Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn2+. Despite compromised integrin activation and adhesion, Mn2+ enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner. DOI: http://dx.doi.org/10.7554/eLife.10130.001


Introduction
Integrins are heterodimeric transmembrane receptors that mediate cell adhesion to the extracellular matrix (ECM) and to other cells (Hynes, 2002). The consequence of integrin-mediated adhesion is the assembly of a large molecular network that induces various signaling pathways, resulting in cell migration, proliferation, survival and differentiation (Winograd-Katz et al., 2014). The quality and strength of integrin signaling is controlled by the interaction between integrins and substrateattached ligands, which is, in turn, regulated by the on-and off-rates of the integrin-ligand binding process. The on-rate of the integrin-ligand binding reaction (also called integrin activation or insideout signaling) is characterized by switching the unbound form of integrins from an inactive (low affinity) to an active (high affinity) conformation. The affinity switch proceeds from a bent and clasped low affinity conformation to an extended and unclasped high affinity conformation with an open ligand-binding pocket (Askari et al., 2010;Springer and Dustin, 2012). This change in affinity is believed to be induced through the binding of talin and kindlin to the b integrin cytoplasmic domain (Moser et al., 2009;Shattil et al., 2010) and divalent cations to distinct sites close to the ligandbinding pocket (Gailit and Ruoslahti, 1988;Mould et al., 1995;Xia and Springer, 2014;Mould et al., 2003).
The stabilisation of integrin-ligand complexes is mediated by integrin clustering and catch bond formation between integrin and bound ligand. The stabilizing effect of clustered integrins is achieved by the ability of dissociated integrin-ligand complexes to rebind before they leave the adhesion site (van Kooyk and Figdor, 2000;Roca-Cusachs et al., 2009), while catch bonds are receptor-ligand bonds whose lifetime increases with mechanical force (Kong et al., 2009;Chen et al., 2010;Kong et al., 2013). Both mechanisms extend the duration and increase the strength of integrin-mediated adhesion and signaling (also called outside-in signaling) (Koo et al., 2002;van Kooyk and Figdor, 2000;Maheshwari et al., 2000;Roca-Cusachs et al., 2009;Coussen et al., 2002), and depend on the association of integrins with the actin cytoskeleton via talin (Roca-Cusachs et al., 2009;Friedland et al., 2009), and probably kindlin (Ye et al., 2013).
The talin family consists of two (talin-1 and -2) and the kindlin family of three isoforms (kindlin-1-3), which show tissue-specific expression patterns (Calderwood et al., 2013;Moser et al., 2009;Shattil et al., 2010). The majority of studies that defined integrin affinity regulation by talin and kindlin were performed on aIIbb3 and b2-class integrins expressed by platelets and leukocytes, respectively. These cells circulate in the blood and hold their integrins in an inactive state until they encounter soluble or membrane-bound agonists (Evans et al., 2009, Bennett, 2005. The prevailing view is that agonist-induced signaling pathways activate talin-1 and the hematopoietic cell-specific kindlin-3, which cooperate to induce integrin activation Han et al., 2006) and clustering (Cluzel et al., 2005;Ye et al., 2013).
Integrin affinity regulation in non-hematopoietic cells such as fibroblasts and epithelial cells is poorly understood. It is not known how integrin activation is induced on these cells (no integrinactivating agonists have been identified) and it is also controversial whether talin and kindlin are required to shift their integrins into the high affinity state. While there are reports showing that talin and kindlin are required for integrin activation in epithelial cells (Montanez et al., 2008;Margadant et al., 2012), it was also shown that in myoblasts and mammary epithelial cells activation of b1 integrins, adhesion and spreading on multiple ECM substrates can proceed in the absence of talin (Conti et al., 2009;Wang et al., 2011). Likewise, it was reported that focal adhesion eLife digest A meshwork of proteins called the extracellular matrix surrounds the cells that make up our tissues. Integrins are adhesion proteins that sit on the membrane surrounding each cell and bind to the matrix proteins. These adhesive interactions control many aspects of cell behavior such as their ability to divide, move and survive.
Before integrins can bind to the extracellular matrix they must be activated. Previous research has shown that in certain types of blood cells, proteins called talins and kindlins perform this activation. These proteins bind to the part of the integrin that extends into the cell, causing shape changes to the integrin that allow binding to the extracellular matrix. However, it is not clear whether talin and kindlin also activate integrins in other cell types.
Fibroblasts are cells that help to make extracellular matrix proteins, and are an important part of connective tissue. Theodosiou et al. engineered mouse fibroblast cells to lack either talin or kindlin, and found that both of these mutant cell types were unable to activate their integrins and as a result failed to bind to an extracellular matrix protein called fibronectin.
Even when cells were artificially induced to activate integrins by treating them with manganese ions, cells lacking talin or kindlin failed to fully activate integrins and hence did not adhere well to fibronectin. This suggests that talin and kindlin work together to activate integrins and to maintain them in this activated state.
When treated with manganese ions, cells that lacked talin were able to flatten and spread out, whereas cells that lacked kindlin were unable to undergo this shape change. Theodosiou et al. found that this cell shape is dependent on kindlin and its ability to bind to and recruit a protein called paxillin to "adhesion sites", where integrins connect the cell surface with the extracellular matrix. Kindlin and paxillin then work together to activate other signaling molecules to induce the cell spreading.
The next challenge is to understand how talin and kindlin are activated in non-blood cells and how they maintain integrins in an active state. kinase (FAK)-deficient fibroblasts develop small, nascent adhesions (NAs) at the edge of membrane protrusions without visible talin and that integrins carrying a mutation in the talin-binding site can still nucleate and stabilize NAs (Lawson et al., 2012). Also fibroblasts lacking talin-1 and -2 were shown to adhere to fibronectin (FN) and initiate isotropic spreading . Another intriguing study demonstrated that overexpression of kindlin-2 in Chinese hamster ovary (CHO) cells inhibits rather than promotes talin head-induced a5b1 integrin activation (Harburger et al., 2009). Given the fundamental importance of talin and kindlin for integrin activation in hematopoietic cells, the findings of these studies are unexpected and imply that either integrin affinity regulation is substantially different in fibroblasts and epithelial cells or the experimental approaches used to manipulate protein expression and localization were imperfect.
To directly evaluate the functions of talin and kindlins for FN-binding integrins on fibroblasts, we used a genetic approach and derived fibroblasts from mice lacking either the Tln1 and -2 or the Fermt1 and -2 genes. We show that integrin affinity regulation depends on both talin and kindlin, and that kindlin has the additional function of triggering cell spreading by binding directly to paxillin in a talin-independent manner.

Kindlins and talins control cell morphology, adhesion and integrin expression
To obtain cells lacking the expression of talin-1 and kindlin-2, we intercrossed mice carrying loxP flanked (floxed; fl) Tln1 or Fermt2 alleles ( Figure 1A), isolated kidney fibroblasts and immortalized them with the SV40 large T antigen (parental fibroblasts). The floxed alleles were deleted by adenoviral Cre recombinase transduction resulting in T1 Ko and K2 Ko fibroblasts. Loss of talin-1 or kindlin-2 expression in fibroblasts was compensated by talin-2 or the de novo expression of kindlin-1, respectively, allowing adhesion and spreading, although to a lesser extent compared with control cells (Figure 1-figure supplement 1A,B). To prevent this compensation, we generated mice with floxed Tln1 and nullizygous Tln2 alleles or with floxed Fermt1 and -2 alleles (Tln Ctr ; Kind Ctr ) from which we isolated, immortalized and cloned kidney fibroblasts with comparable integrin surface levels ( Figure 1A and Figure 1-figure supplement 2). The floxed alleles were deleted by transducing Cre resulting in talin-1, -2 (Tln Ko ) and kindlin-1, -2 (Kind Ko ) deficient cells, respectively ( Figure 1A-C). Since the Tln Ctr and Kind Ctr control cells showed similar morphologies and behaviour in our experiments, we display one control cell line in several result panels. Cre-mediated deletion of the floxed Tln1 or floxed Fermt1/2 genes was efficient ( Figure 1B) and resulted in cell rounding, weak adhesion of a few cells, and reduced cell proliferation despite the immortalisation with the oncogenic large T antigen ( Figure 1C and Figure 1-figure supplement 3). To minimize cell passage-induced abnormalities, we used cells only up to 12 passages after Cre-mediated gene deletions.
To define the adhesion defect, we performed plate and wash assays for 30 min on defined substrates and found that neither Tln Ko nor Kind Ko cells adhered to FN, laminin-111 (LN), type I collagen (COL) and vitronectin (VN) ( Figure 1D). To test whether the inability of Tln Ko and Kind Ko cells to adhere to ECM proteins is due to an integrin activation defect, we bypassed inside-out activation by treating cells with Mn 2+ , which binds to the integrin ectodomain and induces unbending and unclasping of integrin heterodimers (Mould et al., 1995). Treatment with Mn 2+ induced partial adhesion of Tln Ko and Kind Ko cells to FN, while partial adhesion to LN and VN was only induced in Tln Ko cells ( Figure 1D). Time course experiments revealed that Mn 2+ -induced adhesion of Tln Ko and Kind Ko cells to FN was already significantly lower 2.5 min after plating and remained significantly lower compared with control cells ( Figure 1E), suggesting that talin and kindlin cooperate to initiate and maintain normal Mn 2+ -induced adhesion to FN. In line with these findings, dose-response profiles showed that Tln Ko and Kind Ko cells have severe adhesion defects at low (1.25 mg ml -1 ) as well as high (20 mg ml -1 ) substrate concentrations (Figure 1-figure supplement 4).
These findings indicate that talin and kindlin promote integrin-mediated adhesion to FN and proliferation, and that the integrin-activating compound Mn 2+ can only partially substitute for the adhesion promoting roles that talin and kindlin accomplish together. Quantification of cell adhesion on indicated substrates 30 min after seeding by counting DAPI stained cells; n=3 independent experiments, error bars indicate standard error of the mean; t-test significances are calculated between untreated Tln Ko or Kind Ko cells and the corresponding Tln Ctr and Kind Ctr or Mn 2+ -treated Tln Ko or Kind Ko cell lines on same substrates; only significant differences are shown. (E) Quantification of Mn 2+ -stimulated cell adhesion for indicated times on FN; cells were quantified by absorbance measurement of crystal violet staining; n=3 independent experiments; lines represent sigmoidal curve fit; error bars indicate standard deviation; significances for indicated pairs after 2.5 min were calculated by two-tailed t-test and significances for indicated pairs of the overall kinetics were calculated by two-way RM ANOVA. Bar, 10 mm. COL, collagen; DAPI, 4',6-diamidino-2phenylindole; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LN, laminin-111; RM ANOVA, repeated measures analysis of variance; VN, vitronectin. DOI: 10.7554/eLife.10130.003 The following figure supplements are available for figure 1:  Integrin activation and binding to FN requires talin and kindlin-2 The inability of Mn 2+ to fully rescue the adhesion defect of Tln Ko and Kind Ko cells raised the question whether integrin surface levels change after deletion of the Tln1/2 and Fermt1/2 genes. We quantified integrin surface levels by flow cytometry and found that the levels of b1 and b3 were significantly reduced in Kind Ko and unaffected in Tln Ko cells ( Figure 2A and Figure 2-figure supplement 1). The levels of a2 and a3 integrin were reduced in both cell lines, a6 was elevated in Tln Ko and decreased in Kind Ko cells, and the a3 levels were significantly more decreased in Kind Ko than in Tln Ko cells ( Figure 2A) explaining the absent adhesion of both cell lines to COL and their differential adhesion behaviour on LN ( Figure 1D). The b5 levels were similarly up-regulated in Kind Ko and Tln Ko cells, and the a5 and av integrin levels were slightly reduced but not significantly different between Tln Ko and Kind Ko cells ( Figure 2A). The differential adhesion of Mn 2+ -treated Tln Ko and Kind Ko cells to VN ( Figure 1D), despite similar surface levels of av integrins, points to particularly important role(s) for kindlin-2 in av integrins-VN adhesion and signaling (Liao et al., 2015). Serendipitously, the reduced expression of b1-associating a2, a3 and a6 subunits in Kind Ko cells, which impairs adhesion to LN and COL enables a5 to associate with the remaining b1 subunits and leads to comparable a5b1 levels on Tln Ko  Since we excluded different surface levels of FN-binding integrins as a cause for the severely compromised adhesion of Tln Ko and Kind Ko cells to FN, we tested whether talin and kindlin are required to activate FN-binding a5b1 integrins. To directly assess integrin activation, we made use of an antibody against the 9EG7 epitope, which specifically recognizes Mn 2+ and/or ligand activated b1 integrins (Bazzoni et al., 1995). The amount of 9EG7 epitope exposure relative to total b1 integrin exposure corresponds to the integrin activation index, which can be measured by flow cytometry using 9EG7 and anti-total b1 integrin antibodies. These measurements revealed that Tln Ctr and Kind Ctr cells bound 9EG7 antibodies, while Tln Ko and Kind Ko cells lacked 9EG7 binding in the absence of Mn 2+ ( . Moreover, the normalization of the 9EG7 binding to the total b1 integrin surface levels also indicated a significantly lower influence of Mn 2+ and FN-RGD on the integrin activation index of Kind Ko as compared to Tln Ko cells ( Figure 2-figure supplement 3A). These findings confirm that both, talin and kindlin are required for b1 integrin activation and to stabilize Mn 2+ -induced unbending/unclasping of a5b1 integrins.
Our findings so far suggest that talin and kindlin are required to activate FN-binding integrins and maintain Mn 2+ -induced activation of FN-binding integrins. To further analyze whether ligandinduced stabilisation of high-affinity integrin conformations (also termed 'ligand-induced integrin activation; Du et al., 1991) can form in the absence of talin or kindlin, we used atomic force microscopy (AFM)-based single cell force spectroscopy (SCFS). We attached control, Tln Ko or Kind Ko cells to Concanavalin A (ConA)-coated cantilevers, allowed the cells to contact surfaces coated with either wild type FN-III 7-10 (FN-RGD) or an integrin-binding-deficient FN-III 7-10 fragment lacking the RGD binding motif (FN-DRGD) for increasing contact times, either in the absence or presence of Mn 2+ and then detached them from the substrate by lifting the cantilever ( Figure 2B,C). In the absence of Mn 2+ Tln Ctr and Kind Ctr cells developed significant adhesion to FN-RGD within 5 s contact time. After a contact time of 20 s around 2 nN force was required to disrupt adhesion to FN-RGD, and after 50 and 120 s, respectively, 3 and 6 nN were required ( Figure 2B). Tln Ko and Kind Ko cells failed to develop measurable adhesions to FN-RGD after contact times of 5, 20, 50 and 120 s ( Figure 2B). Treatment with Mn 2+ induced a slight and similar increase of force required to disrupt adhesion of    control, Tln Ko and Kind Ko cells to FN-RGD after 5 s contact time ( Figure 2C). However, with increasing contact times, the AFM profiles of Tln Ko and Kind Ko cells differ in the presence of Mn 2+ . While the adhesion force increased concomitantly with longer contact times in Tln Ctr , Kind Ctr and Tln Ko cells, adhesion forces of Kind Ko cells plateaued after 50 s and showed no further increase towards 120 s contact time. The latter finding suggests that kindlin stabilizes integrin-ligand complexes with time, by inducing integrin clustering and/or by modulating the off-rate of integrin ligand bonds, for example, through associating with the integrin-linked kinase (ILK)-Pinch-Parvin (IPP) complex that links kindlin to the F-actin cytoskeleton (Cluzel et al., 2005;Ye et al., 2013;Montanez et al., 2008;Fukuda et al., 2014).
We next tested whether their impaired integrin function affects the assembly of FN into fibrils, which requires association of active a5b1 integrin with the actin cytoskeleton (Pankov et al., 2000), and whether re-expression of talin and kindlin reverts the defects of Tln Ko and Kind Ko cells. While neither Tln Ko nor Kind Ko cells were able to assemble FN fibrils, re-expression of full-length Venustagged talin-1 (Tln1V) in Tln Ko or GFP-tagged kindlin-2 (K2GFP) in Kind Ko cells ( Altogether, our results demonstrate that both talin and kindlin are required (1) for ligand-induced stabilisation of integrin-ligand complexes, (2) to stabilize Mn 2+ -activated a5b1 integrins, and (3) to induce integrin-mediated FN fibril formation.

Tln Ko cells initiate spreading and assemble b1 integrins at protruding membranes
It has been reported that a significant number of talin-2 small interfering RNA (siRNA)-expressing talin-1 -/fibroblasts adhere to FN and initiate isotropic cells spreading . To test whether spreading can also be induced in adherent Tln Ko and Kind Ko cells, we bypassed their adhesion defect with Mn 2+ , seeded them for 30 min on FN and stained with an antibody against total b1 integrin and the b1 integrin activation epitope-reporting 9EG7 antibody. As expected, Tln Ctr or Kind Ctr cells clustered 9EG7-positive b1 integrins in NAs and focal adhesions (FAs), whose frequency and size increased upon Mn 2+ treatment ( Figure 3A,B). In contrast, the sporadic and very weakly adherent Tln Ko and Kind Ko cells were small, round and formed small and finely dispersed b1 integrin aggregates over the entire cell ( Figure 3A) and lacked 9EG7-positive signals ( Figure 3B) in the absence of Mn 2+ treatment. Upon Mn 2+ treatment 37 ± 1% (n=684, mean ± standard deviation of three independent experiments) of the Tln Ko cells showed isotropic membrane protrusions (circumferential lamellipodia) with small, dot-like aggregates of b1 integrin, kindlin-2, paxillin and ILK at the membrane periphery ( Figure 3A and Figure 3-figure supplement 1), which eventually detached from the substrate leading to the collapse of the protruded membrane (Video 1). Furthermore, 9EG7-positive b1 integrins accumulated along the lamellipodial edge and beneath the nucleus of Tln Ko cells ( Figure 3B). The remaining cells were spheroid, with half of them showing short, fingerlike protrusions, which were motile due to their poor anchorage to the substrate. In the case of Kind Ko cells, we analysed 652 cells in three independent experiments and found that only 7 ± 1% (mean ± standard deviation) of the cells established lamellipodia, which formed around the entire circumference in 2 ± 0.4% (mean ± standard deviation) of the cells. Around 93 ± 1% of the Kind Ko cells were spheroid (mean ± standard deviation) and frequently had finger-like, motile protrusions with small dot-like signals containing b1 integrin and talin but rarely paxillin or ILK ( Figure 3A  To further characterize the distribution of b1 integrins in the lamellipodial edges of Tln Ko cells, we visualized them by combining direct stochastic optical reconstruction microscopy (dSTORM) and total internal reflection fluorescence microscopy (TIRFM). Mn 2+ -treated and non-permeablized cells were seeded on FN, stained with anti-total b1 integrin antibodies, and then permeabilized, immunostained for paxillin and imaged with normal resolution TIRFM and dSTORM ( Figure 3C). Each localization detected by dSTORM was plotted as a Gaussian distribution around its centre with an average spatial accuracy of~20 nm (resolution limit of dSTORM imaging). Since two or more localizations from single or multiple dyes in close proximity cannot be distinguished, the number of localizations does not directly reflect integrin numbers. However, all antibody molecules display the same average behaviour with respect to the number of localizations per second in all areas of the cell. This allowed to average the number of localizations per second and mm 2 and to plot them in a heat map representation ( Figure 3D), which directly reflects the density of stained b1 integrin molecules and thus the degree of integrin clustering. The b1 integrin staining of Tln Ctr and Kind Ctr cells revealed small round structures of~50 nm diameter indicating clusters of integrins larger than the resolution limit ( Figure 3C; high magnification; see arrowheads). Furthermore, high numbers of localizations were enriched in paxillin-positive FAs and in NAs at the lamellipodial edge ( Figure 3C; see arrowheads). In these areas, we observed a high average density of 60-120 localizations s -1 mm -2 , while outside of the adhesion sites~0-20 localizations s -1 mm -2 were detected, indicating a high degree of b1 integrin clustering within and a low degree of clustering outside of adhesion sites ( Figure 3D). Tln Ko cells with circumferential lamellipodia showed a high density of blinking with up to 100 localizations s -1 mm -2 at lamellipodial edges ( Figure 3C,D; see arrowheads), which appeared less compact than in control cells. Kind Ko cells showed >120 localizations s -1 mm -2 in the periphery and finger-like membrane protrusions ( Figure 3C,D; see arrowheads), which were also observed in Tln Ko cells that adopted a spheroid rather than an isotropic spread shape (Figure 3-figure supplement 3). The exclusive presence of these large and entangled b1 integrin aggregates on Tln Ko and Kind Ko cells with small, spheroid shapes and protrusions suggests that they were induced by spatial constraints rather than specific signaling.
These findings demonstrate that, in contrast to Kind Ko cells, Mn 2+ -treated kindlin-2-expressing Tln Ko cells induce circumferential membrane protrusions with b1 integrins at the protrusive edges.

Kindlin-2 binds and recruits paxillin to NAs
Our data so far indicate that the expression of kindlin-2 enables initial, isotropic spreading and the accumulation of integrins in lamellipodia of Mn 2+ -treated Tln Ko cells. To identify binding partner(s) of kindlin-2 that transduce this function to downstream effectors, we performed yeast-two-hybrid assays with kindlin-2 as bait using a human complementary DNA (cDNA) library containing all possible open reading frames and a human keratinocyte-derived cDNA library. Among the 124 cDNAs identified from both screenings, 17 coded for leupaxin and 11 for Hic-5. Immunoprecipitation of overexpressed green fluorescent protein (GFP)-tagged paxillin family members, paxillin, Hic-5 and leupaxin in HEK-293 cells with an anti-GFP antibody efficiently co-precipitated FLAG-tagged kindlin-2 (K2flag) ( Figure 4A). Conversely, overexpressed GFP-tagged kindlin family members (kindlin-1, kindlin-2, kindlin-3) co-precipitated Cherry-paxillin ( the interaction between kindlin-2 and paxillin was dramatically reduced in the absence of the Lin-11, Isl-1 and Mec-3 (LIM)1-4, LIM2-4 or LIM3-4 domains of paxillin ( Figure 4B), or the pleckstrin homology (PH) domain (K2DPHGFP; lacking amino acids 380-477) or the N-terminus of kindlin-2 including the F0, F1, and the N-terminal part of the F2 domains (K2NTGFP; terminating at the end of F1; spanning amino acids 1-229) ( Figure 4C). As expected, the interaction between kindlin-2 and ILK (Montanez et al., 2008), which is mediated via a recently identified sequence in the linker domain between the end of the N-terminal F2 and the beginning of the PH domain (amino acids 353-357) (Fukuda et al., 2014;Huet-Calderwood et al., 2014), was abolished by the K2NTGFP truncation but unaffected by the deletion of the PH domain (K2DPHGFP) or the deletion of the N-terminal F0 and F1 domains (K2CTGFP, spanning amino acids 244-680) ( Figure 4C). Importantly, immunoprecipitation of Kind Ctr lysates with antibodies against paxillin co-precipitated kindlin-2 ( Figure 4D), confirming interactions between the endogenous proteins. Pull down experiments with recombinant full-length paxillin or paxillin-LIM3 domain and recombinant kindlin-2 demonstrated that binding to LIM3 and full-length paxillin is direct, Zn 2+ -dependent and abrogated with ethylenediaminetetraacetic acid (EDTA) ( Figure 4E and . Interestingly, mature FAs in K2DPHGFP-expressing cells were prominent after 30 min and contained significant amounts of paxillin, indicating that paxillin is recruited to mature FAs in a kindlin-2-independent manner ( Figure 4F).
These findings indicate that the PH domain of kindlin-2 directly binds the LIM3 domain of paxillin and recruits paxillin into NAs but not into mature FAs.

The kindlin-2/paxillin complex promotes FAK-mediated cell spreading
Our findings revealed that kindlin-2 is required to recruit paxillin to NAs. Paxillin in turn, was shown to bind, cluster and activate FAK in NAs, which leads to the recruitment of p130Cas, Crk and Dock followed by the activation of Rac1 and the induction of cell spreading, and, in concert with growth factor signals, to the activation of Akt-1 followed by the induction of cell proliferation and survival (Schlaepfer et al., 2004;Bouchard et al., 2007;Zhang et al., 2014;Brami-Cherrier et al., 2014). We therefore hypothesized that the recruitment of paxillin and FAK by kindlin-2 triggers the isotropic spreading and expansion of Tln Ko cells. To test this hypothesis, we seeded our cell lines on FN or poly-L-lysine (PLL) in the presence or absence of epidermal growth factor (EGF) and Mn 2+ . We found that EGF induced similar phosphorylation of tyrosine-992 (Y992) of the epidermal growth factor receptor (pY992-EGFR) in control, Tln Ko and Kind Ko cells. The phosphorylation of tyrosine-397 of FAK (pY379-FAK) in Kind Ctr cells was strongly induced after the adhesion of control cells on FN and was not further elevated after the addition of EGF and Mn 2+ ( Figure 5A and Figure 5-figure supplement 1). Tln Ko cells also increased pY397-FAK as well as pY31-Pxn and pY118-Pxn levels upon adhesion to FN, however, significantly less compared to control cells ( Figure 5A and  Figure 5B). Importantly, reexpression of Talin1-Venus in Tln Ko and Kindlin2-GFP and Kind Ko cells fully rescued these signaling defects ( Figure 5-figure supplement 1B,C). Furthermore, stable expression of K2GFP in Kind Ko cells rescued pY397-FAK and pS473-Akt levels ( Figure 5C) and co-precipitated paxillin and FAK with K2GFP ( Figure 5-figure supplement 2). In contrast, stable expression of K2DPHGFP in Kind Ko cells failed to co-precipitate paxillin and FAK ( Figure 5-figure supplement 2) and induce pY397-FAK and pS473-Akt ( Figure 5C).
In line with previous reports showing that the paxillin/FAK complex can trigger the activation of p130Cas (Zhang et al., 2014) and, in cooperation with EGFR signaling, the activation of Akt (Sulzmaier et al., 2014, Deakin et al., 2012, we observed Y410-p130Cas, pT308-Akt, S473-Akt and Altogether, these findings show that the kindlin-2/paxillin complex in NAs recruits and activates FAK to induce cell spreading and increase the strength of Akt signaling.

Discussion
While the functions of talin and kindlin for integrin activation, adhesion and integrin-dependent signaling in hematopoietic cells are firmly established, their roles for these processes in non-hematopoietic cells are less clear. To clarify this issue, we established mouse fibroblast cell lines that lacked either talin-1/2 (Tln Ko ) or kindlin-1/2 (Kind Ko ) and tested whether they were able to activate integrins and mediate substrate adhesion and signaling. In line with previous reports (Bottcher et al., 2012;Margadant et al., 2012), the deletion of Tln1/2 or Fermt1/2 genes changed the surface levels of laminin-and collagen-binding integrins. Since surface levels of a5 and av integrins remained unchanged between Tln Ko and Kind Ko cells, we were able to establish the specific roles of talin and kindlin for the function of FN-binding integrins under identical conditions.
A major finding of our study demonstrates that integrin affinity regulation (activation) is essential for fibroblast adhesion and depends on both talin and kindlin-2 ( Figure 6A,D). The unambiguity of this finding was unexpected in light of several reports showing that integrin activation and integrinmediated adhesion still occurs in talin-depleted cells, or is inhibited when kindlin-2 is overexpressed (Harburger et al., 2009;Wang et al., 2011;Lawson et al., 2012). The previous studies that addressed the functional properties of talin used siRNA-mediated protein depletion, a combination of gene ablation and siRNA technology, or approaches to interfere with talin recruitment to NAs either by ablating the talin upstream protein FAK or by expressing an integrin that harbors a mutation in the talin binding site. Since the majority of approaches deplete rather than eliminate proteins from cells and adhesion sites, the respective cells were most likely recruiting sufficient residual protein to adhesion sites to allow integrin activation, cell adhesion and spreading, and the assembly of adhesion-and signaling-competent NAs. It is possible that not all integrin molecules have to be occupied by talin and therefore low levels of talin suffice, particularly in NAs that were shown by fluorescence correlation spectroscopy to contain only half the number of talin relative to a5b1 integrin and kindlin-2 molecules (Bachir et al., 2014). However, when the entire pool of talin is lost or decreased below certain thresholds (Margadant et al., 2012) integrins remain inactivate and consequently adhesion sites do not form. With respect to kindlin, it was reported that overexpressed kindlin-2 in CHO cells inhibits rather than promotes talin head domain-induced a5b1 integrin activation (Harburger et al., 2009). An integrin inhibiting effect of kindlin-2 is inconsistent with our study,  which identified a crucial role for kindlin in integrin activation, as well as with other studies also demonstrating that kindlin-2 promotes integrin functions (Montanez et al., 2008). It could well be that the reported inhibition of a5b1 by kindlin-2 represents an artifact that arose from protein overexpression.
Integrin activation can be induced with Mn 2+ , whose binding to the ectodomain of b subunits directly shifts integrins into the high affinity state without the requirement for inside-out signals (Mould et al., 1995). We observed that Mn 2+ -treated Tln Ko and Kind Ko cells expressed the activation-dependent epitope 9EG7 and adhered to FN, albeit at significantly lower levels and efficiencies than the normal parental or rescued cells ( Figure 6B,C). This observation strongly indicates that talin and kindlin also cooperate to maintain the extended and unclasped conformation of active integrins. Although it is not known how talin and kindlin keep integrins in an active state, it is possible that they stabilize this conformation by linking the unclasped b integrin cytoplasmic domain to the plasma membrane and/or to cortical actin, which may firmly hold separated integrin a/b subunits apart from each other. The expression of mutant talins and kindlins in our cells should allow us to examine these possibilities in future.
Finally, our study also revealed that Mn 2+ -treated Tln Ko cells began to form large, circumferential lamellipodia that eventually detached from FN, leading to the collapse of the protruded membrane. This initial isotropic spreading was significantly less frequent in Kind Ko cells, and has also been observed in talin-2-depleted talin-1 -/cells on FN, although these cells did not require Mn 2+ for inducing spreading, which is likely due to the presence of residual talin-2 that escaped siRNA-mediated depletion Zhang et al., 2014). These findings strongly suggest that integrin binding to FN enables kindlin-2 in Tln Ko cells to cluster b1 integrins (as shown for aIIbb3 by kindlin-3 in Ye et al., 2013) and to trigger a signaling process that initiates spreading.
To find a mechanistic explanation for the kindlin-2-mediated cell spreading, we used the yeasttwo-hybrid technology to identify paxillin as a novel and direct binding partner of kindlin-2. The interaction of the two proteins occurs through the LIM3 domain of paxillin, which was previously identified as integrin adhesion-targeting site (Brown et al., 1996), and the PH domain of kindlin-2. It is not unusual that PH domains fulfill dual roles by binding phospholipids and proteins, either simultaneously or consecutively (Scheffzek and Welti, 2012). The expression of a PH domain-deficient kindlin-2 in Kind Ko cells rescues adhesion to FN and FA maturation, however, significantly impairs spreading and plasma membrane protrusions. This finding together with the observations that paxillin-null fibroblasts and embryonic stem cells have defects in spreading, adhesion site remodeling and formation of lamellipodia (Hagel et al., 2002, Wade et al., 2002 indicates that the kindlin-2/ paxillin complex induces the elusive signaling process, leading to initial spreading of Tln Ko and talindepleted cells . Indeed, the kindlin-2/paxillin complex in NAs recruits FAK (Deramaudt et al., 2014, Thwaites et al., 2014Choi et al., 2011), which cooperates with growth factor receptors (such as EGFR) to induce signaling pathways that activate Erk and Akt to promote proliferation and survival, as well as Arp2/3 and Rac1 to induce actin polymerization and membrane protrusions ( Figure 6B,E). Kindlin-2 also recruits ILK, which binds in the vicinity of the kindlin-2 PH domain and links integrins to actin and additional signaling pathways ( Figure 6E). The short-lived nature of the initial spreading of Tln Ko and talin-depleted  cells shows that talin concludes the integrin-mediated adhesion process in NAs ( Figure 6F) and induces the maturation of FAs. The formation of paxillin-positive FAs in cells expressing the PH domain-deficient kindlin-2 suggests that the recruitment of paxillin to FAs occurs either in a kindlin-independent manner or through a modification of kindlin in a second binding motif. . Model for the roles of talin and kindlin during inside-out and outside-in signaling of a5b1 integrin. Integrin subunits are modelled according to Zhu et al. (2008), with the a5 subunit in green and the b1 subunit in blue showing the bent and clasped low affinity and the extended and unclasped high affinity conformations; the 9EG7 epitope is marked as red dot at the b1 leg and the FN ligand as beige dimers. (A) a5b1 integrin fails to shift from a bent to an extended/unclasped, high affinity state in the absence of talin-1/2 or kindlin-1/2; the bent/clasped conformation brings the EGF-2 domain of the b subunit in close contact with the calf domain of the a5 subunit and prevents exposure of the 9EG7 epitope. (B) In the absence of talin (Tln Ko ) and presence of Mn 2+ , kindlin-2 allows adhesion by stabilizing the high affinity conformation of a low number of integrins and the direct binding of paxillin, leading to nucleation of integrins, recruitment of FAK, FAK-dependent signaling and lamellipodia formation. (C) In the absence of kindlins (Kind Ko ), talin stabilizes the high affinity conformation of a low number of integrins but does not enable paxillin recruitment and lamellipodia formation. (D) In normal fibroblasts, binding of kindlin and talin to the b1 tail is associated with the stabilisation of the unclasped a5b1 heterodimer and 9EG7 epitope exposure. (E) Kindlin recruits paxillin and FAK through the kindlin-PH domain and ILK/Pinch/Parvin (IPP; not shown) in a talinindependent manner and induces cell spreading, proliferation and survival. (F) The high affinity conformation of a5b1 integrin is stabilized by linkage of the b1 tail to the actin cytoskeleton through talin (and potentially the IPP complex; not shown). The arrow length indicates integrin conformations existing at equilibrium. EGF, epidermal growth factor; FAK, focal adhesion kinase; FN, fibronectin; ILK, integrin-linked kinase; IPP, integrin-linked kinase-Pinch-Parvin; SFK, src family kinases. DOI: 10.7554/eLife.10130.028
The cell lines used in this study are mouse fibroblasts derived from the kidneys of 21 d old mice, immortalized by retrovirally transducing the SV40 large T antigen, cloned (Tln Ctr and Kind Ctr ) and finally infected with an adenovirus to transduce the Cre recombinase resulting in talin-null (Tln Ko ) and kindlin-null (Kind Ko ) cells. The parental cell lines were authenticated based on morphological criteria and the surface experession of specific integrins. All cells were cultured under standard cell culture conditions using Dulbecco's modified Eagle's medium (DMEM) supplemented with 8% fetal calf serum (FCS) and Penicillin/Streptomycin but not subjected to mycoplasma contamination testing.

Flow cytometry
Flow cytometry was carried out with a FACSCantoTMII cytometer (BD Biosciences, Franklin Lakes, NJ, USA) equipped with FACS DiVa software (BD Biosciences) using standard procedures. Data analysis was carried out with the FlowJo program (version 9.4.10). Fibroblasts were incubated with primary antibodies diluted in FACS-Tris buffered saline (FACS-TBS; 30 mM Tris, pH 7.4, 180 mM NaCl, 3.5 mM KCl, supplemented with 1 mM CaCl 2 , 1 mM MgCl 2 , 3% BSA, 0,02% NaN 3 ) for 1 hr on ice, washed twice with cold FACS-TBS and finally incubated with the secondary antibody for 45 min on ice.

Antibodies and inhibitors
The following antibodies or molecular probes were used at indicated concentrations for western blot (WB), immunofluorescence (IF) or flow cytometry (FACS): kindlin-1 (home made),   The FAK inhibitor PF-228 (PZ0117 from Sigma) was dissolved in dimethyl sulfoxide at 10 mM and used at 1:2000.

Immunostaining
For immunostaining, cells were cultured on plastic ibidi-m-slides (80826 from Ibidi, Germany) coated with 20 mg ml -1 FN (Calbiochem). Cells were routinely fixed with 4% paraformaldehyde (PFA) (w/v) in phosphate buffered saline (PBS; 180 mM NaCl, 3.5 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM K 2 H 2 PO 4 ) for 10 min at room temperature (RT) or with -20˚C cold acetone-methanol when indicated. If necessary, cells were solubilized with staining buffer (PBS supplemented with 0.1% Triton X-100 (v/v) and 3% BSA (w/v)) or with -20˚C cold methanol for kindlin-2 staining. Background signals were blocked by incubating cells for 1 hr at RT in staining buffer. Subsequently, they were incubated in the dark with primary and secondary antibodies diluted in staining buffer. Fluorescent images were aquired with a LSM 780 confocal microscope (Zeiss, Germany) equiped with a 100Â/NA 1.46 oil objective and with a DMIRE2-SP5 confocal microscope (Leica, Germany) equiped with a 40Â/NA 1.25 or 63Â/NA 1.4 oil objective using Leica Confocal software (version 2.5 build 1227). Brightfield images were aquired with an Axioskop (Carl Zeiss) 40Â/NA 0.75 objective and DC500 camera with IM50 software (Leica). Z-stack projection and contrast adjustments ImageJ (v1.47) were used for further image analysis.
dSTORM was implemented on a custom built total internal reflection fluourescence (TIRF) system (Visitron Systems, Germany) based on a Zeiss Axiovert 200M with fiber-coupled lasers. Sample were excited with a 640 nm laser in a TIRF mode using a Zeiss a Plan-Fluar 100Â/NA 1.45 oil objective. The emitted light was detected in the spectral range 660-710 nm through a Semrock FF02-685/40-25 bandpass filter (Semrock Inc., Rochester, NY, USA). Images were recorded with a Photometrics Evolve Delta emCCD camera (Photometrics, Huntington Beach, CA, USA), with its EM gain set to 250. Additional magnification by a factor of 1.6 resulted in a pixel size of 100 nm. For each final image, a total of 20,000 frames with an exposure time of 14 ms were recorded.
A standard TIRF imaging of the same sample in the green channel (anti-paxillin) was achieved by illumination with a 488 nm laser and detection in the spectral range 500-550 nm through a Chroma Et 525/50 bandpass filter (Chroma Technology Corporation, Bellows Falls, VT, USA). Simultaneous dual-colour imaging of both the green and the red channel was realized with a Hamamatsu W-View Gemini image splitter (Hamamatsu Photonics, Bridgewater, NJ, USA) mounted between the microscope and the camera. Image analysis was carried out with the ImageJ plugin ThunderSTORM (Ovesny et al., 2014) and standard tools of ImageJ. Heat maps of density of blink events were created using the 2D-Frequency Count/Binning module of OriginPro 9.1 (OriginLab Corporation, Northampton, MA, USA).

AFM-based single-cell force spectroscopy
Tipless, 200 mm long V-shaped cantilevers (spring constants of 0.06 N m -1 ; NP-O, Bruker, Billerica, MA, USA) were prepared for cell attachment as described (Friedrichs et al., 2010). Briefly, plasma cleaned cantilevers were incubated in 2 mg ml -1 ConA (Sigma) in PBS at 4˚C overnight. Polydimethylsiloxan (PDMS) masks were overlaid on glass bottoms of Petri dishes (35 mm FluoroDish, World Precision Instruments, Sarasota, FL, USA) to allow different coatings of the glass surface (Te Riet et al., 2014). PDMS-framed glass surfaces were incubated overnight with 50 mg ml -1 FN-RGD and 50 mg ml -1 FN-DRGD in PBS at 4˚C. Overnight serum-starved fibroblasts (Tln Ctr , Kind Ctr , Tln Ko , Kind Ko ) grown on FN-coated (Calbiochem) 24 well plates (Thermo Scientific, Denmark) to confluency of~80% were washed with PBS and detached with 0.25% (w/v) trypsin/EDTA (Sigma). Detached cells were suspended in single-cell force spectroscopy (SCFS) medium (DMEM supplemented with 20 mM HEPES) containing 1% (v/v) FCS, pelleted and further resuspended in serum-free SCFS medium. Detached cells were left suspended in SCFS media to recover from detachment for~1 hr (Schubert et al., 2014). For the activation or chelation assay, the detached cells were incubated in SCFS media supplemented with 0.5 mM Mn 2+ or 5 mM EDTA, respectively, for~1 hr and SCFS was performed in the presence of the indicated supplement. SCFS was performed using an AFM (Nano-Wizard II, JPK Instruments, Germany) equipped with a CellHesion module (JPK Instruments) mounted on an inverted optical microscope (Zeiss Axiovert 200M). Measurements were performed at 37˚C, controlled by a PetriDish Heater (JPK Instruments). Cantilevers were calibrated using the equipartition theorem (Hutter and Bechhoefer, 1993).
To attach a single cell to the cantilever, cell suspensions were pipetted to the region containing the FN-DRGD coating. The ConA functionalized cantilever was lowered onto a single cell with a velocity of 10 mm s À1 until reaching a contact force of 5 nN. After 5 s contact, the cantilever was retracted from the Petri dish by 50 mm and the cantilever-bound cell was left for incubation for >10 min. For adhesion experiments, the cantilever-bound cell was brought into contact with the FN-D RGD coated support at a contact force of~2 nN for 5, 20, 50 and 120 s and then retracted while measuring the cantilever deflection and the distance travelled. Subsequently, the cell adhesion to the FN-RGD coated support was characterized as described. In case cantilever attached cells showed morphological changes (e.g. spreading) they were discarded. The approach and retract velocity of the cantilever was 5 mm s -1 . The deflection of the cantilever was recorded as force-distance curves. Adhesion forces were extracted from retraction force-distance curves using the AFM data processing software (JPK Instruments).

Immunoprecipitations and recombinant protein pulldown
GFP-IPs were performed using m-MACS anti-GFP magnetic beads (130-091-288 from Miltenyi, Germany). To pulldown recombinant kindlin-2 35 mg of purified His10-LIM3 or 10 mg of purified His10paxillin-FL were incubated with 100 ml of 50% Ni-NTA-Agarose slurry (Qiagen) in pulldown buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM TCEP, 0.05% Tween20) for 1 hr at 4˚C. After a first wash with 20 column volumes (CV) of pulldown buffer supplemented with 1 mM ZnCl 2 and a second wash with 20 CV of pulldown buffer, 14 mg of purified kindlin-2 were added to 100 ml of Ni-NTA-agarose slurry and incubated for 30 min at 4˚C. Subsequently, the Ni-NTA beads were washed three times with 20 CV of pulldown buffer supplemented with 25 mM imidazole and either 1 mM ZnCl 2 or 1 mM EDTA. The beads were eluted with 50 ml pulldown buffer supplemented with 500 mM imidazole and analysed on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
For immunoprecipitation of kindlin-2 or paxillin, control fibroblasts were lysed in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.05% sodium deoxycholate, 10 mM EDTA). Lysates were incubated with kindlin-2 or paxillin antibodies for 2 hr at 4˚C while rotating. Isotypematched IgG was used as a negative control. After this, lysates were incubated with 50 ml protein A/ G Plus Agarose (Santa Cruz) for 2 hr at 4˚C. Following repeated washes with lysis buffer, proteins were eluted from the beads using Laemmli buffer and analyzed by western blotting.
For the immunoprecipitation of a5 integrin from the cell surface of live cells, a5 integrins were labeled with a biotinylated anti-a5 integrin antibody (PharMingen #557446) or an isotype control (eBioscience # 13-4321) for 1 hr on ice. After two washes in ice-cold PBS to remove unbound antibody, cells were lysed in IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM EDTA, and protease inhibitors) and cleared by centrifugation. a5 integrin immuno-complexes were pulled-down by incubation with streptavidin-sepharose (GE Healthcare) overnight at 4˚C with gentle agitation. After several washes with lysis buffer, proteins were subjected to SDS-PAGE and western blot analysis with antibodies against a5 and b1 integrin.

Spreading and adhesion assays
Cells were grown to 70% confluency and then detached using trypsin/EDTA. Suspended cells were serum starved for 1 hr in adhesion assay medium (10 mM HEPES, pH 7.4; 137 mM NaCl; 1 mM MgCl 2 ; 1 mM CaCl 2 ; 2.7 mM KCl; 4.5 g L -1 glucose; 3% BSA (w/v)) before 40,000 cells per well were plated out in the same medium supplemented with 8% FCS, and 5 mM Mn 2+ if indicated. Plastic ibidi-m-slides (Ibidi; 80826) were coated with 10 mg ml -1 FN (Calbiochem) for adhesion or 20 mg ml -1 FN for spreading assays, 10 mg ml -1 LN (11243217001 from Roche, Germany), 10 mg ml -1 COL (5005B from Advanced Bio Matrix, Carlsbad, CA, USA), 10 mg ml -1 VN (07180 from StemCell, Canada) or 0.1% PLL (w/v) (Sigma; P4707) diluted in PBS. Seeded cells were centrifuged at 600 rpm in a Beckman centrifuge for 30 min at 37˚C before they were fixed with 4% PFA (w/v) in PBS and stained with Phalloidin-TRITC and DAPI. For cell adhesion assays, nuclear staining of the whole well was imaged using a 2.5x objective and cell numbers were counted using ITCN plugin for imageJ (Byun et al., 2006). For cell spreading assays, 12 confocal images of different regions of Phalloidin and DAPI stained cells were aquired using a Leica confocal microscope, cell spreading was quantified using imageJ.
For time dependent and ligand concentration dependent adhesion on FN, 40,000 cells were plated on 96-well plates, vigorously washed after the indicated timepoints with PBS and fixed with 4% PFA. Cell attachment was meassured by crystal violet staining (0.1% in 20% methanol) of cells in a absorbance plate reader at the wavelength of 570 nm.

Live cell imaging
A hole of 15 mm diameter was drilled into the bottom of a 35 mm falcon tissue culture dish (353001, Becton Dickinson) and a coverslip (Ø 25 mm, Menzel-Glä ser, Germany), rinsed with ethanol, was glued to the dish with silicon glue (Elastosil E43, Wacker, Germany). After coating coverslips with 20 mg ml -1 FN (Calbiochem) overnight at 4˚C, cells were plated and imaged in an inverted transmission light microscope (Zeiss Axiovert 200 M, Carl Zeiss) equipped with a climate chamber. Phase contrast images were taken with a ProEM 1024 EMCCD camera (Princeton Instruments, Acton, MA, USA) through a Zeiss Plan Neofluar 100x objective (NA 1.3, Ph3). Frames were acquired at 30 sec or 1 min intervals and converted to time lapse movies using ImageJ.

Statistical analysis
Experiments were routinely repeated at least three times and the repeat number was increased according to the effect size or sample variation. Unless stated differently, all statistical significances (*P<0.05; **P<0.01; ***P<0.001; n.s., not significant) were determined by two-tailed unpaired t-test. In the boxplots, the middle line represents the median, the box ends represent the 25th and 75th percentiles and the whisker ends show the 5th and 95th percentiles. Statistical analysis were performed with Prism (GraphPad, La Jolla, CA, USA).