Kindlin-1 Regulates Integrin Dynamics and Adhesion Turnover

Loss-of-function mutations in the gene encoding the integrin co-activator kindlin-1 cause Kindler syndrome. We report a novel kindlin-1-deficient keratinocyte cell line derived from a Kindler syndrome patient. Despite the expression of kindlin-2, the patient’s cells display several hallmarks related to reduced function of β1 integrins, including abnormal cell morphology, cell adhesion, cell spreading, focal adhesion assembly, and cell migration. Defective cell adhesion was aggravated by kindlin-2 depletion, indicating that kindlin-2 can compensate to a certain extent for the loss of kindlin-1. Intriguingly, β1 at the cell-surface was aberrantly glycosylated in the patient’s cells, and its expression was considerably reduced, both in cells in vitro and in the patient’s epidermis. Reconstitution with wild-type kindlin-1 but not with a β1-binding defective mutant restored the aberrant β1 expression and glycosylation, and normalized cell morphology, adhesion, spreading, and migration. Furthermore, the expression of wild-type kindlin-1, but not of the integrin-binding-defective mutant, increased the stability of integrin-mediated cell-matrix adhesions and enhanced the redistribution of internalized integrins to the cell surface. Thus, these data uncover a role for kindlin-1 in the regulation of integrin trafficking and adhesion turnover.


Introduction
Integrins are ab heterodimeric transmembrane glycoproteins that link the extracellular matrix to the cytoskeleton. Integrinligand binding triggers the recruitment of a variety of adaptor, structural, and signalling proteins, and the formation of adhesion complexes such as focal adhesions (FAs) [1,2]. Cell adhesion to the extracellular matrix is crucial for the integrity of tissues, in particular for those that encounter great mechanical stress. In the skin, integrins provide for the attachment of the epidermis to the underlying basement membrane (BM). The main epidermal integrin is the laminin (Ln)-binding integrin a6b4, which is localized in hemidesmosomes and connects to intermediate filaments [3]. In addition, b1-integrins such as the collagen (Col)-binding a2b1, Ln-binding a3b1, and the RGD-binding a9b1 integrins, which connect to the actin cytoskeleton, are expressed in basal keratinocytes [4,5].
Many integrins can tune their affinity for ligand by conformational changes, and the switch from the low-to the high-affinity conformation is called integrin activation [6]. Integrin activation is promoted by the binding of talin-1 or talin-2 and any of the 3 kindlin isoforms to the cytoplasmic tail of the b-subunit [6][7][8]. The kindlins consist of an F0-F3 four-point-one/ezrin/radixin/moesin (FERM) domain, that contains the integrin-binding site in F3, and a pleckstrin homology (PH) domain inserted into F2. Kindlin-1 is expressed at high levels in epithelia, in particular in the epidermis and the gastro-intestinal tract, and loss-of-function mutations in KIND1, the gene encoding kindlin-1, cause Kindler syndrome (KS), a congenital bullous disorder of the epidermolysis bullosatype [9][10][11].
In this paper, we describe a novel kindlin-1-deficient keratinocyte cell line derived from an Italian KS patient, which expresses kindlin-2 but not kindlin-1. We investigated functional redundancy between the kindlins, and identified a role for kindlin-1 in the regulation of adhesion turnover and integrin trafficking.

Results and Discussion
Defects in b1 Integrin Function in Kindler Syndrome Cells that Express Kindlin-2 but not Kindlin-1 We isolated kindlin-1-deficient keratinocytes from a previously described male KS patient from Italy [26]. This patient is homozygous for the mutation c.1161delA within exon 10 (Fig. 1A). We first investigated kindlin-1 protein expression by Western blotting, using an antibody directed against an epitope in the F1 domain [24]. Full-length kindlin-1 was clearly detected at the expected size (,75 kDa) in lysates of normal human keratinocytes (NHK) isolated from a healthy individual [27], but not KS cells (Fig. 1B). In contrast, kindlin-2 expression was detected in both NHK and KS keratinocytes (Fig. 1B). The morphology of the KS cells was highly abnormal, as compared to that of NHK (Fig. 1C). In addition, cell growth was compromised and large numbers of dead cells were regularly observed in KS, but not in NHK cultures ( Fig. 1C and unpublished data). To establish whether the observed abnormalities were due to defects in integrin-mediated adhesion, we measured cell adhesion to Col-1, a ligand for integrin a2b1, and to a Rac-11P cell-derived matrix rich in Ln-332, a ligand for integrin a3b1 [21]. Adhesion of KS cells to these ligands was indeed significantly impaired, and only a fraction of the adherent cells spread properly over the substratum (Figs. 1D,E). Consistent with reduced integrin-mediated adhesion, cell motility was significantly enhanced (Figs. 1F,G). We and other groups have previously observed a similar increase in keratinocyte migration upon loss of a3b1, both in cultured keratinocytes and in the epidermis [21,[28][29][30]. However, it should be noted that conflicting results have been reported with regard to keratinocyte migration in the absence of kindlin-1 [24,31]. Similarly, either impaired or increased migration have been described for keratinocytes isolated from epidermolysis bullosa patients that lack Ln-332 [32][33][34][35][36]. Discrepancies in cell migration between different cell lines may derive from the integrin repertoire expressed on the cell-surface, the relative abundance of a particular integrin, and the density of available ligand. Indeed, as migrating cells must be able to attach and exert traction, but also to detach from the substratum, the velocity of migration is a biphasic function of adhesion strength [37]. Finally, we studied the organization of the actin cytoskeleton and the presence of FAs, using phalloidin and an antibody against phosphotyrosines P(Y). P(Y)-staining was generally weak in KS keratinocytes, and actin filaments appeared abnormal ( Figure 1H). In contrast, the synthesis and deposition of Ln-332 in KS cells was not impaired, although the pattern of deposition seemed different from that of NHK cells (Fig. S1). However, because patterns of Ln-332 deposition can differ considerably between cell lines, which often reflects differences in cell motility [21,29,38], it is uncertain whether the differences observed here are a direct consequence of kindlin-1 loss. In summary, we have isolated a kindlin-1-deficient keratinocyte cell line that displays defects in b1 integrin function, despite the presence of kindlin-2.
Kindlin-1 and Kindlin-2 are Partially Redundant, and b1 Expression is Decreased in Kindlin-1-deficient Keratinocytes and Epidermis We next investigated the cell-surface expression and activation status of b1-integrins in KS and NHK cells by flow cytometry. Interestingly, b1 cell-surface expression was significantly reduced (about two-fold), whereas the activation status, as judged by the ratio of 9EG7 staining over total b1 staining, was slightly (but not significantly) increased ( Fig. 2A). The reduction of b1 levels at the cell surface was accompanied by reduced expression of associated a2 and a3-subunits, while b4 levels were normal (Fig. S2). Decreased expression of b1 in KS cells was further confirmed by Western blotting (Fig. 2B). We then analysed skin biopsies of the same patient. Ln-332 staining revealed BM abnormalities and detachment of keratinocytes in the patient's epidermis ( Fig. 2C; indicated by arrows), typical of KS [24,39], and the expression of b1 was strikingly decreased (Fig. 2C). Thus, whereas there is a clear reduction in b1 expression, both in vivo and in vitro, b1 activation in the KS cells is not impaired. The latter finding is reminiscent of keratinocytes derived from the kindlin-1 knockout mice, in which integrin-mediated cell adhesion and cell spreading were compromised whereas there was no significant reduction in integrin activation, due to the presence of kindlin-2 [40]. We therefore introduced shRNAs directed against kindlin-2 into KS cells by lentiviral transduction. Depletion of kindlin-2 caused massive detachment of KS cells (Fig. 2D). Previous studies have reported both overlapping and distinct functions of kindlin-1 and kindlin-2 in keratinocytes [41,42]. Our results are in line with these findings as kindlin-2 can apparently partially rescue cell adhesion in the absence of kindlin-1, but considerable defects in cell adhesion and spreading remain. In vivo, kindlin-2 cannot completely compensate for the loss of kindlin-1, either in the epidermis of KS patients, or in the colon of kindlin-1(2/2) mice [40,43], which is probably due to differences in subcellular localization [11]. We therefore investigated kindlin-2 distribution in vivo. Consistent with its expression in NHK and KS cells, kindlin-2 was detected both in the patient's epidermis and in the epidermis of a normal individual. In basal keratinocytes kindlin-2 localization was exclusively lateral, while kindlin-1 distribution in normal epidermis was predominantly basal, in line with previous observations (Fig. 2E) [11,43]. Interestingly, kindlin-2 staining at the lateral membranes was weak and occasionally completely absent from the basal keratinocyte layer of the patient ( Fig. 2E; indicated by arrows), which most likely reflects defects in cell-cell contacts as we described previously [44].
Together, these data show that b1 expression is reduced in KS cells and epidermis, and that kindlin-2 compensates only partially for reduced cell adhesion in the absence of kindlin-1.

Stable Re-expression of Kindlin-1 in KS Cells Restores the Defects in Integrin Function
The previous sections have shown that in the absence of kindlin-1, integrin-dependent events are disturbed despite the presence of kindlin-2. To investigate whether the observed defects in KS cells are a direct consequence of the loss of kindlin-1, kindlin-1 expression was restored in KS cells by retroviral delivery of eGFPconjugated kindlin-1 followed by FACS sorting, creating a stable cell line that we designated KSK. Expression of eGFP-kindlin-1 was confirmed by Western blotting (Fig. 3A). Kindlin-1 restored the aberrant morphology of KS cells to normal keratinocyte morphology (Fig. 3B, compare to Fig. 1C), and significantly enhanced cell proliferation (Fig. 3C). Kindlin-1 was diffusely distributed in the cytoplasm, while some enrichment in P(Y)positive FAs was observed. In addition, a re-organization of the actin cytoskeleton into stress fibers and/or circumferential actin bundles was observed in KSK cells (Fig. 3D). Re-expression of kindlin-1 reversed the two-fold decrease in cell adhesion as compared to NHK cells, both to Col-1 and to Ln-332-containing matrix (Figs. 3E, 1D). Moreover, the decrease in the number of spread cells was similarly reversed (Figs. 3F, 1E), and the average surface area of KSK cells was about two-fold greater than that of KS cells ( Figure 3G). Finally, the enhanced migration of KS cells was decreased by re-introduction of kindlin-1 (Figs. 3H,I and Figs. 1F,G). To investigate whether the re-expression of kindlin-1 also normalized integrin expression on the cell-surface, we analyzed b1 cell-surface levels by flow cytometry. Expression of b1 on the plasma membrane was up to two-fold higher in KSK cells than in KS (comparable to those in NHK) (Figs. 3J, 2A). The promoting effect of kindlin-1 on integrin cell-surface levels is in line with several previous studies [45][46][47], and was further supported by the observation that overexpression of kindlin-1 also enhanced b1 cell-surface expression in NHK cells, which was accompanied by increased cell spreading (Fig. S3).
Together, these data show that kindlin-1 expression in KS cells rescues the defects in b1 integrin function, and restores the KS phenotype to that of normal keratinocytes.

Regulation of b1 Expression and Function by Kindlin-1 Requires the F3 Domain
We next investigated whether the effects of kindlin-1 depend on a direct interaction with the integrin b1-tail. The integrin-binding site in kindlin-1 resides in the C-terminal F3 domain, and a mutation that causes the expression of a protein lacking the F3 and part of the F2 has been identified in a KS patient, demonstrating the vital importance of this region [48]. In addition, we have recently isolated a zebrafish mutant with KS-like epidermal defects, which expresses a truncated kindlin-1 protein lacking the F3 domain [44]. To delete the integrin-binding site, we truncated the F3 region after residue 581, and stably expressed eGFPkindlin-1 del581 into KS cells, creating a cell line designated KSK del581 (Fig. 4A). Western blotting revealed a band of the expected size of ,95 kDa in KSK del581 cells (Fig. 4B). Intriguingly, increased expression of mature b1, but not precursor b1, was observed in KSK cells but not in KSK del581 , suggesting that a direct interaction is required for the stimulation of b1 cell-surface expression by kindlin-1. Furthermore, the mobility of mature b1 in gel electrophoresis was reduced in lysates of KS and KSK del581 as compared to that of KSK cells (Fig. 4C). This phenomenon was abolished by treatment of immuno-precipitated b1 with neuraminidase, indicating that kindlin-1 regulates b1 sialylation in a manner dependent on the F3 domain (Fig. 4D). Flow cytometry analysis confirmed that whereas the full-length kindlin-1 increased b1 cell-surface expression to wild-type levels (i.e. as in NHK), mutant kindlin-1 did not (Fig. 4E). Furthermore, expression of fulllength kindlin-1 enhanced cell spreading about two-fold (comparable to that of NHK), but eGFP-kindlin-1 del581 did not promote cell spreading (Fig. 4F). Consistently, FAs seemed less pronounced in KSK del581 than in KSK cells, and the subcellular distribution of mutant kindlin-1 was different from that of full-length kindlin-1; kindlin-1 del581 localization seemed predominantly cytoplasmic, with no clear enrichment in adhesions (Fig. 4G). These results are in line with the observation that wild-type kindlin-1, when overexpressed in fibroblasts, is targeted to FAs and increases cell-surface expression of a5b1, whereas kindlin-1 mutants that do not interact with the b1-tail are unable to do so [45].
Together, these data suggest that a direct interaction between kindlin-1 and b1 is required for the targeting of kindlin-1 to cell-

Targeting of Kindlin-1 to Adhesions and Adhesion Dynamics Depend on the F3 Domain
We next addressed the relationship between kindlin-1 targeting to adhesions and adhesion dynamics in living cells. To this end, we introduced the FA marker vinculin, fused to mCherry, into KS, KSK, and KSK del581 cells by lentiviral transduction. The dynamics of eGFP-kindlin-1 and mCherry-vinculin were then monitored by total internal reflection (TIRF) microscopy. Consistent with the results of the cell migration assays, KS cells were very motile and displayed many rapid shape changes. Imaging of vinculin revealed few FAs that had a high turnover rate (Movie S1 and Fig. 5A). In contrast, KSK cells were considerably more static, in line with the reduced migration speed, and their adhesions were much more stable than those in KS cells (Movie S2 and Fig. 5B).
Interestingly, eGFP-kindlin-1 was clearly enriched in adhesions, some of which were surprisingly large, but many of these clusters did not contain mCherry-vinculin, suggesting that kindlin-1 and vinculin can reside in distinct pools of adhesions (Movie S2 and Fig. 5B). Furthermore, kindlin-1 was strongly concentrated in retraction fibers, consistent with the role of kindlin-1 in delaying cell migration. In KSK del581 cells, we also observed a rapid turnover of mCherry-vinculin-containing adhesions, as well as fast shape changes. Consistent with the images acquired by confocal microscopy, there was some diffuse localization of eGFP-kindlin-1 del581 at the basal cell-surface, but clearly no enrichment in adhesions or retraction fibers (Movie S3 and Fig. 5C).
Thus, kindlin-1 controls the dynamics of integrin-mediated cellmatrix adhesions, which is dependent on an intact F3 region.

Kindlin-1 Interaction with b1 Regulates Integrin Traffic
We next investigated whether integrin trafficking plays a role in the regulation of cell-matrix adhesion dynamics and b1 surface expression by kindlin-1. Integrins undergo continuous internalization, and the recycling of internalized integrins is important for integrin-mediated processes such as cell spreading [49,50]. Internalization and recycling were determined according to a well-established protocol [51]. First, we labeled cell-surface b1 with the antibody K20, conjugated to DyLight 649 (10 mg/ml), which clearly revealed localisation of b1 integrins at the membrane in KS, KSK, and KS cells (Fig. 6, top). The cells were then transferred to serum-free medium at 37uC, which allows internalization but not recycling of internalized integrins. The labeled cell-surface pool underwent internalization in all cell lines, with no apparent differences that can be ascribed to kindlin-1 (Fig. 6, middle panel). Recycling of internalized integrins was subsequently induced by stimulation with 20% FCS, which in the majority of KSK cells (77%) triggered the rapid return of b1 integrins to the plasma membrane and their delivery to peripheral adhesions (Fig. 6, bottom). In contrast, redistribution of the internal integrin pool to the plasma membrane was observed only in a small fraction of KS cells (20%) or KS cells expressing truncated kindlin-1 (29%), indicating that kindlin-1 regulates the redistribution of internalized integrins, which is dependent on the F3 domain. A role for kindlin-2 and kindlin-3 in integrin trafficking has been suggested in previous studies, but the mechanism remains to be elucidated [47,52]. We did not detect kindlin-1 in vesicles, in line with similar observations for kindlin-2 [46,53]. Therefore, kindlin-1 probably regulates integrin routing indirectly, i.e. by sorting integrins at the plasma membrane to a specific internalization and recycling pathway. This is conceivable as the kindlin-binding site in b1 is largely defined by the membrane-distal NPxY motif, which is also a canonical signal for clathrin-mediated endocytosis.
In summary, the results presented here suggest that kindlin-1 regulates the redistribution of internalized integrins, which requires a direct kindlin-integrin interaction.

Antibodies, Plasmids and other Materials
Plasmids encoding eGFP-kindlin-1 or mCherry-Vinculin were generously donated by Dr. Reinhard Fassler and Dr. Johan de Rooij, respectively. Antibodies used in this study were directed The use of skin biopsies and keratinocytes from KS patients for research purposes has been approved by the Local Ethics Committee of the Istituto Dermopatico dell'Immacolata (03/07/ 2007; study: ''Epithelial adhesion disorders: molecular mechanisms, development and validation of diagnostic procedures''). Skin biopsies and primary KS keratinocytes were obtained after written informed consent for use in research from a previously described patient [26], and immortalized by SV40 infection. NHK cells were isolated from human foreskin of a healthy individual, and immortalized by transfection with full-length HPV 16 DNA as described previously [27]. NHK and KS cells were routinely cultured on Col-1 (3 mg/ml) in standard keratinocyte medium (Gibco BRL), supplemented with 50 mg/ml bovine pituitary extract, 5 ng/ml EGF, 100 U/ml penicillin and 100 U/ml streptomycin. Rac-11P cells were cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 U/ml streptomycin. All cells were maintained at 37uC and 5% CO 2 . eGFP-kindlin-1 del581 was generated using eGFP-kindlin-1 in C1. Full-length or truncated kindlin-1 were recloned into LZRS-IRESzeo, and transfected into Phoenix packaging cells using the Calcium Phosphate method. Virus-containing supernatant was isolated 48 hrs later and stable expression in KS cells was achieved by retroviral transduction, followed by selection with zeocin and cell sorting. Expression of mCherry-vinculin was established by lentiviral transduction of the pLV-CMV-mCherry-Vinculin-Ires-Puro-construct, followed by selection with 5 mg/ml puromycin.

Knockdown of Kindlin-2 in KS Cells
Short hairpins against human kindlin-2 (target sequence CGACTGATATAACTCCTGAAT), cloned into pLKO.1, were obtained from the TRC shRNA Open Biosystems library and transfected into HEK 293 FT cells together with the Virapower TM Packaging mix (Invitrogen), using Lipofectamine 2000 according to the manufacturers' instructions. Viral supernatant was harvested 48 hrs later, transduced into KS cells, and positive cells were selected with puromycin.
For Western blotting of whole-cell extracts, cell lysates were cleared by centrifugation at 13,0006g, heated at 95uC in SDS sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% b-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue), and proteins were resolved by SDS-PAGE, after which they were transferred to polyvinylidene difluoride membranes (Millipore) and analyzed by Western blotting followed by ECL using the SuperSignal system (Pierce Chemical Co.).

Microscopy
Phase-contrast images were acquired on a Zeiss microscope (Axiovert 25) at 106 (NA 0.25) or 206 (NA 0.3) magnification, using a Zeiss CCD camera (Axiocam MRC) and Zeiss Mr. Grab 1.0 software. For confocal microscopy, cryosections of human skin or cells cultured on coverslips were prepared as previously described [21], and images were acquired on an inverted confocal Figure 6. Kindlin-1 interaction with b1 regulates integrin trafficking. Cell-surface b1 integrins on KS, KSK, and KSK del581 cells were labelled with DyLight 649-conjugated K-20 at 4uC (top panel), after which they were allowed to internalize in serum-free medium at 37uC for 2 hrs (middle panel). Recycling of the internal pool was induced with 20% FCS for 7.5 min (bottom panel). Cells were then fixed and processed for confocal microscopy. b1 is pseudo-colored green, nuclei were counterstained with DAPI (pseudocolored red). Arrows indicate delivery of recycled b1 to adhesions. Percentages of cells with recycled integrins are shown (from ,120 cells out of 3 independent experiments). Bar, 10 mm. doi:10.1371/journal.pone.0065341.g006 Kindlin-1 Regulates Integrin Dynamics PLOS ONE | www.plosone.org microscope (Leica AOBS) using 206 (NA 0.7) dry, 406 (NA 1.25) oil, and 636 (NA 1.32) oil objectives (Leica). For TIRF microscopy, cells were seeded on glass coverslips and videos were acquired using Leica application suite software on a Leica DMI600B system with a 636 objective (NA 1.47), at 37uC in an atmosphere containing 5% CO 2 . Images and videos were processed using Photoshop 7.0 and ImageJ 1.44.

Adhesion, Migration, Cell Spreading, and Proliferation Assays
For adhesion assays, 96-well plates were coated with 2% BSA or 3 mg/ml Col-1 for 1 hr at 37uC. Ln-332-containing matrix was prepared by growing Rac-11P cells to confluency, prior to overnight detachment with 10 mM EDTA at 4uC. The plates were then washed twice with PBS, blocked with 2% BSA for 1 hr at 37uC, and washed twice with PBS before use. Subconfluent cells were trypsinized and seeded at a density of 3610 4 cells per well. After 30 min at 37uC, nonadherent cells were removed by washing with PBS. The adherent cells were fixed in 4% PFA, washed with H2O, stained for 10 min with crystal violet, washed with H2O, and then lysed in 2% SDS. Absorbance was measured at 490 nm on a microplate reader. Background values (binding to BSA) were subtracted from all other values.
To determine cell spreading, cells were seeded in 12-well plates coated with Col-1 or Rac-11P matrix. Cells were photographed on a Widefield CCD system using 106 and 206 dry lens objectives (Carl Zeiss MicroImaging, Inc.). The number of spread cells was counted and expressed as a percentage of the total number of cells. Alternatively, the surface area was determined using ImageJ. Values shown represent the averages of 3 experiments. In each experiment, approximately 500 cells were analyzed for each condition.
For single-cell migration assays, cells were seeded sparsely on 3 mg/ml Col-1, and phase-contrast images were captured every 15 min at 37uC and 5% CO 2 on a Widefield CCD system using a 106 dry lens objective (Carl Zeiss MicroImaging). Migration tracks were generated using ImageJ 1.44, and the average velocity was calculated from approximately 250 cells out of 3 independent experiments.
Proliferation was investigated by seeding cells in 6-well plates, coated with 3 mg/ml Col-1, at a density of 5610 4 cells per well, whereafter they were trypsinized and counted every day. Values shown represent the averages of 3 experiments.

Integrin Internalization and Recycling Assays
Integrin internalization and recycling was investigated essentially as described earlier with some modifications [51]. Briefly, cells on glass coverslips were incubated for 2 hrs at 37uC in serumfree medium, after which they were washed twice in the same medium at 4uC. Cell-surface b1 was then labeled with DyLight 649-conjugated K-20 (10 mg/ml) for 1 hr at 4uC. Immediately after labeling, some coverslips were fixed, and the rest was transferred to 37uC to undergo endocytosis. After 2 hrs, some coverslips were fixed, and the rest was stimulated with 20% FCS for 7.5 min to stimulate recycling of internalized integrins. The cells were fixed, permeabilized with 0.5% Triton and 0.01% saponin, and then processed for confocal microscopy as described above. The number of cells with recycled integrins was scored from confocal images (,120 cells out of 3 independent experiments). Figure S1 Ln-332 deposition is not impaired in KS cells. Ln-332 deposition (green) in NHK and KS cells. Keratinocytes derived from a Junctional Epidermolysis Bullosa (JEB) patient, carying a mutation in the LAMC2 gene encoding the c2 chain of Ln-332, were included as a negative control. F-actin, red. Bar, 20 mm. Movie S1 Dynamics of mCherry-vinculin in KS cells. Dynamics of mCherry-vinculin at the cell-substratum interface were monitored by TIRF microscopy on Col-1-coated glass coverslips. Penetration depth 90 nm, image interval 30 sec, total time 30 min. ImageJ lookup table 'fire' was used to enhance visibility.