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Kindlin-1 Regulates Integrin Dynamics and Adhesion Turnover


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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.
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Kindlin-1 Regulates Integrin Dynamics and Adhesion
Coert Margadant
, Maaike Kreft
, Giovanna Zambruno
, Arnoud Sonnenberg
1Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands, 2Laboratory of Molecular and Cell Biology, IDI-IRCCS, Rome, Italy
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 b1 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, b1at
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 b1-binding defective mutant
restored the aberrant b1 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.
Citation: Margadant C, Kreft M, Zambruno G, Sonnenberg A (2013) Kindlin-1 Regulates Integrin Dynamics and Adhesion Turnover. PLoS ONE 8(6): e65341.
Editor: Mirjam M. Zegers, NCMLS, Radboud University Nijmegen Medical Center, The Netherlands
Received March 12, 2013; Accepted April 25, 2013; Published June 11, 2013
Copyright: ß2013 Margadant et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by a grant from DEBRA UK. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail:
Integrins are ab heterodimeric transmembrane glycoproteins
that link the extracellular matrix to the cytoskeleton. Integrin-
ligand 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 conforma-
tional 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 [6–8]. 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 bullosa-
type [9–11].
KS is characterized by skin fragility and blistering, photosen-
sitivity and poikiloderma, while some patients also suffer from
colitis [12–17]. Hemidesmosomes appear unaffected in KS
patients and the defects result from compromised function of b1-
integrins. Indeed, the defects are reminiscent of the abnormalities
in mice lacking the a3 or the b1 subunit in the epidermis, as well
as of patients carrying mutations in the ITGA3 gene encoding a3
[12–22]. In vitro, keratinocytes isolated from KS patients or
keratinocytes in which kindlin-1 expression is suppressed, display
several abnormalities related to defects in b1 integrin function,
including reduced cell adhesion, cell spreading, and polarity [23–
In this paper, we describe a novel kindlin-1-deficient keratino-
cyte 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
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(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 [21,28–30]. 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 [32–36].
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 eGFP-
conjugated 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 [45–47], 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.
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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 eGFP-
into KS cells, creating a cell line designated
(Fig. 4A). Western blotting revealed a band of the
expected size of ,95 kDa in KSK
cells (Fig. 4B). Intriguingly,
increased expression of mature b1, but not precursor b1, was
observed in KSK cells but not in KSK
, suggesting that a
direct interaction is required for the stimulation of b1 cell-surface
expression by kindlin-1. Furthermore, the mobility of mature b1in
gel electrophoresis was reduced in lysates of KS and KSK
compared to that of KSK cells (Fig. 4C). This phenomenon was
abolished by treatment of immuno-precipitated b1 with neur-
aminidase, 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 full-
length kindlin-1 enhanced cell spreading about two-fold (compa-
rable to that of NHK), but eGFP-kindlin-1
did not promote
cell spreading (Fig. 4F). Consistently, FAs seemed less pronounced
in KSK
than in KSK cells, and the subcellular distribution of
mutant kindlin-1 was different from that of full-length kindlin-1;
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-
Figure 1. Abnormalities in KS cells. A) Schematic representation of the KIND1 gene (top), indicating the position of the c.1161delA mutation, and
kindlin-1 protein (bottom). Exons are represented by boxes, introns are not to scale. B) Western blot showing the expression of kindlin-1 and kindlin-2
in NHK and KS cells. C) Phase/contrast images of NHK and KS cells. Bar, 20 mm. D) Adhesion of KS cells to Col-1 and Ln-332, expressed relative to that
of NHK. Shown are the averages 6SEM from 3 independent experiments. E) Cell spreading of NHK and KS cells on Col-1. Shown are the averages
6SEM from 3 independent experiments. F) Rose-plots depicting migration tracks of NHK and KS cells. G) Quantification of the velocity of cell
migration (Bars represent averages 6SEM from ,250 cells out of 3 experiments). H) Confocal images of FAs, visualized using an antibody against P(Y)
(green), and F-actin (red). Scale bar, 10 mm.
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matrix adhesions, and for the effects of kindlin-1 on b1 cell-surface
expression and glycosylation.
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
cells by lentiviral transduction. The
dynamics of eGFP-kindlin-1 and mCherry-vinculin were then
monitored by total internal reflection (TIRF) microscopy. Consis-
tent 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
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-
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 cell-
matrix adhesions, which is dependent on an intact F3 region.
Figure 2. Decreased integrin expression in the absence of kindlin-1. A) FACS histograms (top) and quantification (bottom; average 6SEM
from 3 independent experiments) of NHK and KS cells showing cell-surface expression of b1 (left) and active b1 (right), as measured by 9EG7 staining.
AU, arbitrary units. B) Western blot showing the precursor b1 (110 kDa) and the mature form of b1 (130 kDa) in NHK and KS cells. C) Expression of b1
(green) and Ln-332 (red) in the skin of an unaffected individual (normal) and the KS patient. The upper border of the epidermis is indicated with a
white line. Bar, 50 mm. d; dermis, e; epidermis. D) Depletion of kindlin-2 causes detachment of KS cells. The numbers above the blot indicate the
normalized kindlin-2 expression in the remaining (attached) cells, relative to that in untreated cells. Bar, 20 mm. E) Expression of kindlin-1 (top) and
kindlin-2 (bottom) in the skin. Bar, 50 mm. d; dermis, e; epidermis.
Kindlin-1 Regulates Integrin Dynamics
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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 internali-
zation, 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.
Materials and Methods
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
Figure 3. Re-expression of kindlin-1 in KS cells. A) Western blot showing expression of eGFP-kindlin-1 in KS and KSK cells. B) Morphology of KS
and KSK cells. Bar, 20 mm. C) Proliferation of KS and KSK cells. Shown are the averages 6SEM from 3 independent experiments. D) eGFP-kindlin-1
(green), FAs (blue) and F-actin (red) in KSK cells. Bar, 5 mm. E) Cell adhesion to Col-1 and Ln-332 in KS and KSK cells. Bars represent averages 6SEM of
3 independent experiments. AU, arbitrary units. F) Number of KS and KSK cells spread on Ln-332 and Col-1. Shown are the average values 6SEM from
,500 cells out of a representative experiment. G) Surface area of KS and KSK cells on Ln-332 and Col-1. Shown are the averages 6SEM from ,500
cells of a representative experiment. H) Rose-plots depicting migration tracks of KS and KSK cells generated by time-lapse video microscopy. I)
Quantification of the velocity of cell migration (average 6SEM from ,300 cells out of 3 experiments). J) FACS histograms of NHK and KS cells
showing b1 cell-surface expression (left) and quantification (average 6SEM from 3 independent experiments) (right). AU, arbitrary units.
Kindlin-1 Regulates Integrin Dynamics
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against actin (clone C4; Chemicon), a-tubulin (clone B5-1-2;
Sigma-Aldrich), GFP (Covance), the integrin a6-subunit (GoH3),
the integrin b1-subunit (clone TS2/16; Developmental Studies
Hybridoma Bank, clone 9EG7; a kind gift from Dr. Dietmar
Vestweber, clone K-20; a kind gift from Dr. Andre van Agthoven;
and clone 18 from BD Transduction laboratories), the integrin a2-
subunit (10G11), the integrin a3-subunit (J143), the integrin a5-
subunit (SAM-1), the integrin b4-subunit (439-9B), kindlin-1 (KS-
4; a kind gift from Dr. Cristina Has), kindlin-2 (Sigma-Aldrich),
kindlin-2 (a kind gift from Dr. Reinhard Fassler), Ln-332 (a kind
gift from Dr. Takako Sasaki), plectin (a kind gift from Dr. Katsushi
Owaribe), and P(Y) (clone 4G10; a kind gift from Dr. Kevin
Wilhelmsen). Neuraminidase, puromycin and zeocin were from
Sigma-Aldrich. TRITC-, FITC-, and Cy5-conjugated secondary
antibodies, phalloidin, and DAPI were purchased from Molecular
Probes (Eugene, OR), HRP-conjugated secondary antibodies were
from Amersham, and Col-I was from Vitrogen (Nutacon,
Leimuiden, The Netherlands). K-20 was conjugated to DyLight
649 (Thermo Scientific) at the NKI.
Patient Material, Cell Culture, Cloning, Retroviral and
Lentiviral Transductions
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 mecha-
nisms, 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
Figure 4. Regulation of b1 expression and cell spreading by kindlin-1 require the F3 domain. A) Schematic representation of full-length
kindlin-1 (top) and kindlin-1
(bottom). B) Expression of full-length eGFP-kindlin-1 and eGFP-kindlin-1
in KSK and KSK
cells. C)
Expression of precursor b1 (110 kDa) and mature b1 (130 kDa) in KS, KSK, and KSK
cells. Expression of mature b1 was quantified by
densitometry, normalized to actin, and expressed relative to the expression in KSK cells. Shown are the values acquired from a representative blot. D)
Immunoprecipitated b1 was treated with neuraminidase (NANase) and analyzed by Western blotting. E) FACS histograms (left) and averages 6SEM
quantified from 3 independent experiments (right) of b1 cell-surface expression in KS, KSK, KSK
, and NHK, expressed relative to that in KS. F)
Phase-contrast images of KS, KSK, KSK
and NHK on Col-1 (left), and average surface area 6SEM of KS, KSK, KSK
and NHK cells (quantified
from ,250 cells from a representative experiment) (right). Bar, 10 mm. G) Subcellular distribution of eGFP-kindlin-1 and eGFP-kindlin-1
FAs (blue), F-actin (red). Bar, 5 mm.
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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 supple-
mented with 10% FCS, 100 U/ml penicillin and 100 U/ml
streptomycin. All cells were maintained at 37uC and 5% CO
was generated using eGFP-kindlin-1 in C1.
Full-length or truncated kindlin-1 were recloned into LZRS-IRES-
zeo, 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
Packaging mix (Invitrogen), using Lipofectamine 2000 according
to the manufacturers’ instructions. Viral supernatant was harvest-
ed 48 hrs later, transduced into KS cells, and positive cells were
selected with puromycin.
Figure 5. Kindlin-1 targeting to adhesions and adhesion stability depend on the F3 domain. A) Stills from a TIRF movie, showing the
dynamics of mCherry-vinculin in KS cells. B) Dynamics of mCherry-vinculin (top), and eGFP-kindlin-1 (bottom) in KSK cells. C) Dynamics of mCherry-
vinculin (top), and eGFP-kindlin-1 (bottom) in KSK
cells. Look-up table ‘fire’ was used to enhance visibility of adhesions. Shown are images at 0,
7.5, 15, 22.5, and 30 min. Boxed regions are enlarged. Arrows indicate retraction fibers. Bar, 10 mm.
Kindlin-1 Regulates Integrin Dynamics
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Flow Cytometry and Cell Sorting
For flow cytometry and cell sorting, cultured cells were
trypsinized, washed twice in PBS containing 2% FCS, and
incubated with primary antibodies for 45 min at 4uC. Cells were
then washed twice in 2% FCS/PBS, incubated with appropriate
secondary antibodies for 45 min at 4uC, washed twice in 2%
FCS/PBS, and analyzed on a FACS Calibur (BD Biosciences).
Alternatively, the cells were sorted on a MoFlo High Speed Cell
Sorter (Beckman Coulter).
Immunoprecipitations and Western Blotting
Cells were washed in ice-cold PBS and lysed on ice in RIPA
buffer (25 mM Tris/HCl pH 7.6, 150 mM NaCl, 1% NP-40,
0.5% sodium deoxycholate, 0.1% SDS), supplemented with
protease inhibitor cocktail (Sigma). Immunoprecipitation of b1
was performed essentially as described earlier [21], using TS2/16.
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 (Milli-
pore) and analyzed by Western blotting followed by ECL using the
SuperSignal system (Pierce Chemical Co.).
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
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 b1to
adhesions. Percentages of cells with recycled integrins are shown (from ,120 cells out of 3 independent experiments). Bar, 10 mm.
Kindlin-1 Regulates Integrin Dynamics
PLOS ONE | 8 June 2013 | Volume 8 | Issue 6 | e65341
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 636objective (NA 1.47), at 37uCinan
atmosphere containing 5% CO
. Images and videos were
processed using Photoshop 7.0 and ImageJ 1.44.
Adhesion, Migration, Cell Spreading, and Proliferation
For adhesion assays, 96-well plates were coated with 2% BSA or
3mg/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
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 106and 206dry 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
For single-cell migration assays, cells were seeded sparsely on
3mg/ml Col-1, and phase-contrast images were captured every
15 min at 37uC and 5% CO
on a Widefield CCD system using a
106dry 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
Proliferation was investigated by seeding cells in 6-well plates,
coated with 3 mg/ml Col-1, at a density of 5610
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 essen-
tially as described earlier with some modifications [51]. Briefly,
cells on glass coverslips were incubated for 2 hrs at 37uC in serum-
free 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 experi-
Supporting Information
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.
Figure S2 Integrin expression in NHK and KS cells. Cell-
surface expression of a3, a2, a5, and b4 subunits on NHK and KS
cells was measured by flow cytometry.
Figure S3 Overexpression of kindlin-1 in NHK cells
promotes b1 cell-surface expression and cell spreading.
A) Western blot showing overexpression of eGFP-kindlin-1 in
NHK cells (NHK
). B) FACS histogram showing b1 cell-
surface expression on NHK and NHK
cells. C) Phase/
contrast images of NHK and NHK
cells on Col-1 (top) and
quantification of average cell area (bottom). AU, arbitrary units.
Bar, 10 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
Movie S2 Dynamics of mCherry-vinculin and eGFP-
kindlin-1 in KSK cells. Dynamics of mCherry-vinculin (left)
and eGFP-kindlin-1 (right) 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.
Movie S3 Dynamics of mCherry-vinculin and eGFP-
in KSK
cells. Dynamics of mCherry-
vinculin (left) and eGFP-kindlin-1 (right) 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
We are grateful to Andre van Agthoven, Reinhard Fassler, Cristina Has,
Katsushi Owaribe, Johan de Rooij, Takako Sasaki, Dietmar Vestweber,
and Kevin Wilhelmsen for their generous gifts of antibodies or constructs.
We thank Lauran Oomen and Lenny Brocks for their excellent assistance
with TIRF and confocal microscopy, and Anita Pfauth and Frank van
Diepen for expert technical assistance with FACS. Many thanks to Ana
Jimenez Orgaz and Tomas Meijer for technical support.
Author Contributions
Conceived and designed the experiments: CM AS. Performed the
experiments: CM MK AS. Analyzed the data: CM AS. Contributed
reagents/materials/analysis tools: GZ. Wrote the paper: CM AS.
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Kindlin-1 Regulates Integrin Dynamics
PLOS ONE | 10 June 2013 | Volume 8 | Issue 6 | e65341
... We ectopically expressed Kindlin-1 in 168FARN cells and determined the status of β1-integrin activity. Consistent with previous studies [4,6,7], adhesion to extracellular matrix substrates was enhanced in Kindlin-1 expressing cells (hereafter cited as Kind1-cells) as compared to control cells (a three-fold increase on laminin, P < 0.001) ( Figure 1A). We then evaluated the binding of Kind1-cells to a fibronectin fragment (FN7-10), harboring the integrinbinding RGD motif. ...
... Moreover, Kindlin-1 induced the activation of β1-integrin as shown by increased levels of the active form of the protein specifically detected by the 9EG7 antibody on the cell surface ( Figure 1C,D). In agreement with previous works [7], Kindlin-1 enhanced cell spreading as measured by the area of the cells (1240 versus 556 μm 2 , P < 0.001), and Kind1-cells exhibited a reorganized actin cytoskeleton into stress fibers (Supplementary Figure S1A-B). Furthermore, Kindlin-1 triggered the phosphorylation of Src at Tyr416 and the autophosphorylation of FAK at Tyr397, indicating that integrin signaling was enhanced ( Figure 1E). ...
... Kindlin-1 was shown to regulate integrin dynamics and adhesion turnover in keratinocytes [7]. We, therefore, investigated whether Kindlin-1 could influence integrin trafficking in cancer cells. ...
... The measure of the amount of active b1 integrin in comparison to the total b1 integrin is deemed the integrin activation index. The link between a higher integrin activity index and tumor progression or cell migration is well established (13,14,(43)(44)(45)(46)(47)(48). For example, metastatic breast cancer and melanoma have increased activated b1 integrin compared with the respective primary tumor (44). ...
Full-text available
Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier Major Facilitator Superfamily Domain-containing protein 1 (MFSD1) in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in experimental and spontaneous metastasis mouse models. We identified an increased migratory potential in MFSD1 −/− tumor cells which was mediated by increased focal adhesion turnover, reduced stability of mature inactive β1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive β1 integrin and thereby protected β1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, downregulation of MFSD1 expression was observed during the early steps of tumorigenesis, and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient β1 integrin recycling to suppress tumor cell dissemination.
... Using a Förster resonance energy transfer (FRET) based technique, it was shown that upon binding to the extracellular matrix, integrin alpha and beta subunits undergo conformational changes, leading to the extension of the cytoplasmic tail of integrins (Kim et al.,2003). The unraveling of the cytoplasmic domain leads to the binding of adapter proteins talin and kindlin (Calderwood et al.,2002, Margadant et al.,2013, which in turn triggers the crosslinking of integrin to the actin cytoskeleton. ...
Conference Paper
Collective cell migration is involved in a plethora of developmental and physiological processes. One well-studied example is the collective migration of the cephalic neural crest (NC); an embryonic stem cell population that gives rise to a diverse cell lineage in vertebrate embryos. NC migration requires the formation of integrin-based cell matrix adhesions. However, the exact molecular composition of these adhesions and how they regulate cell polarity during NC migration remains unclear. Furthermore, it was recently demonstrated that stiffening of the migratory substrate is required to trigger the onset of NC migration. This finding raises the question of how the neural crest transduces mechanical cues from its substrate to trigger migration. Whilst integrin-based adhesions serve as the mechanosensory unit of adherent cells, their functional relevance in mechanotransduction during NC migration remains unknown. Here, we investigate the role of Crk Associated Substrate (CAS) protein, X-chef-1, within the cephalic neural crest in Xenopus laevis. This scaffolding protein within the integrin signaling pathway is expressed in the neural crest prior to the onset of migration, however its function is unknown. Through loss of function experiments, we investigated the requirement of X-chef-1 during NC migration. Knock down of X-chef-1 inhibited migration in vivo and cell dispersion and motility in vitro. Through the targeted expression of X-chef-1 dominant negative constructs, we observed that the migratory deficit was primarily attributed to the loss of tyrosine phosphorylation of the X-chef-1 substrate domain. Taken together, we propose that tyrosine phosphorylation of the X-chef-1 substrate domain promotes migration through activating the cell polarity effector Rac-1 at the leading edge of the neural crest. Furthermore, preliminary experiments suggested that constitutive phosphorylation of the X-chef-1 substrate domain may rescue NC migration on mechanically non-permissive substrates in vivo. Hence, our results set up the framework for further investigation into the role of X-chef-1 in the response to mechanical cues during NC migration.
Integrin-dependent cell-extracellular matrix adhesion is essential for wound healing, embryonic development, immunity, and tissue organization. Here, we present a protocol for the imaging and quantitative analysis of integrin-dependent cell-matrix adhesions. We describe steps for cell culture; virus preparation; lentiviral transduction; imaging with widefield, confocal, and total internal reflection fluorescence microscopy; and using a script for their quantitative analysis. We then detail procedures for analyzing adhesion dynamics by live-cell imaging and fluorescence recovery after photobleaching (FRAP). For complete details on the use and execution of this protocol, please refer to Margadant et al. (2012),1 van der Bijl et al. (2020),2 Amado-Azevedo et al. (2021).3.
Background The extracellular matrix (ECM) is a vital structure with a dynamic and complex organization that plays an essential role in tissue homeostasis. In the skin, the ECM is arranged into two types of compartments: interstitial dermal matrix and basement membrane (BM). All evidence in the literature supports the notion that direct dysregulation of the composition, abundance or structure of one of these types of ECM, or indirect modifications in proteins that interact with them is linked to a wide range of human skin pathologies, including hereditary, autoimmune, and neoplastic diseases. Even though the ECM’s key role in these pathologies has been widely documented, its potential as a therapeutic target has been overlooked. Aim of review: This review discusses the molecular mechanisms involved in three groups of skin ECM-related diseases - genetic, autoimmune, and neoplastic – and the recent therapeutic progress and opportunities targeting ECM. Key scientific concepts of review This article describes the implications of alterations in ECM components and in BM-associated molecules that are determinant for guaranteeing its function in different skin disorders. Also, ongoing clinical trials on ECM-targeted therapies are discussed together with future opportunities that may open new avenues for treating ECM-associated skin diseases.
The epidermis is a specialized epithelium that constitutes the outermost layer of the skin, and it provides a protective barrier against environmental assaults. Primarily consisting of multi-layered keratinocytes, the epidermis is continuously renewed by proliferation of stem cells and the differentiation of their progeny, which undergo terminal differentiation as they leave the basal layer and move upward toward the surface, where they die and slough off. Basal keratinocytes rest on a basement membrane at the dermal-epidermal junction that is composed of specific extracellular matrix proteins organized into interactive and mechanically supportive networks. Firm attachment of basal keratinocytes, and their dynamic regulation via focal adhesions and hemidesmosomes, are essential for maintaining major skin processes, such as self-renewal, barrier function, and resistance to physical and chemical stresses. The adhesive integrin receptors expressed by epidermal cells serve structural, signaling, and mechanosensory roles that are critical for epidermal cell anchorage and tissue homeostasis. More specifically, the basement membrane components play key roles in preserving the stem cell pool, and establishing cell polarity cues enabling asymmetric cell divisions, which result in the transition from a proliferative basal cell layer to suprabasal cells committed to terminal differentiation. Finally, through a well-regulated sequence of synthesis and remodeling, the components of the dermal-epidermal junction play an essential role in regeneration of the epidermis during skin healing. Here too, they provide biological and mechanical signals that are essential to the restoration of barrier function.
The activation of spinal astrocytes and release of neuroinflammatory mediators are important events in neuropathic pain (NP) pathogenesis. In this study, we investigated the role of Wnt10a/β-catenin signaling in kindlin-1-mediated astrocyte activation using a chronic constriction injury (CCI) NP rat model. Using kindlin-1 overexpression and knockdown plasmids, we assessed hyperalgesia, changes in spinal astrocyte activation, and the release of inflammatory mediators in a NP rat model. We also performed co-immunoprecipitation, western blotting, and real-time PCR to characterize the underlying mechanisms of kindlin-1 in astrocyte cultures in vitro. Kindlin-1 was significantly upregulated in CCI rats and promoted hyperalgesia. Moreover, we observed increased kindlin-1, Wnt10a, and glial fibrillary acidic protein (GFAP; biomarker for astroglial injury) levels and the release of inflammatory mediators in NP rats (P<0.05). Inhibiting GFAP in vitro led to decreased kindlin-1 levels, prevented astrocyte activation, decreased Wnt10a level, and the release of inflammatory mediators (P<0.05). Co-immunoprecipitation showed that kindlin-1 can interact with Wnt10a. We showed that kindlin-1-mediated astrocyte activation was associated with Wnt10a/β-catenin signaling and the downstream release of inflammatory mediators in a CCI NP rat model. Our findings provide novel insights into the molecular mechanisms of kindlin-1-mediated astrocyte activation post-CCI.
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Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier MFSD1 in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in a mouse model. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turn-over, reduced stability of mature inactive β1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive β1 integrin and thereby protected β1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, down-regulation of MFSD1 expression was observed during early steps of tumorigenesis and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient β1 integrin recycling to suppress tumor spread.
Cell migration involves front-rear asymmetric FA dynamics, which facilitates trailing edge detachment and directional persistence. Here we show that kinldin-2 is critical for FA sliding and disassembly in migrating cells. Loss of kindlin-2 markedly reduced FA number and selectively impaired rear FA sliding and disassembly, resulting in defective rear retraction and reduced directional persistence during cell migration. Kindlin-2 deficient cells failed to develop serum-induced actomyosin-dependent tension at FAs. At the molecular level, kindlin-2 directly interacted with myosin light chain kinase (MLCK), which was enhanced in response to serum stimulation. Serum deprivation inhibited rear FA disassembly, which was released in response to serum stimulation. Overexpression of the MLCK-binding kindlin-2 F0F1 fragment (aa 1-167), which inhibits the interaction of endogenous kindlin-2 with MLCK, phenocopied kindlin-2 deficiency-induced migration defects. Inhibition of MLCK, like loss of kindlin-2, also impaired trailing edge detachment, rear FA disassembly and directional persistence. These results suggest a role of kindlin-2 in promoting actomyosin contractility at FAs, leading to increased rear FA sliding and disassembly and directional persistence in cell migration.
Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding β1 and β3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal β1/β3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal β1/β3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.
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Integrins α3β1 and α6β4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; α6β4 is present in hemidesmosomes, while α3β1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in α3β1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in α3β1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from α3β1-deficient skin adhered to laminin-5 through α6 integrins. However, α3β1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for α3β1 and indicating distinct roles for α3β1 and α6β4. Our findings support a novel role for α3β1 in establishment and/or maintenance of basement membrane integrity, while α6β4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.
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The major epidermal integrins are α3β1 and hemidesmosome-specific α6β4; both share laminin 5 as ligand. Keratinocyte culture studies implicate both integrins in adhesion, proliferation, and stem cell maintenance and suggest unique roles for αβ1 integrins in migration and terminal differentiation. In mice, however, whereas ablation of α6 or β4 results in loss of hemidesmosomes, epidermal polarity, and basement membrane (BM) attachment, ablation of α3 only generates microblistering due to localized internal shearing of BM. Using conditional knockout technology to ablate β1 in skin epithelium, we have uncovered biological roles for αβ1 integrins not predicted from either the α3 knockout or from in vitro studies. In contrast to α3 null mice, β1 mutant mice exhibit severe skin blistering and hair defects, accompanied by massive failure of BM assembly/organization, hemidesmosome instability, and a failure of hair follicle keratinocytes to remodel BM and invaginate into the dermis. Although epidermal proliferation is impaired, a spatial and temporal program of terminal differentiation is executed. These results indicate that β1's minor partners in skin are important, and together, αβ1 integrins are required not only for extracellular matrix assembly but also for BM formation. This, in turn, is required for hemidesmosome stability, epidermal proliferation, and hair follicle morphogenesis. However, β1 downregulation does not provide the trigger to terminally differentiate.
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From a forward genetic screen for epidermal defects in zebrafish, we identified a loss-of-function mutation in Kindlin-1, an essential regulator of integrin function. The mutation generates a premature stop codon, deleting the integrin-binding site. The mutant zebrafish develop cell-matrix and cell-cell adhesion defects in the basal epidermis leading to progressive fin rupturing, and was therefore designated rupturing-of-fins (rof). Similar defects were observed in the epidermis of Kindler syndrome patients, carrying a loss-of-function mutation in kindlin-1. Mutational analysis and rescue experiments in zebrafish revealed that residues K610, W612, and I647 in the F3 domain are essential for Kindlin-1 function in vivo, and that Kindlin-2 can functionally compensate for the loss of Kindlin-1. The fin phenotype of rof/kindlin-1 mutants resembles that of badfin mutants, carrying a mutation in Integrin α3. We show here that this mutation impairs the biosynthesis of integrin α3β1, and causes cell-matrix and cell-cell defects in vivo. Whereas both Integrin-linked kinase and Kindlin-1 cooperate with Integrin α3β1 to resist trauma-induced epidermal defects, Kindlin-1 and Ilk surprisingly do not act synergistically but in parallel. Thus, the rof/kindlin-1 mutant zebrafish provides a unique model system to study epidermal adhesion mechanisms in vivo.Journal of Investigative Dermatology accepted article preview online, 2 April 2013; doi:10.1038/jid.2013.154.
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1 integrins are ubiquitously expressed receptors that mediate cell–cell and cell–extracellular matrix interactions. To analyze the function of 1 integrin in skin we generated mice with a keratinocyte-restricted deletion of the 1 integrin gene using the cre–loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of 64 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin-5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal–epidermal junction. In contrast, the integrity of the basement membrane surrounding the 1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of 1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiaton and proliferation, and in the formation and/or maintenance of hemidesmosomes.
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Integrin functions are controlled by regulating their affinity for ligand, and by the efficient recycling of intact integrins through endosomes. Here we demonstrate that the Kindlin-binding site in the β1-integrin cytoplasmic domain serves as a molecular switch enabling the sequential binding of two FERM-domain-containing proteins in different cellular compartments. When β1 integrins are at the plasma membrane, Kindlins control ligand-binding affinity. However, when they are internalized, Kindlins dissociate from integrins and sorting nexin 17 (SNX17) is recruited to free β1-integrin tails in early endosomes to prevent β1-integrin degradation, leading to their recycling back to the cell surface. Our results identify SNX17 as a β1-integrin-tail-binding protein that interacts with the free Kindlin-binding site in endosomes to stabilize β1 integrins, resulting in their recycling to the cell surface where they can be reused.
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Integrin α(3) is a transmembrane integrin receptor subunit that mediates signals between the cells and their microenvironment. We identified three patients with homozygous mutations in the integrin α(3) gene that were associated with disrupted basement-membrane structures and compromised barrier functions in kidney, lung, and skin. The patients had a multiorgan disorder that included congenital nephrotic syndrome, interstitial lung disease, and epidermolysis bullosa. The renal and respiratory features predominated, and the lung involvement accounted for the lethal course of the disease. Although skin fragility was mild, it provided clues to the diagnosis.
The migration of keratinocytes over the wound bed plays an important role in the re-epithelialization of cutaneous wounds. However, the mechanisms by which keratinocytes migrate over extracellular matrix components are unknown. In this study, we sought to determine if the RGD sequences in matrix molecules and recognition of these sequences by keratinocytes played a role in the locomotion of keratinocytes. After allowing the cells to attach to the matrix, RGD containing peptides or control peptides were added to a keratinocyte migration assay. The addition of RGD-containing peptide dramatically inhibited keratinocyte locomotion on a matrix of fibronectin but not on collagen matrices. Therefore, RGD recognition is a critical step for fibronectin-mediated migration but not for collagen-mediated migration. Because the RGD sequences are recognized by cell-surface integrin receptors in a number of cell types, we next examined the roles of integrin receptors in human keratinocyte migration. Using monospecific antibodies that recognize integrin subunits, we found that blocking the β1 subunit inhibited the migration of keratinocytes on matrices of fibronectin, interstitial collagen, and basement membrane collagen. Blocking the α5β1 receptor significantly inhibited migration on fibronectin but not on collagen matrices. Conversely, blocking the α2β1 receptor inhibited migration on collagen matrices but not on fibronectin. Blocking the α3β1 receptor uniquely enhanced migration on fibronectin and collagen matrices. In contrast to cells apposed to matrices without the receptor blocked, the enhanced migration in the presence of anti-α3β1 antibody occurred at the later time points of the migration assay. The enhancement of migration by blocking the α3β1 integrin receptor suggests that the interaction of the α3β1 receptor with matrices is associated with immobility.
In comparison to the internalization pathways of endocytosis, the recycling pathways are less understood. Even less defined is the process of regulated recycling, as few examples exist and their underlying mechanisms remain to be clarified. In this study, we examine the endocytic recycling of integrin β1, a process that has been suggested to play an important role during cell motility by mediating the redistribution of integrins to the migrating front. External stimulation regulates the endocytic itinerary of β1, mainly at an internal compartment that is likely to be a subset of the recycling endosomes. This stimulation-dependent recycling is regulated by ARF6 and Rab11, and also requires the actin cytoskeleton in an ARF6-dependent manner. Consistent with these observations being relevant for cell motility, mutant forms of ARF6 that affect either actin rearrangement or recycling inhibit the motility of a breast cancer cell line.
Integrins are heterodimeric αβ transmembrane receptors that play key roles in cellular physiology and pathology. Accumulating data indicate that the two NPxY motifs in the cytoplasmic domain of the β1 subunit synergistically promote integrin activation through the binding of talin and kindlin. However, it is unclear how the individual motifs regulate integrin function and trafficking. To investigate how the two NPxY motifs individually control integrin α5β1 function and trafficking, we introduced Y > A mutations in either motif. Disruption of the membrane-proximal NPxY completely prevented α5β1-induced morphological changes, cell scattering and migration, and fibronectin fibrillogenesis. In addition, it reduced α5β1 internalization but not its recycling. In contrast, disruption of the membrane-distal NPxY promoted degradation of α5β1 in late endosomes/lysosomes but did not prevent α5β1-dependent cell scattering, migration, or fibronectin fibrillogenesis. Whereas depletion of either talin-1 or kindlin-2 reduced α5β1 binding to fibronectin and cell adhesion, talin-1 depletion recapitulated the loss-of-function phenotype of the membrane-proximal NPxY mutation, whereas kindlin-2 depletion induced α5β1 accumulation in lysosomes and degradation. The two NPxY motifs of β1 play distinct and separable roles in controlling the function and trafficking of α5β1. Whereas talin binding to the membrane-proximal NPxY is crucial for connecting α5β1 to the actin cytoskeleton and thus permit the tension required for fibronectin fibrillogenesis and cell migration, kindlin binding to the membrane-distal NPxY is dispensable for these events but regulates α5β1 surface expression and degradation.