A kindlin-3–leupaxin–paxillin signaling pathway regulates podosome stability

Abstract

Binding of kindlins to integrins is required for integrin activation, stable ligand binding, and subsequent intracellular signaling.How hematopoietic kindlin-3 contributes to the assembly and stability of the adhesion complex is not known. Here we reportthat kindlin-3 recruits leupaxin into podosomes and thereby regulates paxillin phosphorylation and podosome turnover. Wedemonstrate that the activity of the protein tyrosine phosphatase PTP-PEST, which controls paxillin phosphorylation,requires leupaxin. In contrast, despite sharing the same binding mode with leupaxin, paxillin recruitment into podosomes iskindlin-3 independent. Instead, we found paxillin together with talin and vinculin in initial adhesion patches of kindlin-3–nullcells. Surprisingly, despite its presence in these early adhesion patches, podosomes can form in the absence of paxillin or anypaxillin member. In conclusion, our findings show that kindlin-3 not only activates and clusters integrins into podosomes butalso regulates their lifetime by recruiting leupaxin, which controls PTP-PEST activity and thereby paxillin phosphorylationand downstream signaling.

Full text

ARTICLE
A kindlin-3–leupaxin–paxillin signaling pathway
regulates podosome stability
Sarah Klapproth
1
, Thomas Bromberger
1
,ClaraTürk
2
, Marcus Krüger
2
, and Markus Moser
1,3

Binding of kindlins to integrins is required for integrin activation, stable ligand binding,and subsequentintracellular signaling.
How hematopoietic kindlin-3 contributes to the assembly and stability of the adhesion complex is not known. Here we report
that kindlin-3 recruits leupaxin into podosomes and thereby regulates paxillin phosphorylation and podosome turnover. We
demonstrate that the activity of the protein tyrosine phosphatase PTP-PEST, which controls paxillin phosphorylation,
requires leupaxin. In contrast, despite sharing the same binding mode with leupaxin, paxillin recruitment into podosomes is
kindlin-3 independent. Instead, we found paxillin together with talin and vinculin in initial adhesion patches of kindlin-3–null
cells. Surprisingly, despite its presence in these early adhesion patches, podosomes can form in the absence of paxillin or any
paxillin member. In conclusion, our findings show that kindlin-3 not only activates and clusters integrins into podosomes but
also regulates their lifetime by recruiting leupaxin, which controls PTP-PEST activity and thereby paxillin phosphorylation
and downstream signaling.
Introduction
Integrin-mediated cell-matrix adhesions anchor cells on or
within extracellular matrices, sense the physical properties of
the environment, and translate them into biochemical signals
(Hynes, 2002). These signals are combined with those from
other signaling pathways to regulate a variety of cellular func-
tions such as cell proliferation, differentiation, and survival
(Legate et al., 2009;Humphries et al., 2019). The ability of in-
tegrins to efficiently and stably bind to their extracellular li-
gands is regulated by changes in their affinity to ligands and the
concentration of activated integrins into clusters (Iwamoto and
Calderwood, 2015). Both aspects of integrin regulation require
the binding of two intracellular adapter proteins, talin and
kindlin, to the integrin βsubunit cytoplasmic domain (Moser
et al., 2009;Sun et al., 2019). These initial events are followed by
a complex assembly of a multitude of adapter and signaling
molecules, which define the biochemical and physical properties
of the adhesion complex (Harburger and Calderwood, 2009).
Different integrin-mediated cell adhesion complexes exist
depending on the cell type, integrin expression, or matrix
composition (Block et al., 2008). Here we focus on podosomes,
which are adhesion structures that show distinct morphological
characteristics compared with focal adhesions, although they are
composed of almost the same components (Marchisio et al.,
1988;Destaing et al., 2003;Calle et al., 2006). Podosomes
have been observed in myeloid, endothelial, and some
c-Src–transformed tumor cells. Each podosome is organized
into two domains: a dense actin core, which contains actin-
regulatory proteins, and a ring of signaling and adaptor pro-
teins embedded in an actin cloud, which surrounds the actin
core and is connected to the extracellular matrix via integrins
(Linder and Kopp, 2005;Murphy and Courtneidge, 2011). Po-
dosomes are more dynamic than focal adhesions with lifetimes
within the minute scale and are involved in matrix degradation
and cell invasion (Block et al., 2008). Talin-1 and kindlin-3 are
both required for proper podosome assembly (Schmidt et al.,
2011;Zou et al., 2013), most likely due to their essential role in
integrin activation, clustering, and linkage to the actin cyto-
skeleton. But how these two proteins orchestrate the complex
assembly of podosomes and regulate their stability and turn-
over, which are important for processes such as cell adhesion,
migration, matrix degradation, transmigration, and tumor in-
vasion, has not been investigated at the molecular level.
The adapter protein paxillin is one of the earliest proteins to
be detected in nascent adhesions at the leading edge of the cell
(Digman et al., 2008), and accumulation of paxillin is the first
visible step in podosome assembly (Luxenburg et al., 2012). In
.............................................................................................................................................................................
1
Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany;
2
Institute for Genetics, Cologne Excellence Cluster on Cellular Stress
Responses in Aging-Associated Diseases, Cologne, Germany;
3
Institute of Experimental Hematology, Center for Translational Cancer Research (TranslaTUM), Klinikum
rechts der Isar der Technischen Universit¨
at München, Munich, Germany.
Correspondence to Markus Moser: m.moser@tum.de.
© 2019 Klapproth et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the
publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0
International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
Rockefeller University Press https://doi.org/10.1083/jcb.201903109 3436
J. Cell Biol. 2019 Vol. 218 No. 10 3436–3454

addition to its role in the assembly of adhesion structures,
paxillin regulates focal adhesion and podosome disassembly.
Phosphorylation of paxillin at Y31 and Y118 is involved in focal
adhesion turnover and dissolving podosomal belts (Laukaitis
et al., 2001;Ballestrem et al., 2006;Badowski et al., 2008).
Mutation of both tyrosines into non-phosphorylated amino acids
impairs the disassembly of adhesion structures (Webb et al.,
2004). Recently, a direct interaction between kindlin-2 and
paxillin has been reported, which is crucial for paxillin re-
cruitment to newly formed adhesion sites and induction of cell
spreading in fibroblasts (Theodosiou et al., 2016;B¨
ottcher et al.,
2017). Moreover, interaction of paxillin with kindlin-3, the he-
matopoietic member of the kindlin family, was shown to pro-
mote platelet integrin activation (Gao et al., 2017).
In the present study, we aimed to elucidate a signaling
pathway downstream of kindlin-3, which is involved in podo-
some assembly and stability regulation and identified leupaxin,
a member of the paxillin gene family, as a new kindlin-3 in-
teractor. Overall our study shows that kindlin-3 has a dual
function in podosome regulation: besides its essential role in
integrin regulation and proper podosome assembly, it also reg-
ulates podosome turnover by recruiting leupaxin into podo-
somes to control phospho-paxillin levels and thereby increasing
the podosome lifetime.
Results
Low kindlin-3 expression results in reduced podosome lifetime
We have previously shown that kindlin-3–deficient cells fail to
assemble definitive podosomes because of their inability to ac-
tivate, cluster, and recruit integrins (Schmidt et al., 2011). Since
the adhesion structures are not properly formed in the absence
of kindlin-3, the role of kindlin-3 in integrin-mediated outside-
in signaling could not be studied yet. We overcame this problem
by strongly reducing kindlin-3 levels rather than eliminating it.
We achieved this by generating preosteoclasts from kindlin-3
hypomorphic mice (K3
n/−
), which express only 5% of normal
kindlin-3 levels due to the presence of a neomycin resistance
cassette within the kindlin-3 gene locus (Klapproth et al., 2015).
Despite the very low kindlin-3 expression, K3
n/−
cells formed
podosomal clusters, in which plaque proteins such as vinculin
and paxillin, as well as αV integrins, are correctly targeted to the
podosomal ring (Fig. 1, A and B). Their actin core diameter,
however, was reduced (Fig. 1 C). Although fewer K3
n/−
cells
formed podosomal clusters (Fig. 1 D), their cluster size was
similar to that of control preosteoclasts (Fig. 1 E). Importantly
and consistent with its strongly reduced expression, kindlin-3 is
hardly detectable by immunofluorescence (IF) in podosomal
clusters of K3
n/n
(10% kindlin-3) and below detection level in
K3
n/−
preosteoclasts (Fig. 1 F). To further characterize the po-
dosomes of K3
n/−
cells, we determined their turnover by track-
ing individual podosomes of control and K3
n/−
cells by life cell
imaging. We transfected the cells with LifeAct-GFP, which binds
to F-actin and labels the central podosomal actin core (Riedl
et al., 2008). Whereas 50% of WT podosomes could be imaged
for 260 s or longer, half of the podosomes in K3
n/−
cells were
already disassembled after 200 s (Fig. 1 G and Videos 1 and 2).
These data suggest two distinct functions of kindlin-3: (1) the
assembly of podosomes, which requires just a small amount of
kindlin-3 and is presumably due to kindlin-3–mediated integrin
activation (inside-out signaling), and (2) stabilizing podosomes,
which requires high kindlin-3 levels and is probably controlled
by kindlin-3–mediated integrin outside-in signaling. The second
mechanism is unexplored and was the focus of the following
studies.
Identification of leupaxin as a new kindlin-3 interactor
To elucidate the molecular mechanism of kindlin-3–mediated in-
tegrin outside-in signaling, we performed yeast-two-hybrid assays
with kindlin-3 as bait using a mouse spleen cDNA library. In two
screens,fivecDNAswereisolated.OneencodedtheCterminus(CT)
of leupaxin, a cytoskeleton adapter protein belonging to the paxillin
protein family (Brown and Turner, 2004). Leupaxin is preferen-
tially expressed in hematopoietic cells and consists of four leucine-
aspartic acid (LD)–rich motifs in the N terminus (NT) and four
Lin11, Isl-1, and Mec-3 (LIM) domains in the CT (Fig. 2 A).
To verify the interaction between kindlin-3 and leupaxin in
hematopoietic cells, we used Flag–kindlin-3 knockin mice, in
which a triple Flag sequence was inserted afterthe start codon of
the kindlin-3 gene (Fig. S1, A and B). Flag-tagged kindlin-3 was
expressed at normal levels (Fig. S1 C) and localized indistin-
guishably from kindlin-3 in the podosomal ring structure of
macrophages (Fig. S1 D; Ussar et al., 2006). We differentiated
macrophages from bone marrow of WT and Flag–kindlin-3
knockin mice and immunoprecipitated Flag-tagged kindlin-3
(Fig. 2 B). These immunoprecipitates contained leupaxin, sup-
porting the direct binding between the two proteins suggested
by the yeast-two-hybrid interaction.
To further map the leupaxin binding site within kindlin-3 we
used RAW 246.7 (RAW) cells, a murine macrophage/monocyte-
like cell line, in which we deleted the kindlin-3 kursiv gene by
CRISPR/Cas9 technology. Kindlin-3–deficient RAW cells ex-
pressed normal levels of leupaxin, talin, and paxillin in all an-
alyzed clones (Fig. 2 C) and were used for reexpression of
various kindlin-3 mutants and domains (Fig. 2 D). We then
transfected EGFP-tagged WT kindlin-3 and an integrin-binding
mutant kindlin-3 (K3 QA; Moser et al., 2008) into these cells,
performed a GFPimmunoprecipitation, and again confirmed the
interaction between WT kindlin-3 and leupaxin. Since the
kindlin-3 integrin-binding mutant precipitated similar amounts
of leupaxin as the WT kindlin-3 (Fig. 2 E), this indicates that
association of kindlin-3 with leupaxin does not require kindlin-3
to be bound to the integrin tail. Furthermore, the interaction
between kindlin-3 and integrin-linked kinase (ILK), which is
mediated via the F2 domain (Fukuda et al., 2014;Huet-
Calderwood et al., 2014), had no impact on kindlin-3/leupaxin
interaction, as ILK-binding mutant kindlin-3 (K3 LA) showed
normal leupaxin binding (Fig. 2 F). Additionally, we conducted
immunoprecipitation experiments with truncated kindlin-3
constructs, which mapped the leupaxin binding site to the F0
domain. Binding of the F0 domain to leupaxin was rather weak
but was enhanced in the presence of the F1 and F2 domains,
indicating that these domains stabilize either the F0 domain or
its interaction with leupaxin. On the contrary, all kindlin-3
Klapproth et al. Journal of Cell Biology 3437
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

constructs that lacked the F0 domain did not bind (Fig. 2 F). The
strong binding of a kindlin-3 construct lacking the integrin-
binding F3 domain confirmed that association of kindlin-3
with integrins was not required for leupaxin binding.
It has been shown recently that an exposed loop within the
N-terminal F0 domain of kindlins mediates direct interaction
with paxillin (Gao et al., 2017). To investigate whether the same
motif (called the M3 cluster) conveys leupaxin binding, we
substituted the M3 residues of kindlin-3 for alanines (K3 M3)
and compared leupaxin and paxillin binding to WT kindlin-3,
the kindlin-3 M3 mutant, and kindlin-3 lacking the F0 domain
(K3 F1–3). After retroviral transduction into kindlin-3–deficient
RAW cells, immunoprecipitation experiments revealed that
when the F0 domain of kindlin-3 is mutated or absent, binding
of both leupaxin and paxillin is strongly diminished (Fig. 2 G).
To map the kindlin-3 binding site within leupaxin, we gen-
erated leupaxin-deficient RAW cells using the CRISPR/Cas9
system. Loss of leupaxin had no effect on kindlin-3, paxillin,
Hic-5, protein tyrosine phosphatase PEST (PTP-PEST), and talin
expression (Fig. 2 H). Reconstitution of these cells with EGFP-
tagged full-length (FL) leupaxin, its NT or CT, followed by GFP-
immunoprecipitations revealed that endogenous kindlin-3 was
precipitated with the FL and the C-terminal LIM domain–
containing region of leupaxin, confirming the result of the yeast-
two-hybrid assay (Fig. 2 I). Moreover, a specific mutation within
the LIM3 domain of leupaxin (C293R), which disrupts the Zn
finger motif and thereby the structural folding of this domain
(Chen and Kroog, 2010;Robertson and Ostergaard, 2011), abol-
ished kindlin-3/leupaxin interaction (Fig. 2 J). Finally, we
proved direct kindlin-3/leupaxin interaction by GST-pulldown
Figure 1. Low kindlin-3 expression results in reduced podosome lifetime. (A) IF stainings of WT (+/+) and K3
n/−
(n/−) preosteoclasts for vinculin (green),
paxillin (red) and actin (white/blue in merge). Scale bar, 10 µm. (B) IF stainings for vinculin (green), integrin αV (red), and actin (white/blue in merge) in
podosomes of +/+ and n/−preosteoclasts. Scale bar, 20 µm. (C) Diameter of the podosomal actin cores in +/+ and n/−cells. 10 actin cores in two regions of
three cells were measured per experiment. n=5.(D) Percentage of podosome forming +/+ and n/−preosteoclasts. n=4.(E) Quantification of the basal cell
surface area that podosomal clusters cover in +/+ and n/−preosteoclasts. n= 48/50 from five different preparations. (F) Vinculin (green) and kindlin-3 (red) IF
stainings of +/+ and n/−preosteoclasts. Scale bar, 5 µm. (G) Cumulative distribution of podosome lifetimesin +/+ untreated or treated with orthovanadate and
n/−preosteoclasts. 15–20 podosomes were measured per cell. Two to five cells were analyzed in each of six different dishes per genotype. See also Videos
1 and 2. Dotted white lines mark cell borders.
Klapproth et al. Journal of Cell Biology 3438
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

Figure 2. Identification and characterization of leupaxin as new kindlin-3 interactor. (A) Domain structure of leupaxin. A C-terminal leupaxin fragment
bound to kindlin-3 in a yeast-two-hybrid screen. (B) Flag-immunoprecipitation (IP) from lysates of +/+ and Flag-tagged kindlin-3 expressing (Flag/Flag) bone
marrow–derived macrophages to verify interaction with endogenous leupaxin. (C) Western blot analyses of leupaxin, talin, and paxillin expression in +/+ RAW
cells and four different K3
−/−
RAW cell clones. (D) Domain structure of kindlin-3. (E) GFP-IP from lysates of K3
−/−
RAW cells expressing EGFP, EGFP-K3, or the
EGFP-tagged K3 QA mutant analyzed for leupaxin. (F) GFP-IP from lysates of K3
−/−
RAW cells expressing different EGFP-K3 fragments to identify the leupaxin-
interacting domain. (G) GFP-IP from lysates of K3
−/−
RAW cells expressing EGFP, EGFP-K3, EGFP-K3 M3 mutant, or EGFP-K3 F1–3 to investigate the interaction
with leupaxin and paxillin. (H) Western blot analyses of +/+ RAW cells and two leupaxin
−/−
RAW cell clones for their expression of kindlin-3, paxillin, PTP-PEST,
and talin. (I) GFP-IP from lysates of leupaxin
−/−
RAW cells reconstituted with EGFP-tagged FL leupaxin (LPXN), a N-terminal fragment (NT), or its CT to
determine interaction with kindlin-3. (J) GFP-IP from lysates of leupaxin
−/−
RAW cells reconstituted eitherwith EGFP-tagged WT leupaxin or a leupaxin mutant
(C293R) analyzed for kindlin-3 binding. (K) GST-pulldown with GST, GST-leupaxin FL, GST-leupaxin NT, and GST-leupaxin CT incubated with His-Sumo-tagged
K3 F0.
Klapproth et al. Journal of Cell Biology 3439
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experiments with bacterially expressed HisSumo-tagged
kindlin-3 F0 domain incubated with GST-FL leupaxin or the
N- and C-terminal leupaxin LD and LIM domains, respectively
(Fig. 2 K). Altogether, these results indicate that the M3 loop in
the F0 domain of kindlin-3 interacts with the LIM domains of
leupaxin, and this interaction is impaired by disrupting LIM3.
Kindlin-3–mediated leupaxin recruitment into podosomes
reduces paxillin phosphorylation and increases podosome
stability
Leupaxin is a cytoskeleton adapter protein, which localizes in
focal adhesions of cancer cells, podosomes of macrophages, and
sealing zones of osteoclasts (Gupta et al., 2003;Chen and Kroog,
2010;Tanaka et al., 2010). IF staining of preosteoclasts, differ-
entiated from bone marrow cells and retrovirally transduced
with EGFP-K3, revealed colocalization of leupaxin and kindlin-3
in the podosomal ring (Fig. 3 A). We then analyzed whether low
kindlin-3 expression impacts on leupaxin localization and found
strongly reduced leupaxin levels in podosomes of K3
n/−
cells. In
contrast, paxillin was found at comparable levels, indicating
that leupaxin but not its paralog paxillin requires kindlin-3 for
podosomal targeting (Fig. 3 B). In support of this observation,
we detected a slight reduction in total cellular leupaxin levels
to ∼70% in K3
n/−
cells, suggesting that the interaction with
kindlin-3 stabilizes cellular leupaxin, whereas paxillin levels
remained unchanged (Fig. 3, C and D).
Next, we investigated whether the interaction between
kindlin-3 and leupaxin is involved in podosome lifetime reg-
ulation. Previous studies suggested that leupaxin is a central
component of the osteoclast podosomal signaling complex and
functions as a paxillin counterpart by suppressing the tyro-
sine phosphorylation of paxillin in focal adhesions (Lipsky
et al., 1998;Gupta et al., 2003;Sahu et al., 2007a;Tanaka
et al., 2010). Nevertheless, leupaxin-null RAW cells formed
podosome clusters, which were comparable to control cells
(Fig. 4 A). We quantified the fluorescence intensity of vin-
culin, paxillin, and phospho-paxillin Y31 and Y118 within
podosomal units of control and leupaxin-null RAW cells by
confocal microscopy and analyzed paxillin phosphorylation
upon adhesion by Western blotting. Whereas vinculin and
paxillin showed normal expression in podosomes, we mea-
sured an increase in phospho-paxillin Y31 and Y118 signal in
leupaxin-null cells (Fig. 4, B and C;andFig.S2,AandB).As
paxillin phosphorylation at Y31 and Y118 is known to promote
adhesion turnover, podosome disassembly, and reorganiza-
tion (Badowski et al., 2008), we also determined the podo-
some lifetime in leupaxin-null and control cells like we did for
K3
n/−
cells. Whereas 50% of the podosomes in WT RAW cells
can be imaged for ∼250 s or longer, the mean lifetime of po-
dosomes in leupaxin-null RAW cells was reduced to ∼180 s
(Fig. 4 D). To confirm that this reduction is due to elevated
paxillin phosphorylation on tyrosines Y31 and Y118, we
transfected the cells with mCherry-tagged WT or non-
phosphorylatable paxillin (paxillin-2YF) and indeed measured
an extension of the podosomal lifetime of paxillin-2YF ex-
pressing WT and leupaxin-null RAW cells compared with cells
transfected with WT paxillin (Fig. 4 E).
We then investigated whether the reduced podosomal
lifetime of K3
n/−
cells is also due to increased paxillin phos-
phorylation. In fact, we measured strongly increased
phospho-paxillin levels in K3
n/−
podosomes of preosteoclasts
(Fig. 4, F and G; and Fig. S2 C), which could be stabilized again
by expression of a phospho-dead paxillin mutant (paxillin 2YF;
Fig. 4 H). To further confirm our hypothesis that adhesion-
mediated paxillin de-phosphorylation stabilizes podosomes,
which is impaired at low kindlin-3 levels, we directly interfered
with protein phosphorylation experimentally. We treated
WT cells with orthovanadate, a general tyrosine phosphatase
inhibitor. Orthovanadate treatment of WT preosteoclasts re-
sulted in an increase in paxillin phosphorylation (Fig. 4, F and
G) and a decrease in podosome lifetime to a similar extent as in
K3
n/−
cells (Fig. 1 G).
Finally, we tested whether a retroviral overexpression of leu-
paxin rescues paxillin phosphorylation in K3
n/−
cells. Notably,
although overexpressed leupaxin localized to podosomes of K3
n/−
Figure 3. Reduced kindlin-3 expression impairs leupaxin recruitment to
podosomes. (A) IF staining of a K3
−/−
preosteoclast retrovirally transduced
with EGFP-K3 (green), for leupaxin (red) and F-actin (white/blue in merge).
(B) IF stainings of K3
+/+
and K3
n/−
preosteoclasts for paxillin (green), leupaxin
(red), and actin (white/blue in merge). (C) Western blot analyses on lysates of
K3
+/+
,K3
+/n
,K3
n/n
, and K3
n/−
macrophages for leupaxin, paxillin, PTP-PEST,
and talin expression. (D) Densitometric quantification of leupaxin expression
in K3
n/−
macrophages relative to WT cells. All values were normalized to the
corresponding GAPDH signal. n= 6. Scale bars, 10 µm. Dotted white lines
mark cell borders.
Klapproth et al. Journal of Cell Biology 3440
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Figure 4. Loss of leupaxin and reduced kindlin-3 expression result in increased podosomal paxillin phosphorylation and decreased podosome
lifetime. (A) IF stainings of +/+ RAW cells and leupaxin
−/−
RAW cells for vinculin, (green), paxillin (red), and actin (white/blue in merge) Scale bar, 10 µm. (B) IF
stainings of +/+ and leupaxin
−/−
RAW cells for vinculin (green), phospho-paxillin Y31 (red), and actin (white/blue in merge). Scale bar, 5 µm. (C) Quantification
of vinculin, total paxillin, and phospho-paxillin Y31 recruitment to podosome clusters assessed by measuring fluorescence intensity (MFI) of confocal images.
MFIs of +/+ cells were set to 1. In each independent experiment, five podosome regions in each of at least 10 cells were measured. n≥6. (D) Cumulative
distribution of podosome lifetime in +/+ and leupaxin
−/−
RAW cells. 10–30 podosomes were measured per cell. At least two cells were analyzed in each of
eight independent experiments. (E) Control RAW cells and different clones of leupaxin
−/−
RAW cells were cotransfected with WT paxillin-Cherry or a
Klapproth et al. Journal of Cell Biology 3441
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cells, it failed to reduce paxillin phosphorylation, suggesting that
kindlin-3 not only recruits leupaxin into podosomes but also fa-
cilitates leupaxin-mediated paxillin dephosphorylation (Fig. S2, D
and E). In sum, these experiments indicate that low kindlin-3
expression results in impaired leupaxin podosomal targeting, in-
creased paxillin phosphorylation, and high podosomal turnover.
Paxillin dephosphorylation by PTP-PEST depends on
kindlin-3–mediated recruitment of leupaxin into podosomes
How does the reduced recruitment of leupaxin in kindlin-3 hy-
pomorphic cells control paxillindephosphorylation?Wespecu-
lated that the protein tyrosine phosphatase PTP-PEST, which
interacts with both paxillin and leupaxin (Shen et al., 1998;Gupta
et al., 2003), might be involved in this process. In support of this
hypothesis, an anti-GFP immunoprecipitation with lysates from
kindlin-3–deficient RAW cells expressing GFP-tagged kindlin-3
showed PTP-PEST in association with kindlin-3, leupaxin, and
paxillin (Fig. 5 A). Unexpectedly, we detected more PTP-PEST in
podosomal clusters of K3
n/−
preosteoclasts and leupaxin-null RAW
cells (Fig. 5, B–D), probably due to the increased binding of PTP-
PEST to phosphorylated paxillin. This hypothesis was supported
by anti-paxillin coimmunoprecipitation experiments showing
more PTP-PEST associated with paxillin in leupaxin-null cells
compared with control cells (Fig. 5 E). This experiment and an
EGFP-leupaxin immunoprecipitation also showed leupaxin and
kindlin-3 in association with paxillin and PTP-PEST (Fig. 5, E and
F). The finding that both phospho-paxillin Y31 and PTP-PEST
levels were elevated in K3
n/−
and leupaxin-null podosomal clusters
suggests that either PTP-PEST does not dephosphorylate paxillin
or its enzymatic activity requires leupaxin. To address these
possibilities, we lentivirally transduced WT and K3
n/−
pre-
osteoclasts with constructs expressing EGFP as a control, an
EGFP-tagged constitutive active PTP-PEST mutant (S39A), or a
dominant-negative form of the phosphatase (D199A; Garton
et al., 1996;Motohashi et al., 2014) and measured paxillin Y31
phosphorylation levels by confocal microscopy. Constitutive
active PTP-PEST reduced Y31 phosphorylation in podosome
clusters of WT and K3
n/−
cells, and dominant-negative PTP-
PEST increased phosphorylation (Fig. 5, G and H). These data
suggest that in the absence of leupaxin, PTP-PEST can still bind
phospho-paxillin but does not dephosphorylate it.
Paxillin recruitment to primitive adhesion patches of actin
cores precedes podosome maturation and is
kindlin-3–independent
A striking observation of our study was that in contrast to leu-
paxin, paxillin is normally targeted to podosomes at very low
kindlin-3 levels. This suggests that either paxillin recruitment to
podosomes is kindlin-3 independent, or very low kindlin-3
levels are sufficient for paxillin podosomal targeting. To ad-
dress this point, we differentiated preosteoclasts from the fetal
liver of kindlin-3 knockout embryos and transduced them with
WT and kindlin-3 M3 retroviral constructs. Both WT kindlin-3
and kindlin-3 M3mutant rescued podosome formation, which is
also indicated by the restored podosomal actin core diameter
(Fig. 6, A and B). Moreover, they showed colocalization with
paxillin in the podosomal ring structure, demonstrating that
paxillin binding to the kindlin-3 F0 domain is not needed for
podosome targeting (Fig. 6 A). In contrast, leupaxin recruitment
depends on this interaction, and hence kindlin-3 M3 transduced
cells exhibited increased phospho-paxillin levels (Fig. 6, C and
D). However, their podosomes appeared more diffuse, with
kindlin-3 and paxillin aberrantly extending into the actin cores,
suggesting that the individual podosomes are less organized
(Fig. 6, E and F). Moreover, consistent with a kindlin-
3–independent paxillin localization to podosomes, we found
paxillin already strongly accumulated in actin-rich adhesion
patches of kindlin-3–null cells (Fig. 6 A). These adhesion patches
were also enriched for vinculin and talin, but lack α4and
β1 integrins, indicating that paxillin is not recruited via binding
to α4 integrin (Fig. S3, A–D). The lack of kindlin-3 probably
explains the absence of leupaxin within these adhesion patches
and the higher phospho-paxillin levels compared with that
within podosomal clusters of cells expressing WT kindlin-3
(Fig. 6 D). These data on the one hand show that paxillin re-
cruitment to podosomes does not depend on interaction with
kindlin-3 and on the other hand imply that during podosome
formation, initial, actin-rich adhesion patches, which contain
actin cores surrounded by the adapter proteins talin, vinculin,
and paxillin, are formed independently of kindlin-3. Kindlin-3 is
subsequently needed to recruit and cluster integrins as well as to
induce integrin-mediated signaling leading to leupaxin-
dependent regulation of phospho-paxillin levels.
Paxillin family proteins are not crucial for podosome
formation but for their signaling
Based on the described interaction of paxillin with all members
of the kindlin family and its early presence in forming podo-
somes (Luxenburg et al., 2012;Gao et al., 2017), we wondered
whether paxillin instead recruits kindlin-3 to podosomes and
how its deficiency affects podosome formation and dynamics. To
this end, we mutated the paxillin gene by CRISPR/Cas9 in RAW
cells. Paxillin-deficient RAW cells expressed normal levels of
kindlin-3, leupaxin, and PTP-PEST (Fig. 7 A), and showed no
non-phosphorylatable mutant paxillin-Cherry (paxillin 2YF) and LifeAct-GFP. Podosome lifetime was assessed and blotted as cumulative distribution. 10–30
podosomes were measured per cell. At least 2 cellswere analyzed in each of 5 different dishes. (F) IF stainings of +/+ preosteoclasts untreated or treated with
Na
3
VO
4
and K3
n/−
preosteoclasts for phosphorylated paxillin (red). Vinculin (green) and actin (white/blue in merge) served as control stainings. Scale bar,
10 µm. (G) Quantification of the recruitment of vinculin, paxillin and phospho-paxillin Y31 to podosomeclusters of +/+ preosteoclasts untreated or treated with
Na
3
VO
4
and K3
n/−
preosteoclasts by measuring MFI of confocal images. MFIs of untreated +/+ cells were set to 1. At least five podosome regions in each of at
least 10 cells were measured per experiment. n≥5. (H) Control and K3
n/−
preosteoclasts were transfected with LifeAct-GFP, left untreated or treated with
Na
3
VO
4
, or cotransfected with WT paxillin-Cherry or a nonphosphorylatable mutant paxillin-Cherry (paxillin 2YF). The cells were imaged for 10 min with a 15-s
time interval. Podosome lifetime was assessed and blotted as cumulative distribution. 10–30 podosomes were measured per cell. Two to five cells were
analyzed per condition in each of six different dishes. Dotted white lines mark cell borders.
Klapproth et al. Journal of Cell Biology 3442
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

Figure 5. Dephosphorylation of paxillin by PTP-PEST depends on kindlin-3–mediated recruitment of leupaxin into podosomes. (A) GFP-IP from K3
−/−
RAW cells, retrovirally transduced with GFP alone or a N-terminally GFP-tagged kindlin-3 analyzed for leupaxin, paxillin, and PTP-PEST. (B) IF staining of +/+
and n/−preosteoclasts for paxillin (green), PTP-PEST (red), and actin (white/blue in merge). Scale bar, 10 µm. (C) IF stainings shown in B were quantified by
measuring fluorescence intensity. Values from WT cells were set to 1. In each independent experiment, five podosome regions in each of at least 10 cellswere
measured. n=5.(D) Control and leupaxin
−/−
RAW cells stained for paxillin (green), PTP-PEST (red), and actin (white/blue in merge). Scale bar, 20 µm. (E) IP
using a mouse–anti-paxillin antibody or an IgG control with lysates from +/+ and leupaxin
−/−
RAW cells. Binding of PTP-PEST, kindlin-3, and leupaxin was
tested. (F) GFP-IP from lysates of leupaxin
−/−
RAW cells, retrovirally transduced with GFP alone or a N-terminally GFP-tagged leupaxin analyzed for kindlin-3,
Klapproth et al. Journal of Cell Biology 3443
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

induction of the third paxillin family member, Hic-5 (Fig. S4 A).
Surprisingly, paxillin-null RAW cells did form podosomes,
which were organized into kindlin-3–,talin-,andvinculin-
positive rings surrounding an ∼10% smaller actin core com-
pared with control podosomes (Fig. 7, B–E). We often noticed a
more diffuse staining of the plaque proteins, indicating a mild
defect in podosomal organization similar to cells expressing the
kindlin-3 M3 mutant. In addition, we measured a strongly re-
duced podosomal lifetime in paxillin-null cells; while 50% of
control podosomes dissolved after 260 s, paxillin-null cells had a
half-life of only 150 s (Fig. 7 F). Of note, PTP-PEST was virtually
absent from the adhesion patches of paxillin-null cells, consis-
tent with the coelevation of PTP-PEST and phospho-paxillin
when kindlin-3 activity is reduced, and suggesting that
phospho-paxillin is responsible for recruiting PTP-PEST to po-
dosomes (Fig. 7 E). These data indicate that paxillin is dispens-
able for podosome formation, but podosomal organization and
stability depend on the adapter protein paxillin.
The rather mild podosome defect in paxillin-null cells sug-
gested partial functional compensation by leupaxin, which was
indeed found at higher levels in podosomal rings of paxillin-null
cells (Fig. 7, G and H). To further investigate the relationship
between kindlin-3, leupaxin, and paxillin, we retrovirally
transduced paxillin and leupaxin-deficient RAW cells with
EGFP-K3 and performed EGFP-coimmunoprecipitation experi-
ments. In both experiments, loss of one paxillin family member
did not result in increased binding of the other member to
kindlin-3, suggesting a lack of competitive binding (Fig. S4, B
and C). This may be explained by an excess of kindlin-3 relative
to the levels of paxillin and leupaxin, which we confirmed by a
whole proteome analysis of WT RAW cells (Fig. S4 D).
To directly address the potential compensatory effects be-
tween paxillin and leupaxin, we generated paxillin and leupaxin
double knockout (dKO) cells, which showed normal kindlin-3
and talin expression (Fig. 8 A). To our surprise, the dKO cells
were also able to form podosomes and podosome clusters, in
which kindlin-3, talin, vinculin, and β1-integrin localized to the
podosomal ring surrounding an actin core with a similarly
reduced size like in paxillin-null cells (Fig. 8, B–DandF). Po-
dosome lifetime was decreased to a similar extent as in
paxillin
−/−
cells (Fig. 8 E). Notably, these cells showed no in-
duction of Hic-5 expression at either the mRNA or the protein
level (Fig. S4, E and F). We then expressed GFP–Hic-5 in dKO
cells, which showed correct localization in the podosomal ring,
however, it failed to rescue the reduced actin core size of dKO
cells, again showing specific functions for the different paxillin
family members (Pignatelli et al., 2012;Petropoulos et al., 2016;
Fig. S4, G and H). Thus, recruitment of kindlin-3 and talin into
podosomal rings as well as general organization of podosomes
can occur in the absence of paxillin family members.
We then studied whether paxillin family members are criti-
cally involved in podosome/integrin signaling and function.
Consistent with previous studies (Luxenburg et al., 2006,2007),
strong phospho-tyrosine signals were detected in the podosomal
ring of control cells, and also present in paxillin- and leupaxin-
null cells (Fig. 8 F). In contrast, dKO cells revealed a dotted
distribution of phospho-tyrosine signals colocalizing with the
actin core, which was also illustrated by fluorescence intensity
profiles across actin cores (Fig. 8, F and G). In addition, we in-
vestigated protein phosphorylation upon adhesion-mediated
signaling to fibronectin by Western blot analyses (Fig. 8 H).
Consistent with the IF stainings, we measured strong induction
of paxillin phosphorylation at Y31 in leupaxin-null cells and only
a weak increase inkindlin-3 knockout cells, which was probably
due to the low number of adhesion complexes formed by
kindlin-3–null cells. Phosphorylation of the actin core marker
cortactin was comparable between the different cell lines. We
detected a reduction in Pyk2 phosphorylation in leupaxin-null
cells and virtually no Pyk2 phosphorylation at Y402 in dKO and
kindlin-3–null cells, while FAK phosphorylation at Y397 was not
reduced.
Finally, we tested whether these changes in podosome dy-
namics and adhesion signaling affect cell migration and matrix
degradation and found reduced gelatin degradation of paxillin
−/−
and leupaxin
−/−
cells, which was further exacerbated in dKO
cells (Fig. 9, A and B). Cell migration was assessed by Transwell
assays showing reduced transmigration in paxillin
−/−
and dKO
cells, whereas loss of leupaxin increased migration (Fig. 9 C).
Discussion
The role of kindlin-3 in regulating integrin activity in coopera-
tion with talin is well established in hematopoietic cells. The
central aim of this study was to investigate how kindlin-3 reg-
ulates integrin signaling and adhesion structure formation in
hematopoietic cells, which isfar less understood. To this end, we
performed a Y2H screen and identified leupaxin as a new
kindlin-3 interactor. Leupaxin is primarily expressed in hema-
topoietic cells and belongs to the paxillin gene family of adapter
proteins, which also includes paxillin and Hic-5 (Lipsky et al.,
1998). We show that recruitment of leupaxin into podosomes
depends on the interaction between a loop within the F0 domain
of kindlin-3 and leupaxin LIM domains. The same loop also
mediates binding to paxillin, indicating a conserved binding
mode between kindlin and paxillin members (Gao et al., 2017).
The finding that leupaxin binding to kindlin-3 is enhanced by
removal of the F3 domain of kindlin-3 suggests that the F3 do-
main exerts an inhibitory effect via steric hindrance. Such a
model is consistent with the fact that in the cloverleaf domain
structure of kindlins, the C-terminal F3 domain and the
N-terminal F0 domain are in proximity with each other (Li et al.,
2017). Notably, leupaxin binds equally well to WT kindlin-3 and
an integrin binding-defective mutant of kindlin-3 (QA), sug-
gesting that their interaction occurs outside of the adhesion site
paxillin, and PTP-PEST. (G) Control and n/−preosteoclasts lentivirally transduced with EGFP, PTP-PEST S39A EGFP, or PTP-PEST D199A EGFP and stained for
paxillin phosphorylated at Y31 (red) and actin (white/blue in merge). Scale bars, 5 µm. (H) Quantification of paxillin phosphorylation levels observedin G. n=5.
Dotted white lines mark cell borders.
Klapproth et al. Journal of Cell Biology 3444
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

Figure 6. Leupaxin-binding mutant kindlin-3 fails to recruit leupaxin to podosomes and to reduce paxillin phosphorylation. (A, C, and D) IF staining
and confocal imaging of K3
−/−
preosteoclasts retrovirally transduced withEGFP, WT EGFP-K3, or the EGFP-K3 M3 mutant (green) for paxillin(red, A), leupaxin
(red, C), and phospho-paxillin Y31 (red, D) and actin (white/blue in merge). Scale bars, 5 µm (A) and 10 µm (C and D). (B) Actin core diameter of K3
−/−
preosteoclasts expressing EGFP, EGPF-K3, or the EGFP-K3 M3 mutant. 10 actin cores in two regions of three to five cells were measured per experiment. n=5.
(E) Fluorescence intensity profile through three actin-cores (indicated by the black lines in A) of K3
−/−
preosteoclasts expressing EGFP, WT EGPF-K3, or the
EGFP-K3 M3 mutant. (F) Percentage of cells with podosome clusters, which reveal discrete localization of EGFP-K3and EGFP-K3 M3 in the podosomal ring. 50
cells were evaluated per condition in each of three independent experiments. Dotted white lines mark cell borders.
Klapproth et al. Journal of Cell Biology 3445
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

Figure 7. Paxillin-deficient cells form podosomes with smaller actin cores and strongly reduced lifetime. (A) Kindlin-3, leupaxin, PTP-PEST, and talin
expression in +/+ RAW cells and four different paxillin
−/−
RAW cell clones analyzed by Western blotting. (B) IF stainings of +/+ and paxillin
−/−
RAW cells for
vinculin (green), paxillin (red), and actin (white/bluein merge). (C) Diameter of the podosome actin cores in +/+, paxillin
−/−
, and leupaxin
−/−
RAW cells. 10 actin
cores in two regions of five to eight cells were measured in each experiment. n=10/6/7.(D) IF stainings for vinculin (green), kindlin-3 (red), and actin (white/
blue in merge) on +/+ and paxillin
−/−
RAW cells. (E) IF stainings for talin (green), PTP-PEST (red) and actin (white/blue in merge) on +/+ and paxillin
−/−
RAW
cells. (F) Control RAW cells and different clones of paxillin
−/−
RAW cells were transfected with LifeAct-GFP and imaged at a spinning disc microscope for 10 min
with a 15-s time interval. The cumulative distribution of these measurements is shown. 10–30 podosomes were measured per cell. At least four cells were
analyzed in each of six independent experiments. (G) Confocal images of talin (green), leupaxin (red), and actin (white/blue in merge) IF stainings of +/+ and
paxillin
−/−
RAW cells. (H) IF stainings shown in G were quantified by measuring fluorescence intensity. Values from WT cells were set to 1. In each independent
experiment, five podosome regions in each of at least 10 cells were measured. n= 4. Scale bars, 10 µm. Dotted white lines mark cell borders.
Klapproth et al. Journal of Cell Biology 3446
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

Figure 8. Characterization of podosomes from paxillin/leupaxin double deficient RAW cells. (A) Western blot analyses of +/+ RAW cells and four
different clones of paxillin/leupaxin dKO RAW cells for their expression of kindlin-3 and talin. (B and C) IF stainings of +/+ and paxillin/leupaxin dKO RAW cells
for vinculin (green), integrin β1 (red, B),kindin-3 (red, C) and actin (white/blue in merge). (D) Diameter of the podosome actin cores in +/+ and paxillin/leupaxin
dKO RAW cells. 10 actin cores in two regions of five to eight cells were measured in each experiment. n= 9/7. (E) Control RAW cells and different clones of
paxillin/leupaxin dKO RAW cells were transfected with LifeAct-GFP andimaged at a spinning disc microscope for 10 min with a 15-s time interval. The cumulative
distribution of these measurements is shown. 20 podosomes were measured per cell. At least four cells were analyzed in each of six dishes. (F) IF stainings
for talin (green), tyrosine-phosphorylated proteins (red) and actin (white/blue in merge) on +/+, paxillin/leupaxin dKO, paxillin
−/−
and leupaxin
−/−
RAW cells.
Klapproth et al. Journal of Cell Biology 3447
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

and thus that kindlin-3/leupaxin interaction may also have
functions other than regulating adhesion stability (Fig. 10).
Which signals modulate their interaction or whether this in-
teraction is of constitutive nature is unclear yet and requires
further studies.
An important finding of our study is that although leupaxin
and paxillin bind kindlin-3 in a similar manner and have mul-
tiple binding partners such as FAK, Pyk2, and PTP-PEST in
common (Brown et al., 1996;Lipsky et al., 1998;Gupta et al.,
2003;Brown and Turner, 2004;Vanarotti et al., 2016), only
leupaxin recruitment to podosomes is kindlin-3 dependent.
While both paxillin and leupaxin act as scaffolds within adhe-
sion complexes, leupaxin also regulates paxillin activity by
suppressing its tyrosine phosphorylation (Tanaka et al., 2010).
Phosphorylation of paxillin on Y31 and Y118, which occurs in
response to integrin-mediated adhesion signaling, creates new
SH2 docking sites for other signaling proteins such as FAK, Crk,
and p120RasGAP and thereby functions as a molecular switch,
which changes the adhesive and signaling properties of the ad-
hesion complex (Zaidel-Bar et al., 2007;Deakin and Turner,
2008). One important function of paxillin tyrosine phospho-
rylation is to enhance adhesion disassembly (Nakamura et al.,
2000;Webb et al., 2004;Badowski et al., 2008). So kindlin-3 not
only is essential for the assembly of podosomes by activating and
clustering integrins within podosomes (Schmidt et al., 2011) but
also stabilizes and prolongs the lifetime of these adhesions
through recruitment of leupaxin.
How leupaxin regulates paxillin phosphorylation is not clear.
Based on the reported interaction between the phosphatase
PTP-PEST with leupaxin and paxillin (Shen et al., 1998;Sahu
et al., 2007b) and the higher phospho-paxillin levels in PTP-
PEST–null cells (Angers-Loustau et al., 1999), we hypothesized
that PTP-PEST might be critically involved in that process.
Several findings supported this hypothesis. First, we found
more PTP-PEST in podosomes of kindlin-3 hypomorphic and
leupaxin-null cells, which have higher levels of phospho-paxillin,
and PTP-PEST does not localize to podosomes in paxillin-null
cells. Second, immunoprecipitation experiments revealed more
PTP-PEST in association with paxillin in leupaxin-null cells
compared with WT cells, indicating that PTP-PEST primarily
associates with phosphorylated paxillin. Third, overexpression of
a constitutive active form of PTP-PEST reduced paxillin phos-
phorylation. In sum, these findings suggest an impaired tyrosine
phosphatase activity of PTP-PESTincellswhereleupaxinisnot
efficiently recruited to podosomes. However, the molecular de-
tails of how leupaxin regulates PTP-PEST activity, e.g., by direct
interaction or by recruiting a kinase or phosphatase that acts on
PTP-PEST, require further investigations.
Another interesting aspect of our study concerns the role of
paxillin family members in podosome formation and signaling.
The first evidence for a direct interaction between paxillin and
kindlin family members came from recent studies in fibroblasts,
which showed that recruitment of paxillin by kindlin-2 to small,
peripheral forming adhesion sites, also known as nascent ad-
hesions, precedes focal adhesion maturation and is required for
lamellipodia formation and cell spreading (Theodosiou et al.,
2016;B¨
ottcher et al., 2017). Another study reported that pax-
illin binding to both kindlin and talin stabilizes talin binding to
(G) Fluorescence intensity profiles of actin (blue) and phospho-tyrosine (p-Y; red) through three actin cores (indicated by the white lines in E) of +/+, paxillin/
leupaxin dKO, paxillin
−/−
, and leupaxin
−/−
RAW cells. (H) Western blot analyses of the phosphorylation status of paxillin, Pyk2, FAK and cortactin in +/+,
paxillin
−/−
,leupaxin
−/−
,K3
−/−
and paxillin/leupaxin dKO RAW cells kept in suspension or adherent to fibronectin. Scale bars, 10 µm. Dotted white lines mark cell
borders.
Figure 9. Loss of leupaxin and/or paxillin affect matrix degradation and cell migration. (A) IF images of DAPI-stained (magenta) +/+, leupaxin
−/−
,
paxillin
−/−
, and paxillin/leupaxin dKO RAW cells seeded on Oregon Green 488–labeled gelatin (green) for 24 h. Scale bar, 100 µm. (B) Relative degradation
capacity (area of degraded collagen normalized by number of nuclei) quantified from images shown in A. (C) Migration of these and K3
−/−
RAW cells in relation
to +/+ cells assessed by Transwell assays.
Klapproth et al. Journal of Cell Biology 3448
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

the integrin cytoplasmic domain, thereby promoting integrin
activation in CHO cells and platelets (Gao et al., 2017). These
studies suggested that interactions between kindlins and pax-
illin are particularly important during the initial phases of
adhesion formation by stabilizing the activation state of talin-
bound integrin and establishing signaling platforms that further
promote cell adhesion and spreading. Consistent with this idea is
the observation of very early local accumulation of paxillin at
sites where podosomes assemble (Luxenburg et al., 2012).
However, in contrast to the kindlin-2–dependent paxillin re-
cruitment to focal complexes, paxillin accumulation at sites
where podosomes form is kindlin-3 independent, as shown by
normal paxillin localization in podosomes of cells expressing a
paxillin-binding mutant kindlin-3 or in adhesion patches of
kindlin-3–deficient cells. Notably, paxillin together with talin
and vinculin are already present within these initial adhesion
sites of kindlin-3–null cells and surround small, less inter-
connected actin cores that lack a ring of integrins. It is important
to note here that hematopoietic cells only express kindlin-3
(Ussar et al., 2006;Moser et al., 2008), which was also con-
firmed by the whole proteome analysis of RAW cells. Thus,
paxillin in complex with vinculin and talin may already form a
preassembled complex within immature adhesion patches be-
fore kindlin-3 and integrins are recruited to finally assemble
podosomes (Deakin and Turner, 2008). The kindlin-
independent mechanism of paxillin targeting to podosomes is
also consistent with the recent finding that paxillin recruitment
to invadosomes, which share many similarities to podosomes, is
mediated via the N-terminal LD domains, whereas targeting to
focal adhesions is mediated via the LIM2 and LIM3 domains
(Brown et al., 1996). Thus, targeting paxillin family members
during formation of podosomes and nascent adhesions occurs
via different mechanisms.
Finally, our study shows that podosomes can be formed in the
absence of any paxillin family member. Although paxillin-deficient
podosomes appear less organized and higher leupaxin levels sug-
gest compensation, the overall organization of podosomes does not
further deteriorate when both paxillin and leupaxin are absent.
Since Hic-5 is not expressed in RAW cells, these cells obviously find
a way by which other adapter proteins help out to organize the
adhesion structure. Nevertheless, podosomes from dKO cells may
exhibit more subtle defects such as an altered equilibrium between
the podosome actin core and the actin cloud, which may impact on
the mechanical properties of the podosome. These unapparent
defects become evident in functional assays such as cell migration
and matrix degradation, which are affected when one or more
paxillin family members are absent, or at the molecular level when
specific signaling pathways such as Pyk2 phosphorylation are in-
vestigated. In this context, the role of paxillin family members in
the formation and function of invadosomes, which are similar to
podosomes, has recently been investigated in src-transformed fi-
broblasts (Petropoulos et al., 2016). While paxillin deficiency also
affected invadosome assembly, loss of both paxillin and Hic-5
abolished invadosome formation. This contrasts with our study
and might be explained by differences in cell type or the src-driven
process of invadosome assembly, which depends on the presence of
either paxillin or Hic-5. Unfortunately, the role of leupaxin was not
addressed in this study (Petropoulos et al., 2016).
In sum, our study shows that kindlin-3 regulates the turno-
ver and lifetime of podosomes in myeloid cells by recruiting
leupaxin to the adhesion complex, which in turn controls PTP-
PEST enzymatic activity and paxillin phosphorylation (Fig. 10);
that initial adhesion patches containing talin, vinculin, and
paxillin form in the absence of kindlin-3; and that podosomes
can form independent of paxillin family proteins.
Materials and methods
Mice
Kindlin-3–deficient (K3
−/−
) and kindlin-3 hypomorphic (K3
n/+
;
K3
n/n
;K3
n/−
) mice were described previously (Moser et al.,
2008;Klapproth et al., 2015). To generate Flag-tagged kindlin-
3 knockin mice, a targeting vector was cloned in which a triple
Flag sequence was inserted into exon 2 after the codon for me-
thionine at position 4. In addition, we introduced a loxP-flanked
neomycin resistance cassette into intron 2 of the Kindlin-3 gene.
Correctly targeted R1 embryonic stem cells were injected into
C57BL/6 blastocysts and the resulting chimeric mice were
crossed with deleter-Cre mice to remove the loxP-flanked neo-
mycin cassette (Betz et al., 1996).
Figure 10. A kindlin-3/leupaxin complex regulates paxillin
tyrosine phosphorylation and podosome stability. Kindlin-
3/leupaxin interaction is independent of integrin binding.
However, kindlin-3 targets leupaxin into the podosome adhe-
sion complex. In contrast, paxillin recruitment to podosomes
occurs independent of kindlin-3. The recruitment of leupaxin
into podosomes enables the tyrosine phosphatase PTP-PEST to
dephosphorylate paxillin at Y31 and Y118, resulting in increased
podosome lifetime and stability. PH, pleckstrin homology
domain.
Klapproth et al. Journal of Cell Biology 3449
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

Mouse experiments were performed with the approval of the
District Government of Bavaria.
Reagents
Recombinant murine macrophage colony-stimulating factor
(M-CSF) and receptor activator of NF-κB ligand (RANKL) were
obtained from Peprotech. Bovine plasma fibronectin was pur-
chased from Merck Millipore and murine TNF-αfrom R&D
Systems. A list of key resources is shown in Table S1.
Antibodies
The following antibodies wereused for immunostaining of cells:
mouse anti-vinculin antibody, mouse anti-Flag M2-Cy3 antibody
(Sigma-Aldrich); rat anti-integrin αV-PE (Millipore); integrin
α4-PE (BD PharMingen); mouse anti-cathepsin K (Calbiochem);
rabbit anti-paxillin and mouse anti-leupaxin (Abcam); rabbit
anti-paxillin Y31, rabbit anti-paxillin Y118, and mouse anti-PTP-
PEST (Thermo Fisher Scientific); rabbit anti–kindlin-3 antibody
(homemade [Ussar et al., 2006]); rabbit anti-integrin β1(home-
made [Azimifar et al., 2012]); rabbit anti-talin-1 (Abcam); and
mouse anti p-tyrosine (pY99, Santa Cruz Biotechnology). Phal-
loidin dyes and secondary antibodies were obtained from In-
vitrogen (Thermo Fisher Scientific).
The following antibodies were used for Western blotting:
mouse anti-GAPDH (Merck); mouse anti-talin (Sigma-Aldrich);
mouse anti-paxillin, rabbit anti-paxillin Y118 (Thermo Fisher
Scientific); mouse anti-leupaxin (Abcam); mouse anti-PTP-
PEST, rabbit anti-paxillin Y31 (Thermo Fisher Scientific); mouse
anti-Hic-5 (BD Biosciences); rabbit anti-Pyk2, rabbit anti-phos-
pho-Pyk2 Y402,rabbit anti-FAK, rabbit anit-phospho-FAK Y397,
rabbit anti-cortactin, rabbit anti-phospho-cortactin Y421, rabbit
anti-His-tag (all from Cell Signaling Technology); mouse anti-
GFP (homemade cell culture supernatant); and rabbit
anti–kindlin-3 antibody (homemade [Ussar et al., 2006]). HRP-
labeled secondary antibodies were purchased from Jackson
ImmunoResearch Laboratories.
Cell culture
Preosteoclasts were differentiated from bone marrow (K3
+/+
and
K3
n/−
) or from embryonic day 14.5 fetal liver cells (K3
−/−
). Cells
were kept in complete α-MEM supplemented with 20 ng/ml
M-CSF overnight. Non-adherent cells were collected after 24 h.
Leukocytes were isolated from the interface after centrifugation
at 1,000 gfor 20 min in leukocyte separation medium (Labo-
ratories Eurobio), and then washed with α-MEM medium and
seeded at a concentration of 2,000–2,500 cells/mm
2
in osteo-
clast differentiation medium (α-MEM [Gibco] containing 10%
FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 60 ng/ml
M-CSF, and 40 ng/ml RANKL). Cells were cultured at 37°C in 5%
CO
2
for 3–5 d, and medium was changed every second day.
Preosteoclasts were defined as adherent cells that were treated
with osteoclast differentiation medium for at least 3 d but had
not yet rearranged their cytoskeleton and translocated the nu-
clei to the cell periphery.
RAW cells (RAW 264.7) were cultured in D10 medium
(DMEM containing 10% FCS, 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-Glu, and nonessential amino acids; all
from Gibco/Thermo Fisher Scientific). For differentiation into
preosteoclasts, RAW cells were treated with osteoclast differ-
entiation medium for ∼5d.
Bone marrow–derived macrophages were generated by
treating bone marrow cells with R10 medium (RPMI 1640
[Gibco] containing 10% FCS, 25 mM Hepes, 100 U/ml penicillin,
100 µg/ml streptomycin, 2 mM L-Glu, nonessential amino acids,
and 50 µM β-mercaptoethanol) supplemented with M-CSF for
6d.
For transient transfection of RAW cells, Lipofectamine 3000
(Invitrogen) was used according to the manufacturer’s protocol.
Primary preosteoclasts were transfected using the Mouse Mac-
rophage Nucleofector Kit from Lonza.
Plasmids and viral infections
All kindlin-3 constructs were cloned into pEGFP-C1 to express
EGFP-tagged proteins. The subdomains were defined according
to Huet-Calderwood et al. (2014): the F0 domain from aa 1–97,
the F1 domain from aa 98–252, the F2 from aa 253–549, and the
F3 domain from aa 550–665. Point mutations (Q597 to A, L334A,
Q57A, D58A, W59A, S60A, and D61A) were generated by site-
directed mutagenesis.
EGFP, EGFP–kindlin-3, EGFP–kindlin-3 M3 mutant, and
EGFP-leupaxin were directionally cloned into the pCLMFG ret-
roviral vector (Naviaux et al., 1996) using the XhoI and NotI
restriction sites. Vesicular stomatitis virus G–pseudotyped ret-
roviral vectors were produced in 293T (human embryonic kid-
ney) cells. Viral particles were concentrated from cell culture
supernatant as described previously (Pfeifer et al., 2000)and
used for infection of preosteoclasts at day three of
differentiation.
Murine FL leupaxin and the C-terminal part (aa 150–386)
were cloned into pEGFP-C1, and the N-terminal part (1–149) into
pEGFP-N1. The C293R mutation was introduced by site-directed
mutagenesis. Murine Hic-5 was cloned into pEGFP-C1.
LifeAct-GFP vector (Riedl et al., 2008) was provided by M.
Sixt (Institute of Science and Technology, Klosterneuburg,
Austria). Mouse paxillin cDNA was cloned into pCherry-C1.
Mutations (Y31 to F and Y118 to F) were introduced by site-
directed mutagenesis (pCherry-C1 paxillin 2YF).
Human PTP-PEST cDNA was subcloned into pJet to introduce
mutations (S39 to A and D199 to A) and then cloned into a len-
tiviral vector (rrl-CMV-GFP; provided by A. Pfeifer, University
of Bonn, Bonn, Germany).
CRISPR/Cas9 mediated gene ablation
The CRISPR/Cas9 guiding sequence was designed with the help
of the CRISPR design webpage of the Zhang laboratory. The
sequence for the single guide RNA was cloned into the GFP-
expressing vector pX458 (for kindlin-3 targeting: 59-GGTGGC
ACCCACTTTATTCAG-39, for leupaxin targeting: 59-GTCCAA
AGACCTTGTCATCGC-39), and RAW cells were transfected using
Lipofectamine LTX.
For paxillin ablation, gRNA was generated with a GeneArt
Precision gRNA Synthesis Kit (Thermo Fisher Scientific) ac-
cording to the manufacturer’s instructions (paxillin target se-
quence: 59-TGAACTTGACCGGCTGTTAC-39). 10 µg Cas9 protein
Klapproth et al. Journal of Cell Biology 3450
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

(Thermo Fisher Scientific) were combined with 2.5 µg gRNA and
incubated for 5 min. The complexes together with a GFP-
encoding plasmid were electroporated into 10
6
RAW cells us-
ing the NEON transfection system (Thermo Fisher Scientific;
conditions: resuspension buffer R, 1,680 V, 20 ms, 1 pulse, 10
7
cells/ml).
After 2 d, GFP-positive cells were FACS-sorted and seeded at
very low concentrations to allow picking of single cell clones.
Clones were checked by Western blot for the presence or ab-
sence of kindlin-3, leupaxin, and paxillin, respectively.
Immunoprecipitation
For immunoprecipitations, cells were washed twice with PBS,
and 0.5 mM dithiobis(succinimidyl propionate) (Thermo Fisher
Scientific) dissolved in PBS was added for 30 min at RT tocross-
link proximal proteins. The reaction was stopped by incubation
with 50 mM Tris, pH 7.5, for 10 min. Cells were washed with
cold PBS and lysed in mammalian protein extraction reagent
(Thermo Fisher Scientific) supplemented with protease in-
hibitors (Roche Diagnostics) and phosphatase inhibitor cocktails
(Sigma-Aldrich). µMACS GFP and DYKDDDDK (to recognize the
Flag-tag) Isolation kits (Miltenyi) were used according to the
manufacturer’s protocol using 1–2 mg protein lysate. Endoge-
nous paxillin immunoprecipitation was performed using 5 µg
mouse anti-paxillin antibody (BD Biosciences) and Pierce Pro-
tein A/G Magnetic Beads following the user’s guide. Mouse IgG1
(Sigma-Aldrich) was used as control. Immunoprecipitations and
40 µg per loading control were subjected to 10% SDS-PAGE and
subsequent Western blotting.
IF microscopy
Preosteoclasts were stained as previously described (Schmidt
et al., 2011). For most antibody stainings, cells were fixed with
4% PFA for 10 min. For kindlin-3 labeling, cells were fixed with
1% PFA for 10 min followed by a 10-minincubation with ice-cold
aceton. Leupaxin was stained in cells fixed with 1.5% PFA for
12 min. Cells were imaged at RT with a Leica TCS SP5 X confocal
microscope (Leica Microsystems) using 63× NA 1.40 oilobjective
lenses and Leica Confocal Software (LAS AF). Single channels
were imaged sequentially. All pictures were processed with
Photoshop (Adobe Systems).
Protein recruitment to podosome clusters was quantified
using ImageJ software (US National Institutes of Health). Fluo-
rescence intensities were assessed in five randomly chosen areas
(∼10 µm
2
) within podosome clusters per cell. All values were
corrected by background fluorescence. Intensities of control
stainings were measured at the identical areas in the corre-
sponding channels (vinculin/paxillin, vinculin/phospho-paxillin
Y31, and paxillin/PTP-PEST).
To assess podosome lifetime, preosteoclasts transfected with
LifeAct-GFP (Riedl et al., 2008) were imaged with a custom-
made spinning disc confocal microscope (Visitron System)
based on a Zeiss Observer Z1 and a Yokogawa spinning disk,
equipped with a 100× NA 1.45 oil objective and an EVOLVE
EM512 digital camera (Photometrics). Where stated, the cells
were additionally transfected with pCherry-C1 paxillin WT or
pCherry-C1 paxillin 2YF. The cells were either left untreated or
treated with 5 mM Na
3
VO
4
and imaged at the spinning disc
microscope for 10 min with a 15-s time interval. The lifetime of a
single podosome was analyzed from these time-lapse videos by
measuring the time until a podosome present at time point 0
disappeared. 10–30 podosomes were measured per cell. 2–10
cells were analyzed per condition in each of two to eight inde-
pendent experiments. Podosome lifetime was analyzed using
ImageJ software and the MTrackJ plugin (National Institutes of
Health).
Actin core size was measured upon phalloidin staining. Two
podosome containing regions were chosen per cell, and 10 po-
dosome core diameters were measured per area. 4–10 cells were
analyzed in each of five experiments by a person blinded to the
genotype of the cells.
Adhesion signaling
RAW cells were trypsinized and kept in serum-free DMEM in
suspension for 3 h. Then cells were either kept in suspension or
plated on a 6-cm cell culture dish coated with 5 µg/ml fibro-
nectin for 20 min. Cells were carefully washed with ice-cold PBS
and lysed in RIPA buffer, containing protease inhibitors and
phosphatase inhibitor cocktails (Sigma-Aldrich). 40 µg of lysates
was subjected to 10% SDS-PAGE and subsequent Western blot
analyses.
Sample preparation and in-solution digest
Proteins wereextracted from pelletedand snap-frozen cells with
4% SDS, heated for 5 min to 95°C, and afterward precipitated
with ice-cold acetone at −20°C for 2.5 h. The proteins were
dissolved in 300 µl of 8 M urea buffer, and protein concen-
trations were determined using the Pierce 660 nm Protein Assay
(Thermo Fisher Scientific). Per sample, 50 µg of protein was
reduced with 5 mM dithiothreitol for 60 min, followed by car-
bamidomethylation with 40 mM iodoacetamide for 45 min in
the dark at RT. A predigest with the protease LysC (Wako Pure
Chemicals Industries) was performed for 3 h, followed by a di-
gest with trypsin (Promega) for 16 h. The digest was terminated
by the acidification of the sample to 1% formic acid, and the
resulting peptides were separated from salts and detergents
using the C18 based Stop and Go extraction tips (Rappsilber
et al., 2003).
Liquid chromatography–tandem mass spectrometry analysis
and data processing
An Easy nLC 1200 ultra-high-performance liquid chromatogra-
phy coupled to a QExactive HF-X Hybrid Quadrupole-Orbitrap
mass spectrometer (Thermo Fisher Scientific) was used for the
proteomic analysis with the following settings. Peptides were
fractionated using in-house–made 50-cm columns packed with
1.7-µm C18 beads using a binary buffer system, consisting of
Buffer A (0.1% formic acid) and Buffer B (80% acetonitrile in
0.1% formic acid). All samples were analyzed over a 90-min
gradient, raising the content of Buffer B from 4% to 23% over
65 min, then from 23% to 55% over 13 min, followed by washing
with 95% Buffer B. Spectra for full MS were acquired at a res-
olution of 60,000 at 200 m/z, and the automated gain control
target was set to 3 × 10
6
with a maximum injection time of 20
Klapproth et al. Journal of Cell Biology 3451
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

ms. The dynamic exclusion was set to 20 s. The MS2 measure-
ments were acquired using a resolution of 15,000 at 200 m/z
with a top 22 data-dependent mode. Here the automated gain
control target was set to 1 × 10
5
with an injection time of 22 ms.
The normalized collision energy in the higher-energy collisional
dissociation cell was 25.
The raw data were analyzed using the Andromeda search
engine implemented in the MaxQuant software 1.5.3.8 (Cox
et al., 2011). Parameters in MaxQuant were set to default with
trypsin selected as protease for digestion. A mouse database
from Uniprot (16.06.17) with contaminants was used for peptide
and protein identification.
Transwell assay
Cell migration was tested in Transwell cell culture inserts (24-
well, 8-µm pore, polyester membrane; Merck Millipore)
coated with 1 mg/ml fibrinogen in TBS for 2 h at 37°C. 10
5
cells
were stimulated with 2 µg/ml TNF-α(R&D Systems) in
DMEM/F-12 containing 0.1% FCS and added to the upper
reservoir. 1 ml DMEM/F-12 with 0.1% FCS was filled in the
lower reservoir. The experiment was started by adding 1 µg/
ml LPS to the lower reservoir. After 6 h, the cells were fixed
for 15 min with 4% PFA and stained with DAPI. Not-
transmigrated cells were removed with a cotton swab, and
six pictures were taken per Transwell with an Evos FL cell
imaging system (Thermo Fisher Scientific) with a 10× objec-
tive to assess the number of transmigrated cells.
Gelatin degradation assay
12-mm glass coverslips were coated with 10 µg/ml Oregon Green
488–conjugated gelatin (Invitrogen) in PBS for 30 min. Slides
were washed with PBS and fixed with 4% PFA for 15 min. Re-
sidual PFA was removed by extensive washing in PBS and in-
cubation with full medium for 30 min. 2 × 10
5
cells that were
cultured in osteoclast differentiation medium for 24 h were
seeded per coverslip. Cells were fixed with 4% PFA the following
day and stained with DAPI. Five pictures were taken per slide
with a Leica TCS SP5 X confocal microscope using a 40× NA
1.25–0.75 oil objective. Gelatin degradation was quantified by
measuring the degraded area using ImageJ software and corre-
lated to the number of nuclei.
Quantitative real-time PCR
mRNA expression level of Hic-5 was assessed by quantitative
real-time PCR. An RNeasy Mini Kit (Qiagen) was used to isolate
RNA, and reverse transcription was performed using a iScript
cDNA Synthesis Kit (Bio-Rad Laboratories). Quantitative PCR
was done with iQ SYBR Green Supermix (Bio-Rad Laboratories)
in LightCycler 480 Multiwell Plate 96, white (Roche) on a
LightCycler 480 II (Roche Molecular Systems) under standard
conditions. The following primers were used: Hic-5 forward:
59-GTAACCAACCCATCCGACAC-39, Hic-5 reverse: 59-GCTGAGCAT
GGAAATGGTTT-39(as published by Rashid et al., 2017); GAPDH
forward: 59-TCGTGGATCTGACGTGCCGCCTG-39,andGAPDH
reverse: 59-CACCACCCTGTTGCTGTAGCCGT-39. All samples
were measured in quadruplicate. Data were analyzed using the
2
−ΔΔCT
-method (Livak and Schmittgen, 2001).
Recombinant protein expression, purification, and pulldown
FL leupaxin, leupaxin NT, or leupaxin CT cDNAs were cloned into
the bacterial expression vector pGEX-2T, kindlin-3 F0 domain into
pCoofy17 (Scholz et al., 2013). GST, N-terminally GST-tagged
leupaxin FL, NT, or CT and HisSUMO-tagged kindlin-3 F0 do-
main were expressed in Rosetta (DE3)–competent cells. Bacteria
were cultured in 50–100 ml Luria-Bertani medium at 37°C to an
OD
600
of0.6to0.7.Proteinswereexpressedat18°Covernight
upon induction with 0.2 mM IPTG. For leupaxin expression,
cultures were supplemented with 500 µM ZnCl
2
. Protein purifi-
cation, pulldowns, and wash steps were performed in 25 mM Tris
buffer, pH 7.5, containing 150 mM NaCl, 5 mM MgCl
2
, 1 mM DTT,
0.01% NP-40 Substitute (Sigma-Aldrich), protease inhibitors, and
10 µM ZnCl
2
(for leupaxin only). Bacteria pellets were lysed by
incubation with lysozyme (10 µg/ml) and sonication. Lysates were
cleared by centrifugation. HisSUMO–kindlin-3 F0 was batch-
purified using PureCube 100 INDIGO Ni-Agarose beads (Cube
Biotech). GST and GST-tagged leupaxin FL, NT, or CT were bound
to 40 µl Glutathione Magnetic Agarose Beads (Jena Bioscience) for
1 h at RT. After washing extensively, 10 µl of loaded beads per
condition were blocked with 5% BSA for 1.5 h at 4°C, and, sub-
sequently, purified kindlin-3 F0 domain was added for 1 h at 4°C in
the presence of 0.5% BSA. After washing, bound proteins were
eluted by incubation for 5 min at 95°C in L¨
ammli buffer. Samples
were subjected to SDS-PAGE and Western blot.
Statistical analysis
Data are presented as means ± 95% confidence interval. Statistical
analyses were performed using the software GraphPad Prism.
Fluorescence intensities were log-transformed before statistical
analyses, as upon transformation, normal distribution can be as-
sumed. For statistical evaluation of podosome lifetime measure-
ments, we determined the time at which 50% of the observed
podosomes had disappeared for each cell culture dish. These values
were subjected to statistical analyses, assuming normal distribution.
Paired or unpaired Student’sttests were used to compare
two different datasets if normal distribution could be assumed.
AMann–Whitney test was performed to compare two datasets if
normal distribution could not be implied. To evaluate three or
more datasets, one-way ANOVA was performed followed by a
Tukey’s or a Sidak’s multiple comparison test, as recommended
by GraphPad Prism.
Data were tested for significance as follows: paired Student’s
ttest for fluorescence intensity measurements; unpaired Stu-
dent’sttest for podosome lifetime measurements, actin core
diameter, and leupaxin expression; Mann–Whitney test for cells
that form podosomes; one-way ANOVA with Tukey’smultiple
comparison test for fluorescence intensity measurements, actin
core diameter, and gelatin degradation assay; and one-way
ANOVA with Sidak’s multiple comparison test for podosome
lifetime measurements and Transwell assay.
A difference between datasets was considered to be signifi-
cant if P < 0.05.
Online supplemental material
Fig. S1 describes the generation of Flag-tagged kindin-3 knockin
mice, Flag-tagged kindlin-3 protein expression, and its
Klapproth et al. Journal of Cell Biology 3452
Kindlin-3 signaling regulates podosome lifetime https://doi.org/10.1083/jcb.201903109

localization to podosomes. Fig. S2 shows by IF stainings and
Western blotting that loss of leupaxin or a reduced kindlin-3
expression result in increased phosphorylation of paxillin at
Y118. Overexpression of leupaxin in K3
n/−
cells is not sufficient
to lower paxillin phosphorylation at Y31. Fig. S3 shows confocal
images of IF stainings of various podosomal marker proteins in
control and K3
n/−
cells. Fig. S4 shows that Hic-5 expression is
not induced in paxillin/leupaxin dKO cells, and retroviral over-
expression of Hic-5 cannot rescue the podosome defect of pax-
illin/leupaxin dKO RAW cells. Furthermore, immunoprecipitation
and quantitative proteomics experiments reveal that leupaxin and
paxillin do not compete for kindlin-3 binding. Table S1 shows a list
of the main reagents and resources used in the study. Videos 1 and
2show+/+andK3
n/−
preosteoclasts expressing LifeAct-GFP, re-
vealing shorter podosome lifetime at low kindlin-3 expression
levels.
Acknowledgments
The authors thank Soo Jin Min-Weiβenhorn and the people from
the transgenic facility of the Max Planck Institute of Biochem-
istry for help with generating the Flag–kindlin-3 mice. We thank
Reinhard F¨
assler for his generous support and Arnoud Son-
nenberg, Stefan Linder, and Nick Brown for critically reading
the manuscript.
This work was supported by the Deutsche Forschungs-
gemeinschaft (SFB914 TP A01) and the Max Planck Society.
The authors declare no competing financial interests.
Author contributions: S. Klapproth, T. Bromberger, and M.
Moser designed and performed the experiments and analyzed
data. C. Türk and M. Krüger performed mass spectrometry and
analyzed the data. M. Moser supervised the work, and S. Klap-
proth and M. Moser wrote the paper with input from all other
authors.
Submitted: 18 March 2019
Revised: 8 July 2019
Accepted: 5 August 2019
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