple).AU, arbitrary units.c, Intensity ratio of peaks of β 1 -CT in the presence and the absence of ΔKIND2 (top, green), TLN1-F3 (middle, blue) or both (bottom, magenta).Talin-(blue) and kindlin-(green) binding sites are indicated above plots (MP, membrane proximal; MD, membrane distal).The isolated peaks in b are indicated by (1) amino acid numbering, shown below plots, (2) dashed lines and (3) reported mean intensity values for each titration in their respective color code.d, Magnified view of [ 1 H, 13 C]methyl correlations observed for 100 µM 13 C, 15   to adjacent but distinct regions of the TLN1-F3 domain.This finding was confirmed by adding KIND2 to uniformly 15 N-labeled TLN1-F3 in the presence of β 1 -CTs at saturating concentrations, which induced CSPs in the α 1 -helix as well as in the integrin-binding site of TLN1-F3 (Extended Data Fig. 2e-g).
To narrow down the KIND2-binding site, we substituted each amino acid in the TLN1-F3 α 1 -helix individually with alanine or glutamic acid to identify THD1 mutants that affect affinities for β 3 -CT in the presence of near-saturation binding concentrations of KIND2.   N-labeled KIND2-F0 in the absence (black) and the presence of 100 µM TLN1-F3 (blue) or 100 µM TLN1-F3 K402E (red) (h) and 1 H- 15 N HSQC NMR spectra of 70 µM 15 N-labeled KIND2-F0 Y13A in the absence (black) and the presence (purple) of 70 µM wild-type TLN1-F3 (i).j, Ratio of average mean peak intensities (±s.d.) assigned to residues H758-K770 in the α-helical region of 15 N-labeled β 1 -CT at 1:0.8 and 1:0.2 ratios of TLN1-F3 and/or ΔKIND2 (raw data in Fig. 1c and Extended Data Fig. 2l). https://doi.org/10.1038/s41594-023-01139-9 severe line broadening beyond detection in the presence of ΔKIND2 at a molar excess of 2-10-fold.Under these conditions, the substantial molecular weight increase of the TLN1-F3-ΔKIND2 complex (from 11.6 kDa to 65.1 kDa) and potential dynamics in the binding interface led to substantial line broadening beyond detection.However, NMR signals in the TLN1-F3 K402E spectra remain largely unaffected upon ΔKIND2 titration, consistent with the substantially reduced binding of TLN1-F3 K402E to ΔKIND2 (Fig. 2d).
To validate whether disrupting the talin-kindlin interaction influences binding to 15 N-labeled β 1 -CT, we analyzed amide signal line widths in 1 H- 15 N correlation spectra.The signals assigned to the α-helical region of β 1 -CT (from H758 to K770) showed little line broadening (intensity reduction of less than 10%) upon addition of TLN1-F3, ΔKIND2 or ΔKIND2 Y13A at a molar stoichiometry of 1:0.8 compared to 1:0.2 (Fig. 2j), confirming that neither protein interacts with the α-helical region of the β 1 -CT (Fig. 1c and Extended Data Fig. 2n).By sharp contrast, signal intensities in the membrane-proximal α-helix decreased by 50% upon addition of TLN1-F3 together with ΔKIND2, by about 30% upon addition of TLN1-F3 together with ΔKIND2 Y13A and by about 10% upon addition of TLN1-F3 K402E together with ΔKIND2 Y13A (Fig. 2j), indicating that disrupting the talin-kindlin interaction decreases but does not inhibit ternary interactions with β 1 -CT.
To determine whether the association with kindlin induces allosteric activation of talin, followed by increased talin affinity for the β 1 -CT during ternary-complex formation, we titrated the TLN1-F3-ΔKIND2 Y13A fusion protein with β 1 -CT (Fig. 3h).We observed identical binding curves as in titration experiments with the wild-type TLN1-F3-ΔKIND2 fusion protein binding to β 1 -CT (Fig. 3g,h).This result was confirmed in MST affinity measurements, which showed that THD1 affinity for β-CTs remained unchanged in the presence of β-CT-binding-deficient KIND2 Q614A,W615A or KIND2-F0 (Fig. 3i,j), indicating that the association of TLN1 and KIND2 increases the population of the ternary talin-β-CTkindlin complex but not THD1 affinity for β-CTs in the ternary complex.

Ternary-complex formation involves allostery in the β-CT
Because direct talin-kindlin interaction is not involved in the kindlin-mediated increase in talin affinity, we tested whether kindlin binding to the β-CT induces conformational changes that, in turn, increase β-CT affinity for talin.To test this hypothesis, we produced 2 H, 13 C, 15 N-labeled β 1 -CT and partially deuterated TLN1-F3 and ΔKIND2 to record three-dimensional HNCACB spectra of β 1 -CT in the presence and the absence of 1:0.8 TLN1-F3 and/or ΔKIND2.From these spectra, we derived secondary 13 Cα and 13 Cβ chemical shifts (Δδ) that are indicative of secondary structure.The calculated Δδ 13 Cα-Δδ 13 Cβ values (Fig. 4a) are around 0 for disordered regions and are positive for α-helices and negative for β-sheets, with a maximum value of ~6 ppm indicating 100% secondary structure population 23 .In line with published structures 4,11,24 , 13 C secondary chemical shifts of β 1 -CT assigned to membrane-proximal α-helical region were positive and regions around the KIND2-binding site indicate β-strand conformation, reflected by negative values.However, values of around |1| indicate only partial folding of the β 1 -CT in solution (Fig. 4a).Whereas addition of TLN1-F3 or ΔKIND2 alone induced minor changes, addition of both TLN1-F3 and ΔKIND2 induced 13 C secondary chemical shifts pointing to increased α-helical conformation in the region between H758 and K774 and more extended β-type conformation in the region between W775 and V791.Compared to its unbound conformation, β 1 -CT is more structured in the presence of TLN1-F3 and ΔKIND2, suggesting that the ternary complex with talin and kindlin changes or selects a specific β 1 -CT conformation to enhance talin binding.
To test the importance of allosteric coupling of the talin-and kindlin-binding sites (Fig. 4b), we separated the two binding sites by duplicating the intervening sequence with a ten-amino acid spacer (termed β 1 -spacer-CT; Fig. 4c) and found that neither THD1 changed KIND2 affinity nor KIND2 changed THD1 affinity for β 1 -spacer-CT (Fig. 4d and Extended Data Fig. 4a,b) and that the calculated dissociation constants complied with the ternary-complex model: The independent binding of talin and kindlin to β 1 -spacer-CT increases ternary-complex formation, however, without inducing the structural conformational change in the integrin CT that is required for the function of the ternary talin-β-CT-kindlin complex.
To test the function of an integrin tail with decoupled talin-and kindlin-binding sites, we transduced β 1 -integrin complementary DNA encoding wild-type β 1 or β 1 -spacer into β 1 -knockout fibroblasts (Extended Data Fig. 5g).Whereas flow cytometry revealed similar surface levels of total β 1 -integrin, cells expressing the β 1 -spacer displayed reduced levels of the integrin-activation-reporting epitope 9EG7 on their surface compared to cells expressing wild-type β 1 (Extended Data Fig. 5h).Furthermore, FN-seeded β 1 -spacer-expressing cells exhibited profound adhesion and spreading defects (Extended Data Fig. 5i,j), which altogether indicates that formation of functional talin-β 1 -CTkindlin complexes depends on the close proximity of talin-kindlin.

Discussion
Our NMR data indicate that β 1 -CT can assemble a ternary talin-β 1 -CTkindlin complex.The ternary complex, which was shown to form also with the β 3 -CT and the β 2 -CT 14,27 , emerges as a principal molecular setting that ensures cooperativity between talin and kindlin, required to induce and maintain the active conformation of integrins.We expected that the two adaptor proteins augment each other's binding to the β-CT rather than compete or bind independently (noncompetitively) of each other 10 .However, affinity measurements at equilibrium in solution and in the presence of charged lipid membranes point to a unidirectional competition mechanism, in which talin outcompetes kindlin from β 1 -CT or β 3 -CT, while kindlin does not affect talin binding.This observation agrees with single-particle tracking microscopy of live cells assigning kindlin a shorter immobilization time than talin in focal adhesions of FN-seeded fibroblasts 28 and predicts three important consequences.First, the unidirectional competition leading to a drastic decrease in kindlin affinity for the β-CT indicates that the ternary talin-β-CT-kindlin complex is transient and rare.Second, unidirectional competition results from ternary talin-β-CT-kindlin complex formation.Third, assembly of the ternary talin-β-CT-kindlin complex is inconsistent with microscopic reversibility, suggesting that complex conformational change(s) are involved in the asymmetric binding behavior of talin and kindlin.
In search of such conformational changes, we first looked for a direct interaction between talin and kindlin that may influence their affinities for β-CTs.Although a direct interaction between talin and kindlin has not been reported thus far 14 , we detected, by NMR and cross-linking mass spectrometry, an interaction between the C-terminal α-helix of the TLN1-F3 domain and the N-terminal F0 domain of KIND2.Mutational disruption of the interaction decreased the population of ternary talin-β-CT-kindlin complexes by shifting the unidirectional competition toward bidirectional competition between talin and kindlin, in which kindlin decreases the affinity of talin and talin decreases the affinity of kindlin for β-CTs, although the latter less potently when compared with the competition studies in which the talin-kindlin interaction is intact.These findings indicate that, upon assembly of the ternary talin-β-CT-kindlin complex, kindlin induces a major increase in affinity of talin for the β 1 -CT and talin profoundly decreases kindlin affinity for the β 1 -CT.The increase in talin affinity for the β 1 -CT induced by kindlin could indeed be confirmed with a talinkindlin construct connected by a flexible peptide linker.
Because β-CTs deficient for talin or kindlin binding excluded a role of direct talin-kindlin binding for inducing conformational changes in talin or kindlin that account for their asymmetric affinity behavior for the β-CT, we searched for a conformational communication of the talinand kindlin-binding regions in the β 1 -CT that changes a low talin-affinity β-CT (termed 'inactive' β-CT) to a high talin-affinity β-CT (termed 'active' β-CT) conformation (Fig. 6a).We found secondary structure changes in the β 1 -CT region by NMR and could abolish allosteric coupling by introducing a spacer between the talin-and kindlin-binding sites that also abrogates asymmetric binding and curbs adhesion and spreading in cells.Interestingly, the population of ternary complexes is small, which is in line with single-molecule force measurements of RGD-bound α V β 3 -and α 5 β 1 -integrins in cells 29 that identified less than 10% of integrins in focal adhesions as transmitting high forces (exceeding 11 pN) and the remaining integrins, probably occupied only by either talin or kindlin, as transmitting low forces (below 11 pN).
THD1 is a FERM domain, and its crystal structure revealed both an atypical, linear and a canonical, cloverleaf-like conformation 5,30 .The cloverleaf-like conformation displays an interdomain interaction between D125 and E126 of TLN1-F1 and K401 and K402 of the TLN1-F3 domain, the latter of which is directly adjacent to the talin-kindlin interaction site identified in our study (Extended Data Fig. 6).It is conceivable that the linear and cloverleaf conformations of THD1 are in equilibrium, and a preference of KIND2 binding for the linear TLN1 conformation might increase membrane affinity via accessibility of additional membrane contacts by the TLN1-F0 and TLN1-F1 domains.This hypothesis is supported by our FC-RDA experiments, which showed that unidirectional talin-mediated kindlin competition is more pronounced from β 1 -TM-CT-and β 3 -TM-CT-containing nanodiscs than from β-CTs in solution.Hence, the interaction between talin and kindlin may also promote talin activation by increasing talin affinity for the plasma membrane, whereas interactions occurring simultaneously between talin, β-CT and kindlin increase talin affinity for the β-CT.
Our data identify a complex mechanism underlying talin and kindlin cooperativity (Fig. 6b).Based on experimental evidence, we envision that talin-kindlin cooperativity commences with kindlin, which encounters the integrin before talin 31 .Because talin and kindlin compete for the inactive β-CT conformation, direct talin-kindlin interaction may help to overcome low talin affinity for the kindlin-occupied β-CT and keep talin close to the β-CT until the active β-CT conformation is induced.Binding of talin and kindlin induces the 'active' β-CT conformation, which increases talin affinity for the β-CT, reinforces talin-β-CT binding and enhances resistance to actomyosin pulling forces.The fascinating feature of the asymmetric binding behavior is that it is inherently autonomous: elevated talin affinity for β-CT results in a decrease in kindlin affinity for the active β-CT conformation, leading to kindlin unbinding, a decrease in talin affinity and eventually talin dissociation, integrin inactivation and initiation of a new cycle of ternary talin-β-CT-kindlin assembly. https://doi.org/10.1038/s41594-023-01139-9

Cloning
Deletions, mutations and short insertions in complementary DNA were introduced by PCR using PfuUltra II (600670, Agilent Technologies) or Q5 Hot Start polymerase (M0493, New England Biolabs) according to the manufacturer's protocol, followed by DpnI (R0176, New England Biolabs) digestion of template DNA (2 h, 37 °C).After cleanup (QIAquick PCR Purification Kit, 28104, Qiagen), the PCR product was phosphorylated using T4 PNK (M0201, New England Biolabs), followed by another cleanup, ligated (Fast-Link DNA Ligation Kit, Epicenter) for 30 min at room temperature and transformed into Escherichia coli OmniMAX (Promega) or NEB 5-alpha competent E. coli (New England Biolabs).
Long insertions were introduced using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, E5520S) according to the manufacturer's protocol.The assembled constructs were transformed into E. coli OmniMAX or NEB 5-alpha competent E. coli.
DNA encoding the β 1A -CT was synthesized by Eurofins Genomics.

Protein expression and purification
Kindlin-2.KIND2 and ΔKIND2 were expressed and purified as described earlier 1  N isotopes were determined by mass spectrometry and were >98%.
After the final chromatography step, the purity, integrity and identity of recombinant KIND2 and talin proteins were controlled by SDS-PAGE, high-resolution mass spectrometry and dynamic light scattering (DLS).Integrin TM-CT peptides were controlled by SDS-PAGE and high-resolution mass spectrometry.Peptides were controlled by high-resolution mass spectrometry.

Fluorescent labeling and biotinylation of proteins
Before labeling, proteins were transferred into a buffer suitable for the intended labeling reaction using desalting columns.Integrin CTs were labeled with ATTO 488 N-hydroxysuccinimide (NHS) at their N terminus during chemical synthesis (by the MPIB core service facility), integrin TM-CTs were labeled with biotin-maleimide, whereas kindlin and talin were labeled with ATTO 565 NHS or Alexa 647 NHS or maleimide.For maleimide labeling, the pH value was adjusted to 7.0-7.5 using 1 M Tris, pH 7.5, and cysteines were reduced by adding TCEP to a final concentration of 2 mM before the reaction.Next, thiol-reactive dye was added at a molar excess of 10-20× and incubated at room temperature for 2 h in the dark or at 4 °C overnight.For amino-reactive dyes, the protein was first transferred to NHS labeling buffer (PBS with 10 mM NaHCO 3 , pH 9.0) and then mixed with dye at a molar excess of 2.5×.The labeling reaction was carried out for 1 h at room temperature or overnight on ice in the dark.Excessive dye was removed with desalting columns.
For nanodisc assembly, integrin TM-CT, MSP2N2 scaffold protein (obtained from the MPIB Biochemistry Core Facility) and lipid stock solution were mixed 1:1:330 to obtain on average one integrin TM-CT per nanodisc.Before adding lipids to the mixture, cholate buffer was added to obtain a final cholate concentration between 10 and 20 mM and to avoid precipitation of lipids.The samples were then dialyzed at least three times against 1 l of nanodisc buffer (20 mM HEPES, pH 7.5, 150 mM sodium chloride, 0.5 mM EDTA) at room temperature, filter sterilized and purified with a Superdex 200 Increase 10/300 GL (GE Healthcare) or an SEC 650 (Bio-Rad) column to separate assembled nanodiscs from non-assembled components.The elution fractions were analyzed by SDS-PAGE with silver staining and flow cytometry for talin and/or kindlin binding.

Microscale thermophoresis measurements
All MST measurements were performed as published 34 on a Monolith NT.115 red-blue machine (NanoTemper) using premium coated capillaries to reduce nonspecific interaction of proteins with the glass surface.Both interaction partners (ligand and receptor) were transferred into MST buffer (20 mM Tris, pH 7.5, 200 mM sodium chloride, 1 mM TCEP, 0.05% Tween-20) to avoid artifacts derived from buffer mismatches.ATTO 488-labeled integrin-β-CTs (50-200 nM, synthesized by the MPIB Core Facility) were used as ligands.Measurements were carried out at 10-20% LED power and 20% and 40% MST power.Data were analyzed using MO.Affinity Analysis Software (NanoTemper) as shown in Extended Data Fig. 1e.MST figures display data from individual titrations that were pooled (circles) and fitted with a global one-site-binding curve (lines).Affinity data in the figure insets (bar charts), in Extended Data Table 1 and in the text are mean ± s.d. of the replicates, which are given as n.
For data analysis, beads were gated in FlowJo and used as the negative control without protein and nanodiscs.MFIs of the samples were determined and exported to OriginPro 2019b.Here, data were fitted to a dose-response curve, setting top and bottom asymptotes to the values measured for the positive and negative controls, respectively.The positive control was measured in the presence of nanodiscs but in the absence of competitor, while, in the negative control, competitor and nanodiscs were absent.We report IC 50 values of these fits of individual experiments with different nanodisc preparations, which were recorded on different days as mean ± s.d.

Dynamic light scattering
Before conducting a DLS measurement, protein samples were centrifuged for 15 min at 21,000g and 4 °C.DLS measurements were performed in triplicate on a DynaPro NanoStar instrument (Wyatt) at 20 °C with laser power set to auto-attenuation, an acquisition time of 5 s and 15 acquisitions.The hydrodynamic radius of the particles in the sample was calculated with Dynamics software (Wyatt).

Circular dichroism spectroscopy
CD spectra were acquired using a quartz cuvette with a path length of 1 cm and a sample volume of 300 µl in a Jasco J-715 spectropolarimeter.Before the measurements, a buffer reference and protein samples (concentration, 0.1 mg ml −1 ) were prepared with PBS buffer.First, a CD spectrum of the buffer sample was acquired from 190 nm to 250 nm at 25 °C.Afterward, buffer was removed from the cuvette, and the protein sample was added and measured using the same parameters.The buffer reference spectrum was subtracted from the protein spectrum to eliminate effects caused by the buffer.

Thermal stability
Thermal stability was measured on a Prometheus NT.48 (NanoTemper) using a temperature gradient from 20 to 95 °C with an increase in temperature of 1 °C min −1 while measuring internal tryptophan fluorescence at λ = 330 nm.

NMR spectroscopy
For triple-resonance experiments with the β 1 -CT, M9 medium was supplemented with 0.5 mg ml −1 [ 15 N]ammonium chloride or with 2 mg ml −1 [ 13 C]glucose.All experiments were performed in NMR buffer consisting of 20 mM Tris, 200 mM NaCl, 1 mM DTT, pH 7.5 and 5-10% D 2 O for the NMR lock signal.For deuterated β 1 -CT samples, cells were adapted to deuterated M9 minimal medium in steps of 0%, 50% and 80% D 2 O supplemented with 0.5 mg ml −1 [ 15 N]ammonium chloride and 2 mg ml −1 [ 13 C]glucose, whereas ΔK2-and T1-F3-expressing cells were grown in 67% D 2 O M9 minimal medium.All datasets were acquired from Bruker Avance III spectrometers at a proton frequency of 600-800 MHz equipped with triple-resonance cryoprobes using TopSpin 3.2-3.5 software.Data were processed with TopSpin or NMRPipe and analyzed using CcpNmr Analysis software.Sample concentrations ranged from 70 µM to 700 µM, with all NMR experiments carried out at 298 K.
Protein backbone resonance assignments of β 1 -CT were obtained using 3D HNCO, HNcaCO, HNCACB and CBCAcoNH experiments.Assignments for β 1 -CT methyl resonances were performed using 3D (H)C(CCO)NH and H(CCCO)NH.Titrations were performed by forming binary or ternary complexes of either 15 N-or 13 C, 15 N-isotope-labeled integrin β1-CT mixed with unlabeled TLN1-F3 and ΔKIND2 at the indicated stoichiometries.For each titration point, 1 H- 13 C constant time HSQC or 1 H-15 N HSQC spectra were recorded.
For NMR titration measurements, wild-type TLN1-F3 and the TLN1-F3 K402E mutant were expressed in M9 medium supplemented with 0.5 mg ml −1 [ 15 N]ammonium chloride.Backbone assignments for wild-type TLN1-F3 were transferred from a previously published study in the Biological Magnetic Resonance Data Bank (BMRB 7061).Titrations were performed by forming binary or ternary complexes of 15 N-labeled wild-type TLN1-F3 or TLN1-F3 K402E protein with unlabeled β 1 -CT or ΔKIND2 at the indicated stoichiometry.For KIND2-F0, assignments were transferred from the Biological Magnetic Resonance Data Bank (BMRB 30659).About 47% of the backbone amide chemical shifts could be transferred by comparing 1 H-15 N correlation spectra.Other signals exhibited some chemical shift differences or could not be unambiguously identified (Supplementary Table 1).Titrations involving wild-type KIND2-F0 or KIND2-F0 Y13 were performed at the indicated stoichiometries.For all measurements, 1 H-15 N HSQC spectra were recorded at each point of the titration, the chemical shift changes of amide resonances in the fast-exchange regime were measured, and the reported weighted-average values of 15 N and 1 H chemical shift changes are given by equation ( 3): https://doi.org/10.1038/s41594-023-01139-9

Cross-linking of proteins
The TLN1-F3-β1-CT fusion protein was mixed with KIND2 at a molar excess of 7.5-fold and concentrated to a final concentration of 60 µM TLN1-F3-β1-CT and 450 µM KIND2 in 120 µl TBS.The protein mixture was rebuffered against PBS using a 0.5-ml Zeba Spin column according to the manufacturer's protocol and subjected to chemical cross-linking using the GraFix method as described earlier 35 .In brief, 5-20% sucrose gradients were generated in SW 40 ultracentrifuge tubes using a Gradient Master station (model IP, Biocomp) with 0.5 mM DSS in the heavy solution.Concentrated proteins were added to the tubes on top of the gradients, and the setup was centrifuged in an SW 40 Ti swing bucket rotor at 40,000 r.p.m. for 16 h (Beckman Coulter).After centrifugation, the gradients were fractionated on the Gradient Master station coupled to a Bio-Rad fraction collector, and fractions were analyzed for complex-containing fractions by SDS-PAGE and Coomassie staining.Fractions of interest were analyzed by mass spectroscopy.

Cross-linking mass spectrometry
Cross-linked protein pellets were incubated in digestion buffer (1:1; 1% SDC, 40 mM CAA, 10 mM TCEP, 50 mM Tris) for 20 min at 37 °C and then diluted with water (VWR) and finally digested at 37 °C overnight with 2 µg trypsin (Promega).The peptide mixture was acidified, desalted with Sep-Pak C18 1 cc vacuum cartridges (Waters), dried in a vacuum and dissolved in buffer A (0.1% formic acid, at a concentration of 400 ng µl −1 ).The peptides (400 ng) were separated with the Thermo EASY-nLC 1200 System (Thermo Fisher Scientific; flow rate of 250 nl min −1 ), equipped with a 30-cm analytical column (inner diameter, 75 µm; packed in house with ReproSil-Pur C18-AQ 1.9-µm beads, Dr. Maisch) coupled to the benchtop Orbitrap Q Exactive HF (Thermo Fisher Scientific) mass spectrometer, with an increasing gradient of buffer B (80% acetonitrile, 0.1% formic acid).The raw data were processed with Proteome Discoverer (version 2.5.0.400) with XlinkX/PD nodes integrated 36 .DSS or BS3 was set as a cross-linker, cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation and protein N-terminal acetylation were set as dynamic modifications.'Trypsin/P' was specified as the protease, and up to two missed cleavages were allowed.Identifications were only accepted with a minimal score of 40 and a minimal delta score of 4. Filtering at a false discovery rate of 1% was calculated with the XlinkX Validator node with the setting 'simple'.

Generation of the structural model
As structural data are unavailable for critical regions of the proteins used in our study, we generated the structural model manually.To this end, we used the crystal structure of the β1D-CT-TLN2 complex (PDB 3G9W) because the structures of neither β 1A -CT nor β 1A -CT in complex with TLN1 (β 1A -CT-TLN1) have been solved yet.Because the kindlin-binding site of β 1D -CT differs from that of β 1A -CT and is not resolved in the structure, we used the β 1A -CT-ΔKIND2 crystal structure (PDB 5XQ0) and aligned the resolved amino acids of β 1A -CT-ΔKIND2 and β 1D -CT-TLN2.Furthermore, structural information of the flexible N terminus of the KIND2-F0 domain is also not resolved in any of the published KIND2 crystal structures.Therefore, we aligned the NMR structures of the individual KIND2-F0 domain (PDB 6U4N) with the model.Subsequently, we mapped the intramolecular and intermolecular cross-links obtained in our cross-linking mass spectrometry experiments onto these assembled published structures (TLN2-F2F3-β 1D 11 , ΔKIND2-β 1A 4 and KIND2-F0 (ref.37)) using the XMAS plugin 38 for ChimeraX 39 .We colored cross-links of the relevant distance of 5-30 Å in yellow and all remaining cross-links in red and then iteratively adjusted the orientation of TLN1, KIND2 and β1-CT toward each other until the maximal number of cross-links were of the relevant distance.

Cell culture
Cells were grown and maintained in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin on 10-cm Petri dishes.At about 80% confluency, cells were washed with 5 ml PBS and detached with 1 ml 0.05% trypsin-EDTA in PBS at room temperature before adding 5 ml warm DMEM with 10% FBS.Cells were sedimented by centrifugation (5 min, 350g, room temperature), the supernatant was removed, and the cells were resuspended in 5 ml warm DMEM with 10% FBS.Cells were stained with Trypan blue to determine cell count and viability (EVE, NanoEnTek).Next, 200,000-400,000 cells were seeded in 10 ml DMEM with 10% FBS and 1% penicillin-streptomycin in 10-cm Petri dishes.All applied cell lines regularly tested negative for mycoplasma contamination.
The plasmid encoding murine TLN1-YPet in the retroviral vector pLPCXmod has been described previously 40 .The TLN1 K402E mutation was introduced by site-specific mutagenesis as described in Cloning.The plasmid encoding mCherry-KIND2 is based on EGFP-KIND2 in the retroviral vector pRetroQ-AcGFP-C1 (Clontech) as published earlier 1 .The plasmid encoding human β 1 -integrin in the retroviral vector pLZRS was described earlier 41 .

Flow cytometry
Around 400,000 cells were seeded into a well of a six-well cell culture plate the day before performing flow cytometry.The cells were detached from the culture plates with 500 µl trypsin and EDTA in PBS, trypsin was neutralized with 500 µl DMEM medium supplemented with 10% FBS, and samples were transferred into 4 ml DMEM supplemented with 10% FBS and split into different FACS tubes or 96-well plates.The medium was removed by centrifugation (5 min, 350g, 4 °C), and cells were washed twice with cold PBS and incubated with primary antibodies (9EG7 monoclonal antibody, which binds extended β 1 -integrin (1:100), or total β 1 -integrin (1:200), diluted in adhesion buffer (PBS, 3% BSA, 4.5 g l −1 glucose, 1 mM calcium chloride, 1 mM magnesium chloride)) for 30 min on ice.After washing with cold PBS, secondary antibodies (anti-rat 647 and streptavidin-720) were diluted 1:500 in adhesion buffer and added to cells for 30 min on ice.The cells were washed again with cold PBS and suspended in PBS supplemented with 3% BSA.For integrin profiling, cells were incubated after washing with PBS at a 1:50 dilution of PE-labeled anti-β 1 -integrin, anti-β 3 -integrin, anti-α 5 -integrin, anti-α V -integrin antibodies or the corresponding isotype controls in PBS supplemented with 3% BSA for 15 min at room temperature.All measurements were performed with an LSRFortessa X-20 flow cytometer (BD Biosciences).Data were analyzed with FlowJo 10 and OriginPro 2019b.Binding of the 9EG7 antibody was normalized to total β 1 -integrin levels (9EG7 signal divided by total β 1 signal).For integrin profiling, the MFI for each integrin staining was first corrected for its corresponding isotype control, and then data from TLN1-YPet K402E dKO and qKO cells were normalized to wild-type TLN1-YPet dKO and qKO cells.Each data point shown originates from an experiment on an individual day.Bar charts show mean ± s.d.https://doi.org/10.1038/s41594-023-01139-9

Focal adhesion analysis
Eight-well glass slides (tissue culture treated, 0030 742.036,Eppendorf) were coated with 5 µg ml −1 FN (341635, Merck) in PBS for at least 30 min at 37 °C, followed by blocking with 3% BSA in PBS for at least 30 min at 37 °C.After washing the wells with PBS and DMEM, 5,000-10,000 cells serum-starved for 4 h in DMEM were seeded and incubated for 40 min at 37 °C.The medium was removed, and the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature, followed by DAPI staining (1:10,000 in PBS) for 5 min at room temperature and three washing steps with PBS.The cells were imaged on a custom-made TIRF microscope (VisiTIRF, Visitron Systems) based on an Observer Z1 microscopy stand (Carl Zeiss).TIRF illumination was performed with a ×100 TIRF objective (Plan-Apochromat ×100/1.46Oil, Carl Zeiss).Focal adhesion properties were analyzed based on TIRF images recorded in the 561-nm laser channel for the mCherry-KIND2 signal using the Focal Adhesion Analysis Server, setting the threshold to 2 and the minimal adhesion size to five pixels 42 .

Spreading
Eight-well slides (µ-Slides, 80826, ibiTreat, ibidi) or six-well plates with hydrogels of different stiffnesses (Cell Guidance Systems) were coated with 5 µg ml −1 FN (341635, Merck) in PBS, 2.5 µg ml −1 VN (07180, Stemcell Technologies) or 10 µg ml −1 laminin-111 in PBS (L2020, Sigma-Aldrich) for at least 30 min at 37 °C, followed by blocking with 3% BSA in PBS for at least 30 min at 37 °C.After washing the wells with PBS and DMEM, 5,000-10,000 cells serum-starved for 4 h in DMEM were seeded and incubated for different times.When working with eight-well slides, the medium was afterward removed, and the cells were fixed with 4% PFA in PBS for 10 min at room temperature, followed by DAPI staining (1:10,000 in PBS) for 5 min at room temperature and three washing steps with PBS.When working with six-well plates and hydrogels, cells were incubated for 24 h, washed with PBS and immediately imaged to avoid artifacts from overfixation.Cells were imaged on an EVOS FL Auto 2 microscope (Invitrogen).The area of at least 50 individual cells was determined with Fiji ImageJ using the fluorescent signal of the reconstituted TLN1-YPet constructs.

Single-cell force spectroscopy
For cantilever functionalization, cantilevers (NP-0, Bruker) were first plasma cleaned (PDC-32G, Harrick Plasma) and then incubated overnight in PBS containing concanavalin A (2 mg ml −1 , Sigma-Aldrich) at 4 °C 43 .For substrate coating, 200-µm-thick four-segmented polydimethylsilane masks were fused to glass surfaces of Petri dishes (WPI) 44 .Polydimethylsilane-framed glass surfaces were incubated overnight with an FN fragment (FNIII7-10 RGD , 50 µg ml −1 in PBS) at 4 °C.A NanoWizard II AFM equipped with a CellHesion module (both from JPK Instruments) mounted on an inverted fluorescence microscope (Observer Z1, Zeiss) was used for SCFS.The temperature was controlled at 37 °C by a PetriDishHeater ( JPK Instruments).Tipless V-shaped silicon nitride cantilevers (200 µm long, NP-0) having nominal spring constants of 0.06 N m −1 were used.Each cantilever was calibrated before measurement by determining its sensitivity and spring constant using the thermal noise analysis of the AFM.
Fibroblasts were grown to a confluency of ~80%, washed with PBS, detached with trypsin for 2 min, suspended in SCFS medium (bicarbonate-free DMEM supplemented with 20 mM HEPES) containing 1% (vol/vol) FCS and centrifuged, and the sedimented cells were resuspended in serum-free SCFS medium.Fibroblasts were allowed to recover from trypsin detachment for at least 30 min 45 .Afterward, suspended fibroblasts were pipetted onto substrate-coated Petri dishes, and the functionalized cantilever was lowered onto a single fibroblast with a speed of 10 µm s −1 until a force of 5 nN was recorded.After 5 s of contact, the cantilever was retracted at 10 µm s −1 until the cell was completely detached from the substrate, and the cantilever-bound fibroblast was incubated for 3-5 min on the cantilever to ensure firm Bacterial strains Merck Millipore, 70954-3 https://doi.org/10.1038/s41594-023-01139-9 binding to the cantilever.The morphological state of the fibroblast was monitored by optical microscopy throughout adhesion experiments.Round, cantilever-bound fibroblasts approached the substrate at 5 µm s −1 until a contact force of 1 nN was recorded.Throughout the contact times of 5, 20, 60, 120 or 240 s, the height of the cantilever was maintained constant and subsequently retracted at 5 µm s −1 for 100 µm until the fibroblast was fully separated from the substrate.Before another experimental cycle was initiated, the cantilever-bound fibroblast was allowed to recover for a time period equal to the contact time.A single fibroblast was used to probe the adhesion force for all contact times once or until morphological changes (that is, spreading) were observed.The sequence of contact times and area on the substrate were varied.Adhesion forces were determined from force-distance curves as the maximum downward deflection of the cantilever after baseline correction using JPK software ( JPK Instruments).

Plate-and-wash assay
Untreated 96-well plates were coated with 100 µl of 10 µg ml −1 FN or a 1:10 dilution of poly-l-lysine (Sigma) in 20 mM Tris, pH 8.8, overnight at 4 °C.Next, 200 µl of 3% BSA in PBS was added to the wells for at least 30 min at 37 °C.The plates were washed with 200 µl PBS and 200 µl DMEM medium before seeding 40,000 serum-starved cells in 100 µl DMEM.After incubating for 30 min at 37 °C, the whole 96-well plate was washed by immersing in PBS three times, followed by fixation in 4% PFA for 10 min at room temperature.The cells were stained with 75 µl crystal violet solution for 30 min at room temperature and washed three times by immersing with dH 2 O.The wells were incubated with 100 µl 2% SDS while shaking at room temperature, until all crystal violet was dissolved.Absorption was measured at λ = 595 nm in a SpectraMax ABS plate reader (Molecular Devices).The data for every cell line were first blanked to those of control wells coated with only BSA and then corrected for the plated cell count obtained from poly-l-lysine-coated wells.Finally, data were normalized to those of the wild-type β 1 -expressing cell line.

Reagents
Reagents used in the study are detailed in Table 1.

bFig. 6 |
Fig. 6 | Model of ternary talin-β-CT-kindlin complex assembly and disassembly.a, Ternary complexes forming between talin, β-CT and kindlin in the presence or the absence of talin-kindlin binding.Because talin and kindlin compete for the inactive β-CT conformation, direct talin-kindlin interaction may help to overcome low talin affinity for the kindlin-occupied β-CT during ternary talin-β-CT-kindlin complex assembly.b, Talin-kindlin cooperativity involves a switch from an inactive to an active β-CT conformation.Kindlin interacts with inactive (bent-closed) and active (extended-open) integrins.Binding of kindlin to the extended-open integrin activates the β-CT, leading to high talin affinity and high resistance to actomyosin pulling forces.High talin affinity in turn decreases kindlin affinity for the β-CT, resulting in kindlin dissociation from the β-CT, β-CT inactivation, a decrease in talin affinity and the integrin bent-closed state.Talin-kindlin interaction at the β-CT maintains a low population of ternary talin-β-CT-kindlin complexes by helping to overcome low talin affinity for the kindlin-occupied β-CT until the active β-CT conformation is induced and the ternary complex is assembled.The low population of ternary complexes in focal adhesions is able to transmit high forces, while the remaining integrins, occupied by only talin or kindlin, transmit low forces.
Two samples were compared to each other using a two-sample two-tailed Student's t-test (Figs.1e,f, 3l,j and 5a and Extended Data Figs.1g,h,l,j,m, 3k-n and 4a,b) or a two-tailed Mann-Whitney test (Fig.5d,e).For comparisons with more than two samples, one-way repeated-measures ANOVA with Tukey's post hoc test was used (Fig.5c,f,g and Extended Data Fig.5e,f,I,j).Normalized data were tested with a one-sample two-tailed Student's t-test for a difference from 1 for comparison with the reference sample (Fig.5aand Extended Data Fig.5d,h,i).P values from statistical tests are written in the figures over the corresponding bar chart or box plot in black, whereas n is written in the same color as the chart or plot.Unless stated otherwise, bar charts represent average values and error bars represent standard deviations of individual experiments or analyzed single cells.In box plots, the boxes show the upper and lower quartiles and the median and the error bars show standard deviations of individual experiments.

Table 1 | Mean affinities with standard deviation measured in this study
1 WT and β 1 -spacer cells 30 min after seeding on FN in absence and presence of MnCl 2 and/or cilengitide (CGN).Data normalized to values from β 1 WT cells cultured in absence of MnCl 2 and CGN (n = 3).j) Spreading area of serum-starved β 1 knockout (KO), β 1 WT and β 1 -spacer cells 1 h after seeding on FN in absence and presence of MnCl 2 and/or CGN.Cells were stained with DAPI and CellMask orange and areas of ≥50 cells were analyzed on three different days.