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RESEARCH ARTICLE
Prickle1 promotes focal adhesion disassembly in cooperation
withthe CLASP–LL5β complex in migrating cellsBoonCheng Lim1, Shinji
Matsumoto1, Hideki Yamamoto1, Hiroki Mizuno2,3, Junichi Kikuta2,3,
Masaru Ishii2,3 andAkira Kikuchi1,*
ABSTRACTPrickle is known to be involved in planar cell polarity,
includingconvergent extension and cell migration; however, the
detailedmechanism by which Prickle regulates cellular functions is
not wellunderstood. Here, we show that Prickle1 regulates
front-rearpolarization and migration of gastric cancer MKN1 cells.
Prickle1preferentially accumulated at the cell retraction site in
close proximityto paxillin at focal adhesions. Prickle1 dynamics
correlated with thoseof paxillin during focal adhesion disassembly.
Furthermore, Prickle1was required for focal adhesion disassembly.
CLASPs (of which thereare two isoforms, CLASP1 and CLASP2, in
mammals) and LL5β(also known as PHLDB2) have been reported to form
a complex atcell edges and to control microtubule-dependent focal
adhesiondisassembly. Prickle1 was associated with CLASPs and LL5β,
andwas required for the LL5β-dependent accumulation of CLASPs at
thecell edge. Knockdown of CLASPs and LL5β suppressed
Prickle1-dependent cell polarization and migration. Prickle1
localized to themembrane through its farnesyl moiety, and the
membranelocalization was necessary for Prickle1 to regulate
migration, tobind to CLASPs and LL5β, and to promote microtubule
targeting offocal adhesions. Taken together, these results suggest
that Prickle1promotes focal adhesion disassembly during the
retraction processesof cell polarization and migration.
KEY WORDS: Prickle, Focal adhesion, Polarity, Migration,CLASP,
LL5β
INTRODUCTIONPrickle was first identified as a protein that
regulates planar cellpolarity (PCP) in Drosophila imaginal discs
(Gubb et al., 1999).Loss of Prickle results in phenotypes affecting
the stereotypicalarrangement of sensory bristles and cellular hairs
on the wing,abdomen and thorax, as well as ommatidia in the eye.
These aresimilar to phenotypes resulting from loss of dishevelled
(Dsh) andfrizzled (fz), which are also known to encode core PCP
proteins(Devenport, 2014; Veeman et al., 2003a; Yang andMlodzik,
2015).In addition to these genes, flamingo (Fmi), Strabismus (Stbm)
andDiego (Dgo) are required for acquisition of epithelial polarity
in theDrosophila eye, wing and epidermis. At the cellular
level,asymmetric localization of core PCP proteins at the apical
cortex
is important to establish PCP (Peng and Axelrod, 2012;
Strutt,2002). Fz, Dsh and Dgo concentrate on the one face of a
cell, andStbm and Prickle1 concentrate on the opposite; Fmi is
present atboth. Stbm is required for the recruitment of Prickle1 at
proximalcell ends in the pupa wing and promotes the degradation of
excessPrickle1 to maintain its asymmetrical localization (Strutt et
al.,2013). Arf1 and adaptor protein-1 (AP-1) are required for the
planarpolarized enrichment of core PCP proteins along the
proximal–distal axis (Carvajal-Gonzalez et al., 2015).
These core PCP proteins are conserved in vertebrates,
functioningas regulators of cell morphology and behavior – in
processes such aspolarized cilia localization, sensory hair
polarization, body hairorientation, neural tube closure and long
bone cartilage elongation –during tissue organization (Goodrich and
Strutt, 2011; Gray et al.,2011; Simons and Mlodzik, 2008; Singh and
Mlodzik, 2012;Zallen, 2007). The specific function of core PCP
proteins invertebrates is to regulate the convergent extension
movement duringgastrulation that results in anterior–posterior
elongation of the bodyaxis (Gray et al., 2011; Heisenberg et al.,
2000). Furthermore, loss-of-function Wnt5 mutations or Prickle1
knockdown in zebrafish aswell as Wnt5a knockdown in Xenopus impair
convergent extensionand axis elongation in embryos (Kilian et al.,
2003; Veeman et al.,2003b), suggesting that Wnt5 and Prickle1
cooperatively regulatethe vertebrate PCP pathway.
Among the four Prickle (Prickle1 to Prickle4) proteins found
inmice and humans, Prickle1 and Prickle2 have been relatively
wellstudied. Prickle1 and Prickle2 have three conserved LIM
domains, aPET domain and a C-terminal farnesylation site
(Maurer-Stroh et al.,2007; Sweede et al., 2008). Both Prickle1 and
Prickle2 areexpressed mainly in neuronal cells during mouse
embryogenesis(here, the mouse genes have been referred to as mpk1
and mpk2,respectively) (Okuda et al., 2007; Tissir and Goffinet,
2006).Knockout mouse studies have revealed that mpk1 is required
for themaintenance and establishment of epiblast apical and
basolateralpolarity (Tao et al., 2009). Knockdown and
overexpression ofPrickle1 reduces and induces, respectively,
neurite outgrowth ofmouse neuroblastoma cells (Fujimura et al.,
2009; Okuda et al.,2007), suggesting that Prickle1 might be
involved in neuronpolarization; however, these cells do not show
the polaritycharacteristics of neurons. Prickle2 localizes to the
postsynapticdensity of asymmetric synapses in the adult mouse brain
and forms acomplex with PSD-95 and NMDA receptors (Hida et al.,
2011).Furthermore, loss-of-function Prickle mutations in flies or
Prickle1mutations in mice and humans result in epileptic
phenotypes(Bassuk et al., 2008; Tao et al., 2011). Prickle
organizes microtubulepolarity, thereby influencing axon growth in
Drosophila neurons(Ehaideb et al., 2014). However, it is not clear
whether these Pricklefunctions are associated with Wnt
signaling.
Prickle1 is also expressed in non-neural tissues, such as in
thedeveloping limbs of mice (Bekman and Henrique, 2002).Received 29
December 2015; Accepted 22 June 2016
1Department of Molecular Biology and Biochemistry, Graduate
School of Medicine,Osaka University, 2-2 Yamada-oka, Suita, Osaka
565-0871, Japan. 2Department ofImmunology and Cell Biology,
Graduate School of Medicine, Osaka University, 2-2Yamada-oka,
Suita, Osaka 565-0871, Japan. 3WPI-Immunology Frontier
ResearchCenter, Osaka University, Yamadaoka 3-1, Suita, Osaka
565-0871, Japan.
*Author for correspondence
([email protected])
A.K., 0000-0003-3378-9522
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Disrupting Prickle1 function in mice results in altered
geneexpression in skeletal condensates and is associated with
analtered apoptosis pattern in digit tips and interdigital
membranes,resulting in shorter and wider bones with disrupted
chondrocytepolarity (Yang et al., 2013). Therefore, Prickle1 might
havepreviously unknown functions in regulating polarity
andmigration in non-neuronal mammalian cells. Xenopus Prickle2
inmulticiliated epithelial cells controls the asymmetric
localization ofa subset of core PCP family proteins (Butler and
Wallingford,2015). The underlying mechanism of Prickle-mediated
non-neuronal polarity and migration, however, remains to be
defined.Cell migration is indispensable for animal development
and
tissue remodeling. Cell migration is dependent on traction
forcesgenerated within cells. Focal adhesions, which consist of
integrinsand adaptor proteins, connect the cytoskeleton to the
extracellularmatrix and transmit intracellular forces to the cell
exterior (Parsonset al., 2010). These forces promote focal adhesion
assembly,resulting in mature focal adhesions (Gardel et al., 2010).
For cells topolarize and to migrate, focal adhesions must be
disassembled.Microtubules play an essential role in focal adhesion
disassemblyduring migration (Stehbens and Wittmann, 2012).
Microtubuleplus-end-tracking proteins (+TIPs) bind to the growing
microtubuleplus end (Schuyler and Pellman, 2001). CLIP-associating
proteins(CLASPs, of which there are two isoforms CLASP1 and
CLASP2in mammals) play an essential role in the regulation of
microtubuledynamics (Akhmanova et al., 2001; Akhmanova and
Steinmetz,2008) and tether microtubules to focal adhesions,
facilitating focaladhesion disassembly (Stehbens et al., 2014).
LL5β (also known asPHLDB2) localizes to the cell membrane through
its pleckstrinhomology (PH) domain, and directly binds to CLASPs,
resulting inthe recruitment of CLASP-bound microtubules to focal
adhesions(Dowler et al., 2000; Lansbergen et al., 2006). Here, we
demonstratethat Prickle1 is involved in focal adhesion disassembly
incooperation with the CLASP–LL5β complex, thereby regulatingcell
polarity and migration.
RESULTSPrickle1 localizes adjacent to focal adhesions and
promotescell migrationBecause we have previously used MKN1 gastric
cancer cells as amodel to analyze cell migration and focal adhesion
turnover(Kurayoshi et al., 2006; Yamamoto et al., 2009),
morphologicalchanges of MKN1 gastric cancer cells were monitored
using time-lapse analyses. MKN1 cells formed lamellipodia randomly
until30 min after cell seeding, after which cells began to
polarize.Polarization was completed between 60 and 70 min
post-seeding, atwhich point lamellipodia consistently formed at one
location(Fig. 1A). One hour after seeding, approximately 30% of
MKN1cells showed a polarized morphology in which actin and stress
fibersaccumulated at the periphery of the cells. Caveolin then
accumulatedin a linear manner opposite to the site of actin
accumulation(Fig. S1A). Actin and caveolin are considered to mark
frontprotrusion (leading) and rear retraction (trailing) edges,
respectively(Gardel et al., 2010; Navarro et al., 2004). Thus,
these cells had asingle front protrusion and single rear site. This
morphological typeof cell was designated as being polarized here.
The remaining 70%of the MKN1 cells exhibited pleomorphic shapes,
and those cellsshowed multiple protrusions and retraction sites.
Caveolin localizedto punctate structures in the central region of
the cells (Fig. S1A). Inthis study these cells were designated as
non-polarized cells.Knockdown of Prickle1 with small interfering
RNA (siRNA) #1,
which targets the 3′-untranslated region of PRICKLE1 mRNA,
decreased the ratio of polarized to non-polarized cells,
whereasstable expression of hemagglutinin (HA)-tagged Prickle1
increasedthis ratio (Fig. 1B,C; Fig. S1B,C). Prickle1 siRNA #2,
which targetsthe open reading frame of PRICKLE1 mRNA, also
suppressed cellpolarization to a similar level as Prickle1 siRNA #1
(Fig. S1C).Furthermore, stable expression of HA–Prickle1 rescued
thephenotype induced by Prickle1 siRNA #1, indicating that
siRNAsagainst Prickle1 did not induce off-target effects (Fig.
1C).
Random migration of single cells was analyzed with
time-lapseimaging (Pankov et al., 2005). All trajectory paths of at
least 60randomly selected cells are shown together; the beginning
of eachcell path was placed at the intersection of the x and y
axes, andblack dots indicate the ends of the cell paths (Fig. 1D).
Euclideandistance (linear distance between start and end position)
wascalculated as an indicator of cell migration activity. Knockdown
ofPrickle1 by siRNA #1 decreased MKN1 cell migration (43.6%decrease
in mean Euclidean distance); however, HA–Prickle1-expressing cells
migrated faster than control cells (Fig. 1D)(106.2% increased in
mean Euclidean distance). The accumulateddistance (total distance
of trajectory path between start and endposition) was also
decreased and increased by Prickle1 knockdownand HA–Prickle1
expression, respectively; however, there was noclear difference in
directionality (Fig. 1D). Prickle1 siRNA #2 alsosuppressed cell
migration to a similar level as Prickle1 siRNA #1(Fig. S1D).
HA–Prickle1 expression rescued the decrease inmigration induced
with Prickle1 knockdown using siRNA #1(Fig. 1D) (144.1% increase in
mean Euclidean distance). Takentogether, these data suggest that
Prickle1 is involved in thepolarization and migration of MKN1
cells.
To understand the role of Prickle1 in cell migration,
subcellularlocalization of Prickle1 was examined. In the cell
retraction site ofnon-polarized cells, where caveolin had
accumulated, HA–Prickle1was observed as small punctate structures
and accumulated inlocalized areas adjacent to paxillin in focal
adhesions (Fig. 1E).HA–Prickle1 was rarely observed to be near
focal adhesions in theprotrusion site, where F-actin was enriched,
cortactin was presentand lamellipodia were formed (Fig. 1E; Fig.
S1E). In polarizedcells, HA–Prickle1 accumulated in a linear manner
along theretraction site and partially localized close to focal
adhesions(Fig. S1F). Close localization of Prickle1 to focal
adhesions in theretraction site was more evident in non-polarized
cells, probablybecause focal adhesion structures were highly
dynamic andtransient in polarized cells. HA–Prickle1 localization
adjacentto focal adhesions was observed to a similar extent in
HeLaS3cervical cancer cells (Fig. S1G). Endogenous Prickle1
wasimmunohistochemically observed adjacent to focal adhesions
inU251-MG malignant glioblastoma cells, which expressed
higherlevels of Prickle1 as compared to MKN1 cells (Fig. 1F).
Mouse Prickle1 (mpk1) and Prickle2 (mpk2) are expressed
inembryos at an early developmental stage, and knockout of the
mpk1gene results in early embryonic lethality (Tao et al., 2009).
Incontrast, mpk2-null mice do not show a strong phenotype(K.
Minegishi, H. Hamada et al., Graduate School for
FrontierBiosciences, Osaka University, Osaka, Japan,
personalcommunications). To confirm the role of Prickle1 in
cellmigration, a pair of mpk1+/−;mpk2−/− mice were crossed,
andmouse embryonic fibroblasts (MEFs) were prepared from
theresulting embryos. mpk1+/−;mpk2−/− MEFs and mpk1−/−;mpk2−/−
MEFs had reduced Euclidean distances (31.2% and 34.9%
decrease,respectively, expressed as mean Euclidean distances for
comparison)as compared with mpk1+/+;mpk2−/− MEFs (Fig. S2A). When
theseMEFs were subjected to a wound healing assay,
mpk1+/−;mpk2−/−
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MEFs and mpk1−/−;mpk2−/− MEFs showed reduced migration
ascompared to mpk1+/+;mpk2−/− MEFs (Fig. S2B). HA–Prickle1
alsolocalized close to paxillin inMEFs, similar to the localization
patternseen inMKN1 and HeLaS3 cells (Fig. S2C). These data
demonstrate
that the role of Prickle1 in polarized cell migration is not
limited toMKN1 cells.
Previous studies have revealed that Wnt5a, and van gogh 1 andvan
gogh 2 (VANGL1 and VANGL2, respectively; vertebrate
Fig. 1. Prickle1 promotes cell polarization andmigration. (A)
MKN1 cells were observed by performing time-lapse imaging using
phase-contrast microscopy atthe indicated time points after the
start of imaging. (B) Lysates of MKN1 cells that had been
transfected with control siRNA or siRNAs against Prickle1 (#1 and
#2)were probed with anti-Prickle1 and anti-Hsp90 antibodies. Hsp90
was used as a loading control. (C) Control MKN1 (MKN1/neo) or MKN1
cells expressingHA–Prickle1 (MKN1/HA-Prickle1) were treated with
control or Prickle1 siRNA #1 and cultured for 1 h. Cells were
stained with an anti-caveolin antibody andphalloidin. The number of
cells with polarizedmorphology or non-polarizedmorphologywas
counted (n≥120). Results are expressed as the percentage of each
celltype counted. (D) Control MKN1 or MKN1/HA-Prickle1 cells were
treated with control or Prickle1 siRNA #1, then observed with
time-lapse imaging using phase-contrast microscopy for 10 h. The
right panel indicates Euclidean distance (µm; n≥60 cells) and
accumulated distance (µm; n≥60 cells) together with
thedirectionality index (left). For box plots, small closed circles
indicate means, lines in the middle of the boxes indicate median,
whiskers indicate maximumand minimum values, and the ends of the
boxes indicate upper and lower quartiles. (E) MKN1/HA-Prickle1
cells were stained with anti-HA, anti-paxillin and anti-caveolin
antibodies, and phalloidin.White arrowheads indicate protrusion
sites, andwhite arrows indicate retraction sites. The four images
in the right-handpanel areenlarged from boxes P (protrusion) and R
(retraction) in the middle panel. (F) U251-MG cells were stained
with anti-Prickle1 and anti-paxillin antibodies, andphalloidin.
Enlarged images in the right bottom panel are extracted from arrow
numbers 1, 2 and 3. The left bottom panel shows endogenous Prickle1
expression inMKN1 and U251-MG cells. Results are expressed as
mean±s.d. *P
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homologs of strabismus) are genetically linked to PRICKLE1 inPCP
phenotypes (Kilian et al., 2003; Veeman et al., 2003b).
Wnt5a,Vangl1 and Vangl2 were knocked down using siRNAs to
determinewhether they play a role in Prickle1 in localization in
MKN1 cells(Fig. S3A). Additionally, MKN1 cells were treated with
IWP2 toblock the secretion of endogenous Wnt proteins, resulting
ininhibition of Dvl2 (one of the mammalian homologs of
Dsh)phosphorylation (Chen et al., 2009) (Fig. S3B).
Prickle1localization adjacent to focal adhesions was unaffected by
Wnt5aknockdown, Vangl1 and Vangl2 knockdown, or IWP2 treatment(Fig.
S3C,D). Thus, Prickle1 localization close to focal adhesionsand
Prickle1-induced migration might be independent of Wntsignaling and
the PCP pathway.It has been demonstrated that in Drosophila Prickle
and Dsh
mutually inhibit localization and function of each other (Jenny
et al.,2005; Tree et al., 2002), and that in mice Smurf ubiquitin
ligasesbind to Dvl and degrade Prickle proteins, thereby
controllingconvergent extension and cochlea hair cell polarity
(Narimatsuet al., 2009). Knockdown of all Dvl proteins (Dvl1, Dvl2
and Dvl3)neither affected the close localization of HA–Prickle1 to
focaladhesions nor inhibited Prickle1-dependent migration in
HA–Prickle1-expressing MKN1 cells, although knockdown of the
threeDvl proteins suppressedmigration of controlMKN1cells (Fig.
S3E,F).Because it is well known that Dvl regulates the cytoskeleton
throughRho and Rac, it is reasonable to assume that Dvl is involved
in basallevels of cell migration activity. Transiently expressed
FLAG–Dvl2showed small punctate structures, and they did not affect
thelocalization of HA–Prickle1 (Fig. S3G). Thus, it is likely that
Dvlproteins are not involved in the Prickle1 functions observed in
thisstudy.
Prickle1 promotes focal adhesion turnoverNext, the role of
Prickle1 in focal adhesion turnover wasinterrogated. GFP–paxillin
was expressed in MKN1 cells, and itsdynamics were visualized in
red, green, blue and magenta at 0, 15,30 and 45 min after the start
of imaging respectively (Fig. 2A). Theimages showing the basal
surface of the cell were superimposed,and dynamic adhesions were
defined as areas containing more thanone color (Yamana et al.,
2006). In Prickle1-depleted MKN1 cells,the focal adhesion size was
enlarged, and areas of paxillinlocalization are shown in white,
indicating that focal adhesiondynamics were decreased (Fig. 2A). In
contrast, focal adhesiondynamics were increased in
HA–Prickle1-expressing MKN1 cells(Fig. 2A). These data indicate
that Prickle1 plays a positive role infocal adhesion turnover.
mCherry–Prickle1 was localized toprojection-like structures at the
cell periphery; these structuresreadily protruded and retracted
during the observation period. Akymograph assay revealed that
mCherry–Prickle1 accumulated atcell retraction sites but not to
protrusion sites (Fig. 2B; Movie 1),suggesting that Prickle1 is
involved in the retraction process at thecell periphery.In the cell
periphery of non-polarized cells, GFP–paxillin-
positive focal adhesions gradually turned over for up to about40
min after the start of the retraction process (Fig. 2C).
mCherry–Prickle1 showed a similar turnover time course (Fig.
2C),suggesting that the appearance and loss of Prickle1 is
associatedwith those of paxillin at the retraction sites. In
comparison withfixed cells, mCherry–Prickle1 in live cells gave a
rather smearedappearance and partially colocalized with
GFP–paxillin.It has been previously shown that microtubules are
guided to
substrate adhesion sites in order to induce contact release and
celledge retraction (Kaverina et al., 1999). Therefore, the effects
of the
microtubule depolymerizing agent nocodazole on
Prickle1localization and dynamics were investigated.
Nocodazoletreatment stabilized and enlarged sites of GFP–paxillin,
whichwas not lost from sites until about 105 min after treatment,
andmCherry–Prickle1 sites also enlarged and persisted to a similar
timeas GFP–paxillin (Fig. 2D), suggesting that microtubule
disassemblyextends the lifetime of Prickle1 and focal adhesions,
probablyowing to the inhibition of their degradation, and that
intactmicrotubules are not required for Prickle1 assembly but
arenecessary for Prickle1 disassembly. Rho, Rho kinase and
myosin-mediated cell contractility are required for the maintenance
ofmature focal adhesions (Katoh et al., 2001; Rottner et al.,
1999).Treatment with the Rho-kinase inhibitor Y-27632 led to the
rapidloss of GFP–paxillin and mCherry–Prickle1 (Fig. 2E).
Y-27632treatment after nocodazole treatment also induced
disassembly ofGFP–paxillin and the associated mCherry–Prickle1
(Fig. 2D).These results suggest that Prickle1 assembly depends on
focaladhesion structures and that intact microtubules are not
required forPrickle1 disassembly when actomyosin contraction has
beeninhibited. Thus, the data indicate that Prickle1 dynamics
areassociated with focal adhesion turnover.
Prickle1 forms a complex with CLASPs and LL5βAs shown in Fig.
1E, HA–Prickle1 closely localized but did notcompletely overlap
with paxillin. Prickle1 localization seemed tobe similar to that of
CLASPs and LL5β (Lansbergen et al., 2006).In addition,
focal-adhesion-associated localization of CLASPsand LL5β has been
shown to depend on focal adhesion structures,similar to Prickle1
localization (Stehbens et al., 2014). CLASPshave been shown to
associate with the plus end of growingmicrotubules and to attach to
the cell cortex through the interactionwith LL5β, and LL5β-mediated
CLASP recruitment facilitatesfocal adhesion turnover (Lansbergen et
al., 2006; Stehbens et al.,2014). HA–Prickle1, endogenous LL5β and
endogenous CLASP1colocalized, and these proteins localized adjacent
to GFP–paxillinin focal adhesions (Fig. 3A). Consistent with these
results, HA-Prickle1 formed a complex with GFP–CLASP1,
GFP–CLASP2and GFP–LL5β in HEK293T kidney epithelial cells (Fig.
3B).HA–Prickle1 did not form a complex with GFP–LL5α (Fig. 3B).In
addition, endogenous Prickle1 bound to GFP–LL5β, andendogenous
CLASP1 bound to HA–Prickle1 (Fig. S4A). Deletionmutant analyses
showed that the PET-LIM domain of Prickle1(amino acid residues
1–313) was necessary and sufficient for theinteraction between
Prickle1 and LL5β or CLASP1 in HEK293Tcells (Fig. S4B).
In controlMKN1 cells, endogenous CLASP1 and LL5β localizedclose
to paxillin in distal areas of the cell cortex (Fig. 3C).
InPrickle1-depleted MKN1 cells, CLASP1 was distributed evenly
indistal and sub-distal areas, although LL5β still localized in
distalareas, and colocalization of LL5β and CLASP1 was lost (Fig.
3C).In MEFs and MKN1 cells, CLASP1 and LL5β colocalized adjacentto
focal adhesions. In mpk1−/−;mpk2−/− MEFs, LL5β accumulatedclose to
focal adhesions in distal areas of the cell cortex. CLASP1was
diffusely distributed in distal and sub-distal areas (Fig.
S4C).Knockdown of CLASP1 and CLASP2, or LL5β in MKN1 cellsdid not
affect paxillin localization at focal adhesions (Fig. 3D;Fig. S4D).
Knockdown of LL5β, but not of CLASP1 and CLASP2,impaired close
localization of HA–Prickle1 to paxillin (Fig. 3D).Therefore,
localization of Prickle1 adjacent focal adhesions mightplay an
important role in the formation of a complex between LL5βand
CLASPs, and might be required for cortical CLASPlocalization.
Furthermore, knockdown of CLASP1 and CLASP2
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or LL5β reduced HA–Prickle1-dependent cell polarization
andmigration (Fig. 3E,F). Thus, Prickle1 could be involved in
focaladhesion turnover through its interaction with the
CLASP–LL5βcomplex.
Propermembrane localization of Prickle1 is necessary for
itsfunctionMouse Prickle1 ismodifiedwith a farnesylmoiety at
cysteine residue829 (Cys829) (Fig. 4A). Farnesylation modification
is necessary
Fig. 2. See next page for legend.
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for Prickle localization to cell membranes as well as its
function inDrosophila melanogaster wings (Strutt et al., 2013).
Wehypothesized that Prickle1 farnesylation might play a role inits
cortical localization to focal adhesions as well as in
Prickle1-mediated regulation of cell migration and polarization.
Cellfractionation analysis revealed that HA–Prickle1 is mainly
presentin the membrane fraction; however, when Cys829 was mutatedto
serine (HA–Prickle1-C829S), Prickle1 was found in thecytosolic
fraction (Fig. 4B). Consistent with these
results,immunocytochemical analysis showed that
HA–Prickle1-C829Swas distributed diffusely throughout the cytosol
and was notlocalized close to focal adhesions (Fig. 4C).
HA–Prickle1-C829Sexpression did not promote cellmigration or
polarization (Fig. 4D,E).To determine whether membrane localization
is sufficient for
Prickle1 activity, a myristic-acid-binding site (Zhou et al.,
1994)was fused to the N-terminal region of HA–Prickle1-C829S
togenerate Myr–HA–Prickle1-C829S, which was then stablyexpressed in
MKN1 cells. Similar to HA–Prickle1, Myr–HA–Prickle1-C829S was
present in the membrane fraction and localizedto the entire cell
periphery; however, Myr–HA–Prickle1-C829S didnot localize to the
vicinity of focal adhesions (Fig. 4B,C).Furthermore,
Myr–HA–Prickle1-C829S did not promote cellmigration or polarization
(Fig. 4D,E). Neither HA–Prickle1-C829S nor Myr–HA–Prickle1-C829S
associated with GFP–CLASP1 or GFP–LL5β (Fig. 4F). These results
suggest thatproper membrane localization of Prickle1 adjacent to
focaladhesions through C-terminal farnesylation is necessary for
cellpolarization and migration, as well as its ability to form a
complexwith CLASPs and LL5β.
Proper membrane localization of Prickle1 is necessary
formicrotubule targeting to focal adhesionsAs shown in Fig. 2C,
Prickle1 dynamics were closely correlatedwith paxillin dynamics.
Prickle1 overexpression promoted GFP–paxillin disassembly (Fig.
5A). Microtubules that have beentargeted to focal contact sites
promote focal adhesion disassemblyin living fibroblasts (Kaverina
et al., 1999). In non-polarizedMKN1cells, microtubules and focal
adhesions were visualized using RFP–
tubulin and GFP–paxillin, respectively; a single
focal-adhesion-targeting event was defined as the overlap of the
microtubule tipwith paxillin (Fig. 5B; Movie 2). The frequency of
targeting eventswas assessed within a 20-min period. In control
MKN1 cells, themajority of focal adhesions were targeted an average
of 9.7 times(Fig. 5C; Fig. S4E). Enforced expression of HA–Prickle1
increasedthe frequency of targeting events to an average of 14.1
times,whereas that of HA–Prickle1–C829S and Myr–HA–Prickle1-C829S
did not affect the frequency (Fig. 5C; Fig. S4E). Prickle1knockdown
reduced the frequency of targeting to an average of 7.5times from
11.3 times in control; HA–Prickle1 expression rescuedthis
phenotype, as demonstrated by an increase in the number oftargeting
events to an average of 14.2 times, but Prickle1 mutantsonly
partially rescued the Prickle1 knockdown phenotypes(Fig. 5D; Fig.
S4F).
Paxillin is known to be ubiquitylated and degraded
duringmesodermal cell migration in Xenopus laevis gastrulation
(Iiokaet al., 2007). It has also been shown that Prickle1 is
degraded by theproteasome through the action of an SCF E3 ubiquitin
ligase inDrosophila pupal wings (Strutt et al., 2013). Indeed,
HA–Prickle1was rapidly (within 2 h) degraded in MKN1 cells in the
presence ofthe protein synthesis inhibitor cycloheximide; when MKN1
cellswere treated with the proteasome inhibitor MG132 in addition
tocycloheximide, Prickle1 degradation was suppressed (Fig.
S4G).Nocodazole treatment suppressed HA–Prickle1 degradation(Fig.
S4H), suggesting that intact microtubules are required forPrickle1
degradation as well as for Prickle1 disassembly.
Overexpression of HA–Prickle1-C829S or Myr–HA–Prickle1-C829S did
not affect GFP–paxillin disassembly (see Fig. 5A).These Prickle1
mutants were also degradation resistant (Fig. S4H).Taken together,
these data indicate that proper membranelocalization of Prickle1
through farnesylation is necessary for theubiquitin-dependent
Prickle1 degradation; thus, Prickle1degradation, focal adhesion
disassembly and cell migration mightbe functionally linked.
Prickle1 mediates EGF-dependent cell signaling andmigrationIt
has been shown that EGF induces focal adhesion disassembly(Xie et
al., 1998). Indeed, EGF promoted paxillin disassembly, andPrickle1
knockdown using siRNA #1 suppressed EGF-mediatedpaxillin
disassembly in MKN1 cells (Fig. 6A). EGF also increasedthe ratio of
polarized cells and promoted MKN1 cell polarization(Fig. 6B,C).
Knockdown of Prickle1 using siRNA #1 suppressedEGF-dependent
polarization and migration; these phenotypes wererescued by
HA–Prickle1 expression (Fig. 6C,D). EGF activatedEGF receptor
(EGFR), AKT and JNK1 and JNK2 in MKN1 cells,as indicated by their
phosphorylation in response to EGF treatment.Prickle1 knockdown
using siRNA #1 had no effect on EGFRand AKT phosphorylation, but
inhibited JNK1 and JNK2phosphorylation (Fig. 6E). This suggests
that Prickle1 actsdownstream of EGFR and AKT, but upstream of JNK1
and JNK2.
Consistent with these results, HA–Prickle1 expression inHEK293T
cells activated JNK1 and JNK2 (Fig. 6F). The JNKinhibitor SP600125
suppressed HA–Prickle1-induced polarization,but the inhibitor of
p38 MAPKs SB203580 and the MEK inhibitorU0126 did not affect
HA–Prickle1-induced polarization (Fig. 6G).Consistent with these
results, SP600125 suppressed EGF-dependent polarization (Fig.
6H).
Interestingly, HA–Prickle1-C829S and Myr–HA–Prickle1-C829S also
activated JNK1 and JNK2 to a similar extent as HA–Prickle1 (Fig.
6F). However, HA–Prickle1-C829S and Myr–HA–
Fig. 2. Prickle1 promotes focal adhesion turnover. (A) The
dynamics ofGFP–paxillin in control MKN1 cells, Prickle1-depleted
MKN1 cells usingPrickle1 siRNA #1 (MKN1/Prickle1 siRNA #1) andMKN1
cells expressing HA–Prickle1 (MKN1/HA-Prickle1) were visualized.
The percentage of adhesionturnover within 45 min was calculated for
20 different focal adhesions (FAs) percell; at least 20 cells were
counted. The results are quantified and are shown inthe right-hand
panel. For box plots, lines in the middle of the boxes
indicatemedian, whiskers indicate maximum and minimum values, and
the ends of theboxes indicate upper and lower quartiles. (B) MKN1
cells expressingmCherry–Prickle1 were observed with time-lapse
imaging. The kymograph in the middlepanel was constructed using
data from point ‘a’ to point ‘b’ for 60 min. The right-hand panel
is an illustration of the protrusion and retraction stages shown in
thekymograph. The arrows indicate accumulation of mCherry–Prickle1
duringthe retraction stage. (C–E) MKN1 cells that transiently
expressed GFP–paxillin (green) and mCherry–Prickle1 (red) were
observed with time-lapseimaging in the absence (C) or presence of
10 µM nocodazole at t=0 minfollowed by treatment with 20 µM Y-27632
at t=105 min (D), or presence of20 µM Y-27632 (E). Top panels
indicate turnover dynamics of GFP–paxillinfocal adhesions
andmCherry–Prickle1. Numbers represent the minutes afterthe start
of imaging. Fluorescence intensity profiles were measured
usingImageJ software (NIH) and plotted as a function of time, and
were normalizedto the maximum fluorescence intensity. For C,
normalized intensity data werealigned based on points of maximum
intensity for GFP–paxillin. (n=20 focaladhesions in C; n=4 focal
adhesions in D,E; from one to three cells). Resultsare expressed as
mean±s.d. *P
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Fig. 3. See next page for legend.
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Prickle1-C829S did not rescue the effects of Prickle1
knockdownon of EGF-induced cell polarity (Fig. 6I). These data
suggest thatproper Prickle1 membrane localization is not necessary
for JNKactivation but is required for EGF-dependent cell
polarization.Thus, these gain- and loss-of-function experiments
suggest thatEGF-dependent JNK activation through Prickle1 is
involved infocal adhesion turnover and cell polarization.
DISCUSSIONMigrating cells continuously form and disassemble
cell–substrateadhesions, not only at the leading edge but also at
the center and thetrailing edge (Broussard et al., 2008). Once
formed, focal adhesionsmust be released for directional movement in
a process termedadhesion turnover (Webb et al., 2002). The
coordinated asymmetryof assembly and disassembly is necessary for
directional migration,and microtubules play a key role in
asymmetric adhesion dynamics(Kaverina et al., 1999). Microtubule
targeting to focal adhesionsoccurs behind the leading edge and at
the trailing edge, resulting infocal adhesion disassembly. We found
that Prickle1 is located withCLASPs and LL5β adjacent to focal
adhesions in the retractingsites, that it promotes front–rear
polarization and cell migration, andthat microtubules are targeted
to focal adhesions through the tertiarycomplex of Prickle1, CLASP
and LL5β. Taken together with theobservations that alternative
Prickle protein isoforms control theorientation of microtubule
network (Olofsson et al., 2014), wesuggest Prickle1 is involved in
cell migration through microtubuledynamics.These findings are not
confined to MKN1 cells because Prickle1
was observed in punctae close to paxillin in HeLaS3, MEFs
andU251 cells. Prickle1 knockout also suppressed MEF migration.
Theeffects of homozygous knockout were similar to those
ofheterozygous knockout. The exact reason is not known
currently,but one possibility is that MEF migration is less
dependent onPrickle1 than MKN1 cell migration, and that
Prickle1haploinsufficiency results in the inhibition of migration
to asimilar level to Prickle1 homozygous knockout. AlthoughPrickle1
is considered to be one of the proteins that regulates the
PCP pathway (Devenport, 2014; Gray et al., 2011; Singh
andMlodzik, 2012; Veeman et al., 2003a), our results show
thatPrickle1 localization close to focal adhesions does not depend
onDvls, Wnt or Vangl1 and Vangl2. Therefore, the regulation of
focaladhesion turnover would be a new function of Prickle1,
independentof the Wnt and PCP pathway.
Peripheral membrane localization of Prickle1
throughfarnesylation was dependent on focal adhesion structure and
LL5βlocalization. Focal adhesions recruit CLASPs independently
ofmicrotubules through the interaction of CLASPs and
LL5β(Lansbergen et al., 2006). This recruitment was dependent
onproper membrane localization of Prickle1, as evidenced by
theinability of the farnesylation-deficient Prickle1 mutant
(Prickle1-C829S) to promote complex assembly and microtubule
targeting.Furthermore, N-terminal myristolyation through the
addition of amyristic-acid-binding site to Prickle1-C829S
(Myr–Prickle1-C829S)did not rescue these functions, indicating that
the membranelocalization of Prickle1 was not sufficient to form a
complex withCLASPs and LL5β, or to target microtubules to focal
adhesions. Thissuggests that C-terminal farnesylation-dependent
Prickle1 membranelocalization is substantively different from
N-terminalmyristoylation-dependent localization.
Farnesylation-mediatedPrickle1 membrane localization might lead
Prickle1 to form a tightcomplex with LL5β to recruit CLASPs,
thereby making a tertiarycomplex of Prickle1, CLASPs and LL5β;
alternatively, farnesylation-mediated Prickle1 membrane
localization might recruit additionalproteins that regulate focal
adhesion turnover. LL5β localizes close tofocal adhesions, probably
through the binding to phosphatidylinositol(3,4,5)-trisphosphate
and integrins (Stehbens and Wittmann, 2012),and its localization
might not depend on Prickle1. By contrast,CLASP localization close
to focal adhesions depends on theinteraction with LL5β (Lansbergen
et al., 2006) and Prickle1 (thisstudy). Therefore, Prickle1 would
support the binding between LL5βand CLASP1 adjacent to focal
adhesions, leading to the promotion ofmicrotubule targeting.
Prickle1 localization to the correct membrane position wasalso
required for its degradation, as evidenced by the
observeddegradation resistance of Prickle1-C829S and
Myr–Prickle1-C829S. It has been previously reported that Prickle1
is degradedin a ubiquitin-dependent manner by a Cullin E3 ligase
inDrosophila (Cho et al., 2015; Strutt et al., 2013) and by Smurf
inmammals (Narimatsu et al., 2009). Strabismus (the
invertebratehomolog of vangl proteins) promotes the recruitment of
farnesylatedPrickle to the membrane, as well as its degradation, in
Drosophila(Strutt et al., 2013). Precisely regulated Prickle
protein levelsestablish polarity by modulating internalization and
removal ofStrabismus and Flamingo (Cho et al., 2015). Our data
indicate thatLL5β could perform a similar function to Strabismus in
mammals.Prickle1 degradation might contribute to the maintenance of
theasymmetric distribution of PCP proteins by controlling the
amountof Prickle1 protein present at focal adhesions. Our results
suggestthat Prickle1 degradation is associated with focal
adhesiondisassembly. Microtubule disassembly stabilized Prickle1,
anddegradation-resistant Prickle1 mutants were unable to
rescuemicrotubule targeting to focal adhesions in
Prickle1-depletedcells. Therefore, proper Prickle1 membrane
localization mightplay an important role in microtubule targeting
to focal adhesionsand in Prickle1 degradation, leading to focal
adhesion disassembly.
It is well established that growth factor receptor signaling
andintegrin signaling merge on focal adhesions to regulate
cellularproliferation, adhesion and migration through the
activation of bothSrc-family kinases and integrin-linked kinases
(Dedhar et al., 1999;
Fig. 3. Prickle1 forms a complex with CLASPs and LL5β. (A) MKN1
cellsexpressing HA–Prickle1 (MKN1/HA-Prickle1) and GFP–paxillin
were stainedwith anti-HA, anti-LL5β, anti-CLASP1 and anti-GFP
antibodies. Dashed whiteboxes indicate areas shown in enlarged
images. (B) Immunoprecipitation (IP)was performed on lysates of
HEK293T cells that expressed the indicatedproteins, using anti-GFP
antibody. Immunoprecipitates were probed with theindicated
antibodies. GFP-CLASP1/2, detection with an antibody
recognizingboth GFP-tagged CLASP isoforms. (C) MKN1 cells that had
been transfectedwith control or Prickle1 siRNA #1 were stained with
anti-LL5β, anti-CLASP1and anti-paxillin antibodies. Enlarged images
of sub-distal and distal areas inthe right-hand top panels were
enlarged from areas indicated with dashedwhite boxes. Bottom panels
are quantifications of the relative intensities ofCLASP1 and LL5β
fluorescence from distal and sub-distal areas. (D) MKN1cells that
had been transfected with control siRNA, or siRNA targeting
CLASP1and CLASP2 (CLASP1/2 siRNA) or LL5β were stained with anti-HA
and anti-paxillin antibodies. (E) MKN1 (MKN1/neo) or
MKN1/HA-Prickle1 cells weretransfected with control siRNA or siRNA
targeting CLASP1 and CLASP2 orLL5β, then the numbers of polarized
cells were counted. The ratio of polarizedcells was expressed as a
percentage of all MKN1 or MKN1/HA-Prickle1 cellsobserved. (F) Cell
migration was observed and analyzed by performing time-lapse
imaging for 10 h in the same cells used for E. The right-hand
panelindicates quantification of Euclidean distance (µm; n≥60
cells). Results areexpressed asmean±s.d. For box plots, lines in
themiddle of the boxes indicatemedian, whiskers indicate maximum
and minimum values, and the ends of theboxes indicate upper and
lower quartiles. *P
-
Fig. 4. See next page for legend.
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Parsons and Parsons, 1997). It has also been shown that
integrinsco-operate with EGFR in several cell types to regulate
multiplesignaling pathways (Streuli and Akhtar, 2009). In addition,
theinteraction between Fz PCP signaling and the transcription
factorFos, which acts downstream of EGFR and JNK signaling, have
beenshown to regulate the Drosophila photoreceptor cell fates
andommatidial polarity (Weber et al., 2008). Consistent with
theseprevious studies, our results revealed that Prickle1 mediates
EGFsignaling and EGF-dependent polarization and migration.
Propermembrane localization of Prickle1 through farnesylation was
alsorequired for EGF-dependent polarization. This supports
thehypothesis that signaling molecules that regulate
EGF-dependentcellular functions are localized together at focal
adhesions. JNK hasbeen shown to phosphorylate paxillin and to
regulate cell migration(Huang et al., 2003). In the present study,
Prickle1 led to JNKactivation downstream of EGFR. Therefore,
Prickle1-dependentJNK activation might be important for
EGF-dependent cellpolarization. However, Prickle1-C829S and
Myr–Prickle1-C829Swere still able to activate JNK. This indicates
that Prickle1-dependent JNK activation is not sufficient for cell
polarization andmigration. Proper membrane localization of Prickle1
therefore mustact through additional pathways to control cellular
functions.It has been reported that Prickle1 activates AKT to
regulate focal
adhesion turnover and promotes breast cancer cell migration
throughthe interaction with the mammalian target of rapamycin
complex 2(mTORC2) (Daulat et al., 2016). Upregulation of Prickle1
in basalbreast cancers is correlated with poor prognosis,
suggesting thatPrickle1 has tumor progressive functions in vivo. To
confirm theinvolvement of Prickle1 in cancer cell migration in
vivo, weexamined migration of MKN1 cells in skin xenografts using
two-photon intravital microscopy. Consistent with previous
observationsthat the majority of cancer cells, other than melanoma
or leukemiacells, are immotile in vivo (Clark and Vignjevic, 2015),
MKN1 cellswere immobile for several hours in skin xenografts (data
not shown).In vivo roles of the Prickle1–CLASP–LL5βmodule in
developmentand tumorigenesis need to be examined in future
experiments.In conclusion, we have identified new functions of
Prickle1 in
focal adhesion turnover and mammalian cell migration.
Thesefunctions are dependent on its localization to the retraction
sitethrough C-terminal farnesylation. The proper trafficking
ofPrickle1 to the cell surface is involved in its association
withCLASPs and LL5β. Similarly, its proper localization increases
thefrequency of contact between microtubules and focal
adhesions,
leading to focal adhesion turnover through Prickle1
degradation.These new functions of Prickle1 could be important for
cellpolarization and migration.
MATERIALS AND METHODSCell culture and transfectionMKN1 gastric
cancer cells were kindly provided by Dr Wataru Yasui(Hiroshima
University, Hiroshima, Japan). MKN1 cells were grown inRPMI1640
supplemented with 10% fetal bovine serum (FBS)
andpenicillin-streptomycin (Yokozaki, 2000). HEK293T cells
weremaintained in Dulbecco’s modified Eagle’s medium (DMEM):Ham’s
F12(1:1) supplemented with 10% FBS, 0.065 g/l penicillin and 0.1
g/lstreptomycin. U251-MG cells were maintained in Eagle’s
minimalessential medium supplemented with 10% FBS, 1% non-essential
aminoacids (NEAA) and 1 mM sodium pyruvate (NaP). For live-cell
imaging,cells were plated on fibronectin-coated glass-bottomed
dishes.
MKN1 cells stably expressing HA–Prickle1 and mutants were
generatedby co-transfecting cells with pPGK-neo and
pCGN-HA-Prickle1, pCGN-HA-Prickle1-C829S or
pCGN-Myr-HA-Prickle1-C829S. Cells wereselected and maintained in
medium containing 400 μg/ml G418. Totransiently express proteins,
cells were transfected with plasmids usingLipofectamine LTX or
Lipofectamine 2000 (Life Technologies, ThermoFisher Scientific)
according to the manufacturer’s protocol.
For the preparation of MEFs, a pair of mpk1+/−;mpk2−/− mutant
micewere crossed, and MEFs were prepared using a 3T3 protocol
(Todaro andGreen, 1963) from the resulting embryos at E13.5 that
were obtained fromDrs Katsura Minegishi and Hiroshi Hamada
(Graduate School for FrontierBiosciences, Osaka University, Osaka,
Japan), and maintained in DMEMsupplemented with 10% FBS.
ImmunocytochemistryCells that had been grown on
fibronectin-coated glass coverslips were fixedfor 10 min at room
temperature in phosphate-buffered saline (PBS)containing 4% (w/v)
paraformaldehyde. Immunocytochemistry wasperformed according to a
previous paper (Matsumoto et al., 2010).Images were taken with a
LSM510, LSM710 or LSM880 confocalmicroscope (Carl Zeiss, Jena,
Germany).
Polarized cells were defined as cells that had formed a
singlelamellipodium (protrusion) at the leading edge and in which
caveolin hadlocalized in a linear manner to the single retraction
site, or as cells in whichcaveolin had localized to the single
retraction site in a linear manner but thathad no apparent
lamellipodium formation. Non-polarized cells were definedas cells
that had formed multiple lamellipodia (protrusions) and
multiplefinger-like retraction sites surrounding the cell
periphery.
Cell migration assaysCells were seeded for 30 min. Time-lapse
imaging was performed using anIX81-ZDC microscope (Olympus, Tokyo,
Japan). For EGF treatment,25 ng/ml of EGF was added to growth
medium. For inhibitor treatment,inhibitors were added to growth
medium 2 h before cell seeding. Imageswere captured every 5 min for
10 h, and movies were exported usingMetaMorph software (Molecular
Devices). Cell tracking and data analysiswas done using the Manual
Tracking and Chemotaxis Tool in ImageJsoftware [National Institutes
of Health (NIH)].
To perform wound healing assays, MEFs were plated onto
collagen-coated coverslips. The monolayer of MEFs was then manually
scratchedwith a plastic pipette tip. Cells were washed with PBS
three times, then thewounded cell monolayers were allowed to heal
for 2, 4, 6 and 8 h inRPMI1640 medium containing 10% FBS (Kobayashi
et al., 2006). Thewound size was measured to determine the distance
traveled by MEFs usingAxioVision 4.8.2.0 (Carl Zeiss).
Knockdown of protein expression with siRNAssiRNA target
sequences used in this study are described in Table S2. Cellswere
transfected with 40 nM siRNA using Lipofectamine RNAiMAX
(LifeTechnologies, Thermo Fisher Scientific) according to the
manufacturer’sinstructions.
Fig. 4. Plasma membrane localization of Prickle1 is necessary
for itsfunction. (A) Schematic representation of Prickle1 mutant
constructs used inthis study. (B) Total homogenates ‘T’ of MKN1
cells that stably expressed HA–Prickle1 (MKN1/HA-Prickle1) wild
type (WT), HA–Prickle1-C829S (C829S)and Myr–HA–Prickle1-C829S
(Myr+C829S) were fractionated into cytosol ‘C’and membrane ‘M’
fractions. Samples were probed for the indicated proteins.β-tubulin
and transferrin receptor (TfR) were used as cytoplasm andmembrane
markers, respectively. (C) The same cells described in B
werestained with anti-HA and anti-paxillin antibodies. (D) The
number of polarizedcells, using the same cells that are described
in B, was counted. The ratio ofpolarized cells is expressed as a
percentage of all MKN1 cells expressingHA–Prickle1 or its mutants.
(E) Cell migration was observed and analyzedby performing
time-lapse imaging for 10 h, using the same cells as thosedescribed
in B. The right panel indicates quantification of Euclidean
distance(µm; n≥60 cells). For box plots, lines in the middle of the
boxes indicatemedian, whiskers indicate maximum and minimum values,
and the ends of theboxes indicate upper and lower quartiles. (F)
Immunoprecipitation (IP) wasperformed on lysates of HEK293T cells
that expressed the indicated proteinsusing an anti-GFP antibody.
The immunoprecipitates were probed for theindicated proteins.
Results are expressed as mean±s.d. *P
-
For rescue experiments, we expressed HA–Prickle1 (wild type)
inMKN1 cells that had been transfected with Prickle1 siRNA #1
targetingthe 3′-untranslated region of Prickle1. In the case of
Prickle1-knockdown experiments, Prickle1 siRNA #1 was used in
all
experiments, and Prickle siRNA #2 was used in Fig. 1B andFig.
S1C,D. Transfection efficiency of siRNA was almost 100% inMKN1
cells when it was assessed with Cy3-labeled siRNA (Takara,Tokyo,
Japan).
Fig. 5. Proper membrane localization of Prickle1 is necessary to
target microtubules to focal adhesion sites. (A) Left panels: focal
adhesion (FA)disassembly was visualized using time-lapse imaging of
control MKN1 cells and MKN1 cells expressing HA–Prickle1
(MKN1/HA-Prickle1) that also transientlyexpressed GFP–paxillin.
Results are shown as representative images from three independent
experiments. Times represent minutes after the start of
imaging.Right panel: focal adhesion disassembly time (min) in MKN1
(MKN1/neo), MKN1/HA-Prickle1 (wild type; WT),
MKN1/HA-Prickle1-C829S (C829S) andMKN1/Myr-HA-Prickle1-C829S
(Myr+C829S) cells was measured by performing time-lapse imaging for
5 h and quantified (n≥40 focal adhesions). Disassemblytime is shown
as the time for focal adhesions with size≥4 µm to completely
disappear. Dashed white boxes indicate areas shown in enlarged
images. (B) MKN1cells expressing RFP–tubulin and GFP–paxillin were
observed using time-lapse imaging at an interval of 30 s to
quantify the frequency of microtubulecontact to focal adhesions.
Arrowheads indicate colocalization of RFP–tubulin and GFP–paxillin
(targeting event). Dashed white box indicates area enlarged
inimages. (C) The frequency of microtubule targeting to a focal
adhesion per focal adhesion (n≥35 focal adhesions) was quantified
in MKN1, MKN1/HA-Prickle1,MKN1/HA-Prickle1-C829S and
MKN1/Myr-HA-Prickle1-C829S cells expressing RFP–tubulin and
GFP–paxillin. Interval 30 s, t=20 min. Small closed circlesindicate
means. For box plots, lines in the middle of the boxes indicate
median, whiskers indicate maximum and minimum values, the ends of
the boxes indicateupper and lower quartiles, and small closed
circles in C,D indicate means. (D) The cells described in C were
transfected with control or Prickle1 siRNA #1, and thesame
experiments were performed. *P
-
Focal adhesion turnover assayIn Fig. 2A, the images at 0, 15, 30
and 45 min are depicted inred, green, blue and magenta,
respectively, and were superimposed.
Non-dynamic adhesions were defined as spots appearing in
whiteafter superimposition; dynamic adhesions were defined as spots
thatdid not appear in white after superimposition. Analyses
were
Fig. 6. See next page for legend.
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performed according to the previous paper (Matsumoto et al.,
2010;Yamana et al., 2006).
In Fig. 2C–E, to determine paxillin and Prickle1 turnover,
time-lapseimages of MKN1 cells were acquired for 150 min at 3-min
intervals.Regions of interest were defined as areas surrounding
individual focaladhesions. Where indicated, cells were treated with
10 µM nocodazoleand/or 20 µM Y-27632. For turnover dynamics of
GFP–paxillin focaladhesions and surrounding mCherry–Prickle1,
fluorescence intensityprofiles were measured using ImageJ software
(NIH) and plotted as afunction of time, and were normalized to the
maximum fluorescenceintensity. For Fig. 2C, normalized intensity
data were aligned based onpoints of maximum intensity for
GFP–paxillin (n=20 focal adhesions inFig. 2C; n=4 focal adhesions
in Fig. 2D,E; from one to three cells).
In Fig. 5A, to observe and quantify focal adhesion disassembly,
time-lapse images of MKN1 cells were acquired for 5 h at 5-min
intervals.Regions of interest were defined as areas surrounding
individual focaladhesions. Focal adhesion disassembly time was
calculated as the timebetween when the focal adhesions reached
maximum size (≥4 µm) andwhen focal adhesions had completely
disappeared. Quantification focusedon those focal adhesions at cell
retraction sites.
Imaging of focal adhesion turnover was performed using
time-lapsefluorescence spinning-disc microscopy with an Observer.Z1
invertedmicroscope equipped with a Yokogawa confocal scanner unit
CSU-W1(Yokogawa Electric Corporation, Tokyo, Japan).
Quantification of the distribution of CLASP1 and LL5β in
MKN1cells and MEFsTo examine CLASP1 and LL5β accumulation at distal
and sub-distalregions of cells, the mean fluorescence intensity in
a 5 nm×5 nm area at the
distal (cell edge) and the sub-distal (center between cell edge
and nucleus)regions was measured using ZEN software (Carl Zeiss)
(n≥17 regions fromfive cells).
Microtubule and focal adhesion targeting assayIn Fig. 5B,
RFP–tubulin was stably expressed in MKN1 cells and MKN1cells
expressing HA–Prickle1, HA–Prickle1-C829S or Myr–HA–Prickle1-C829S
cells. Where indicated, cells were transfected with control siRNA
orsiRNA against Prickle1 together with GFP–paxillin, and then
visualized byusing spinning-disc microscopy. Images were taken at
30-s intervals for30 min. A single targeting event was defined as
an overlap betweenmicrotubules and paxillin. Only focal adhesions
at the retraction sites weremonitored, and at least 35 focal
adhesions were observed and quantified foreach cell line and
treatment.
Prickle1 kymographsTime-lapse imaging was performed for 2 h at
1-min intervals using anIX81-ZDC microscope (Olympus). Kymographs
were constructed usingMetaMorph software (Molecular Devices).
Materials and chemicalsGFP–CLASP1, GFP–CLASP2, GFP–LL5α,
GFP–LL5β, and RFP–tubulinexpression vectors were kindly provided by
Dr Yuko Mimori-Kiyosue(RIKEN Center for Life Science Technologies,
Kobe, Japan). pPGK-neowas provided by Dr Shinji Takada (National
Institutes of Natural Sciences,Okazaki, Japan).
Details on primary antibodies used in this study are described
in Table S1.Other materials were purchased from commercial
sources.
DNA constructsStandard recombinant DNA techniques were used to
construct pCGN-HA-Prickle1, pCGN-HA-Prickle1(1-313),
pCGN-HA-Prickle1(308-832) andpCDNA/FLAG-Dvl2.
pCGN-HA-Prickle1-C829S was generated byintroducing a point mutation
at amino acid 829 to change thecysteine codon (TGT) into a serine
codon (TCT). pCGN-Myr-HA-Prickle1-C829S was generated by inserting
the Src myristoylationsequence
(5′-GGGAGCAGCAAGAGCAAGC-CCAAGGACCCCAGCC-AGCGCGCC-3′; amino acid
sequence, GSSKSKPKDPSQRA) betweenthe HA tag and the first ATG
sequence of pCGN-HA-Prickle1-C829S.
Protein expression using lentivirus and adenovirusTo generate
lentiviruses, lentiviral expression vectors were transfected
intoHEK293T (Lenti-X 293T) cells with the packaging vectors
pCAG-HIV-gpand pCMV-VSV-G-RSV-Rev using FuGENEHD (Roche Applied
Science,Basel, Switzerland). To generate MKN1 cells expressing
RFP–tubulin,parental cells (5×104 cells/well in a 12-well plate)
were transduced withconditioned medium containing lentiviral
particles and 10 µg/ml polybrene.The cells were then centrifuged at
1200 g for 1 h, replated and incubated foran additional 24 h.
To generate adenoviruses, the pAd/CMV/GFP-paxillin plasmid
waslinearized with PacI, phenol-chloroform extracted and then
transfected into293A cells (5×105 cells/well in a 6-well plate)
using Lipofectamine 2000(Life Technologies, Thermo Fisher
Scientific). At 36 h after transfection,cells were replated in a
10-cm dish and further incubated until ready forharvest (typically
7 to 10 days after transfection).
Cell fractionationMKN1 cells expressingHA–Prickle1 (wild type)
or itsmutantswere suspendedin 200 µl of homogenization buffer (20
mM Tris-HCl, pH 7.5, 150 mM NaCland 1 mM dithiothreitol) containing
2 µg/ml leupeptin, 2 µg/ml aprotinin and1 mM PMSF. These
suspensions were homogenized using the sonichomogenizer (Ultrasonic
homogenizer VP-5S, TAITEC, Saitama, Japan).After total homogenate
had been centrifuged at 100,000 g for 30 min, thesupernatantwas
reserved as the cytosol fraction. Theprecipitatewas extracted in200
µl of Laemmli sample buffer after washing with PBS. These samples
wereused as the membrane fraction. Aliquots (20 µl each) were
probed for HA,transferrin receptor (membrane marker) and β-tubulin
(cytosol marker).
Fig. 6. Prickle1 mediates EGF-dependent cell polarization and
migration.(A)MKN1 cells that had been transfectedwith control or
Prickle1 siRNA#1weretreatedwith or without 25 ng/ml EGF for 1 h.
Cellswere then subjected to a focaladhesion disassembly assay. (B)
MKN1 cells were cultured on a fibronectin-coated glass coverslip
with or without 25 ng/ml of EGF for 1 h. Cells werestained with
anti-caveolin and anti-cortactin antibodies, and phalloidin.
Whitearrows indicate polarized cells. The bottom panel indicates
quantification ofpolarized and non-polarized MKN1 cells (n≥120
cells). (C) MKN1 (MKN1/neo)cells or MKN1 cells expressing
HA–Prickle1 (MKN1/HA-Prickle1) weretransfected with control or
Prickle1 siRNA #1 and treated with or without 25 ng/ml EGF for 1 h.
Cells were stained with an anti-caveolin antibody and
phalloidin,and the percentage polarized cells was subsequently
calculated (n≥120 cells).(D) MKN1 or MKN1/HA-Prickle1 cells that
had been transfected with control orPrickle1 siRNA #1 were treated
with or without 25 ng/ml EGF and tracked withtime-lapse imaging for
10 h. Cell migration was tracked with ImageJ, andEuclidean distance
(µm) was calculated (n≥60 cells). (E) MKN1 cells that hadbeen
transfected with control or Prickle1 siRNA #1 were treated with or
without25 ng/ml EGF for 15 min. Lysates were probed for the
indicated proteins. JNKactivation was indicated by the
phosphorylation of JNK1 and JNK2 at Thr183and Tyr185 residues,
respectively. AKT activation was indicated by thephosphorylation of
AKT1, AKT2 and AKT3 at Ser473, Ser474 and Ser472residues,
respectively. p-, phosphorylation of the indicated protein.(F)
HA–Prickle1, HA–Prickle1-C829S or Myr–HA–Prickle1-C829S
wastransiently expressed in HEK293T cells. Lysates were probed for
the indicatedproteins. (G) MKN1 or MKN1/HA-Prickle1 cells were
cultured with the indicatedinhibitors for 1 h. Cells were stained
with an anti-caveolin antibody andphalloidin, and the percentage
polarized cells was calculated (n≥120 cells).(H) MKN1 cells were
pre-treated with or without 20 µM SP600125 and thentreated with or
without 25 ng/ml EGF for 1 h. Cells were stained with anti-caveolin
antibody and phalloidin, and the percentage polarized cells
wascalculated (n≥120 cells). (I) Control MKN1 cells and MKN1 cells
that stablyexpressed HA–Prickle1, HA–Prickle1-C829S or
Myr–HA–Prickle1-C829Swere transfected with control siRNA or
Prickle1 siRNA #1, then incubated withor without 25 ng/ml of EGF
for 1 h. Cells were stained with an anti-caveolinantibody and
phalloidin, and the percentage polarized cells was calculated(n≥120
cells). Results are expressed as mean±s.d. For box plots, lines in
themiddle of the boxes indicatemedian, whiskers
indicatemaximumandminimumvalues, and the ends of the boxes indicate
upper and lower quartiles. *P
-
mRNA and protein analysisQuantitative reverse-transcription PCR
(RT-PCR) was performed using aStepOne Real-Time PCR system (Applied
Biosystems, Life Technologies,Thermo Fisher Scientific). Forward
and reverse primers used are describedin Table S3. Western blot
data shown in the figures is representative of atleast three
independent experiments.
Statistical analysisAll experiments were performed at least
three times, and data are expressedas mean or mean±s.d. Statistical
analyses were performed using a pairedStudent’s t-test. For
experiments with more than two conditions, weemployed ANOVA test
with Bonferroni or Dunnett correction. P-values lessthan 0.05 were
considered statistically significant. Quantification of
proteinexpression by using western blotting was performed using
densitometryanalysis with ImageJ software (NIH). Protein signals
are expressed asrelative area and intensity.
AcknowledgementsThe authors would like to thank the Center of
Medical Research and Education,Graduate School of Medicine, Osaka
University for providing CSU-W1 microscopysystem. The authors would
also like to thank Drs Yuko Mimori-Kiyosue, WataruYasui, Shinji
Takada, Katsura Minegishi and Hiroshi Hamada for donating
cells,plasmids and Prickle-knockout mice.
Competing interestsThe authors declare no competing or financial
interests.
Author contributionsB.C.L. and S.M. designed experiments,
performed cell experiments and wrote themanuscript. H.Y. performed
biochemical analyses of Prickle1 subcellularlocalization. H.M.,
J.K. and M.I. performed in vivo imaging analysis. A.K.
designedexperiments and wrote the manuscript.
FundingThis work was supported by Grants-in-Aid for Scientific
Research (Japan Society forthe Promotion of Science) to A.K.
(2013–2015) [grant number 25250018] and S.M.(2013–2014) [grant
number 25860211]; by Scientific Research on InnovativeAreas
(Ministry of Education, Culture, Sports, Science, and Technology)
to A.K.(2012–2016) [grant number 23112004]; and by grants from the
Uehara MemorialFoundation (2014).
Supplementary informationSupplementary information available
online
athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.185439.supplemental
ReferencesAkhmanova, A. and Steinmetz, M. O. (2008). Tracking
the ends: a dynamic proteinnetwork controls the fate of microtubule
tips. Nat. Rev. Mol. Cell Biol. 9, 309-322.
Akhmanova, A., Hoogenraad, C. C., Drabek, K., Stepanova, T.,
Dortland, B.,Verkerk, T., Vermeulen, W., Burgering, B. M., De
Zeeuw, C. I., Grosveld, F.et al. (2001). CLASPs are CLIP-115 and
-170 associating proteins involved in theregional regulation of
microtubule dynamics in motile fibroblasts. Cell 104,923-935.
Bassuk, A. G., Wallace, R. H., Buhr, A., Buller, A. R., Afawi,
Z., Shimojo, M.,Miyata, S., Chen, S., Gonzalez-Alegre, P.,
Griesbach, H. L. et al. (2008). Ahomozygous mutation in human
PRICKLE1 causes an autosomal-recessiveprogressive myoclonus
epilepsy-ataxia syndrome. Am. J. Hum. Genet. 83,572-581.
Bekman, E. and Henrique, D. (2002). Embryonic expression of
three mouse geneswith homology to the Drosophila melanogaster
prickle gene. Mech. Dev. 119Suppl. 1, S77-S81.
Broussard, J. A., Webb, D. J. and Kaverina, I. (2008).
Asymmetric focal adhesiondisassembly in motile cells. Curr. Opin.
Cell Biol. 20, 85-90.
Butler, M. T. and Wallingford, J. B. (2015). Control of
vertebrate core planar cellpolarity protein localization and
dynamics by Prickle 2. Development 142,3429-3439.
Carvajal-Gonzalez, J. M., Balmer, S., Mendoza, M., Dussert, A.,
Collu, G.,Roman, A.-C., Weber, U., Ciruna, B. and Mlodzik, M.
(2015). The clathrinadaptor AP-1 complex and Arf1 regulate planar
cell polarity in vivo.Nat. Commun.6, 6751.
Chen, B., Dodge, M. E., Tang, W., Lu, J., Ma, Z., Fan, C.-W.,
Wei, S., Hao, W.,Kilgore, J., Williams, N. S. et al. (2009). Small
molecule–mediated disruption ofWnt-dependent signaling in tissue
regeneration and cancer. Nat. Chem. Biol. 5,100-107.
Cho, B., Pierre-Louis, G., Sagner, A., Eaton, S. and Axelrod, J.
D. (2015).Clustering and negative feedback by endocytosis in planar
cell polarity signaling ismodulated by ubiquitinylation of prickle.
PLoS Genet. 11, e1005259.
Clark, A. G. and Vignjevic, D. M. (2015). Modes of cancer cell
invasion and the roleof the microenvironment. Curr. Opin. Cell
Biol. 36, 13-22.
Daulat, A. M., Bertucci, F., Audebert, S., Sergé, A., Finetti,
P., Josselin, E.,Castellano, R., Birnbaum, D., Angers, S. and Borg,
J.-P. (2016). PRICKLE1contributes to cancer cell dissemination
through its interaction with mTORC2.Dev. Cell. 37, 311-325.
Dedhar, S., Williams, B. and Hannigan, G. (1999).
Integrin-linked kinase (ILK): aregulator of integrin and
growth-factor signalling. Trends Cell Biol. 9, 319-323.
Devenport, D. (2014). The cell biology of planar cell polarity.
J. Cell Biol. 207,171-179.
Dowler, S., Currie, R. A., Campbell, D. G., Deak, M., Kular, G.,
Downes, C. P. andAlessi, D. R. (2000). Identification of
pleckstrin-homology-domain-containingproteins with novel
phosphoinositide-binding specificities. Biochem. J. 351,19-31.
Ehaideb, S. N., Iyengar, A., Ueda, A., Iacobucci, G. J.,
Cranston, C., Bassuk,A. G., Gubb, D., Axelrod, J. D., Gunawardena,
S., Wu, C.-F. et al. (2014). pricklemodulates microtubule polarity
and axonal transport to ameliorate seizures inflies. Proc. Natl.
Acad. Sci. USA 111, 11187-11192.
Fujimura, L., Watanabe-Takano, H., Sato, Y., Tokuhisa, T. and
Hatano, M.(2009). Prickle promotes neurite outgrowth via the
Dishevelled dependentpathway in C1300 cells. Neurosci. Lett. 467,
6-10.
Gardel, M. L., Schneider, I. C., Aratyn-Schaus, Y. and Waterman,
C. M. (2010).Mechanical integration of actin and adhesion dynamics
in cell migration. Annu.Rev. Cell Dev. Biol. 26, 315-333.
Goodrich, L. V. and Strutt, D. (2011). Principles of planar
polarity in animaldevelopment. Development 138, 1877-1892.
Gray, R. S., Roszko, I. and Solnica-Krezel, L. (2011). Planar
cell polarity:coordinating morphogenetic cell behaviors with
embryonic polarity. Dev. Cell 21,120-133.
Gubb, D., Green, C., Huen, D., Coulson, D., Johnson, G., Tree,
D., Collier, S. andRoote, J. (1999). The balance between isoforms
of the prickle LIM domain proteinis critical for planar polarity in
Drosophila imaginal discs. Genes Dev. 13,2315-2327.
Heisenberg, C.-P., Tada, M., Rauch, G.-J., Saúde, L., Concha,
M. L., Geisler, R.,Stemple, D. L., Smith, J. C. and Wilson, S. W.
(2000). Silberblick/Wnt11mediates convergent extension movements
during zebrafish gastrulation. Nature405, 76-81.
Hida, Y., Fukaya, M., Hagiwara, A., Deguchi-Tawarada, M.,
Yoshioka, T.,Kitajima, I., Inoue, E., Watanabe, M. and Ohtsuka, T.
(2011). Prickle2 islocalized in the postsynaptic density and
interacts with PSD-95 and NMDAreceptors in the brain. J. Biochem.
149, 693-700.
Huang, C., Rajfur, Z., Borchers, C., Schaller, M. D. and
Jacobson, K. (2003). JNKphosphorylates paxillin and regulates cell
migration. Nature 424, 219-223.
Iioka, H., Iemura, S.-i., Natsume, T. and Kinoshita, N. (2007).
Wnt signallingregulates paxillin ubiquitination essential for
mesodermal cell motility. Nat. CellBiol. 9, 813-821.
Jenny, A., Reynolds-Kenneally, J., Das, G., Burnett, M. and
Mlodzik, M. (2005).Diego and Prickle regulate Frizzled planar cell
polarity signalling by competing forDishevelled binding. Nat. Cell
Biol. 7, 691-697.
Katoh, K., Kano, Y., Amano,M., Kaibuchi, K. and Fujiwara, K.
(2001). Stress fiberorganization regulated by MLCK and Rho-kinase
in cultured human fibroblasts.Am. J. Physiol. Cell Physiol. 280,
C1669-C1679.
Kaverina, I., Krylyshkina, O. and Small, J. V. (1999).
Microtubule targeting ofsubstrate contacts promotes their
relaxation and dissociation. J. Cell Biol. 146,1033-1044.
Kilian, B., Mansukoski, H., Barbosa, F. C., Ulrich, F., Tada, M.
and Heisenberg,C.-P. (2003). The role of Ppt/Wnt5 in regulating
cell shape and movement duringzebrafish gastrulation. Mech. Dev.
120, 467-476.
Kobayashi, T., Hino, S.-i., Oue, N., Asahara, T., Zollo, M.,
Yasui, W. and Kikuchi,A. (2006). Glycogen synthase kinase 3 and
h-prune regulate cell migration bymodulating focal adhesions. Mol.
Cell. Biol. 26, 898-911.
Kurayoshi, M., Oue, N., Yamamoto, H., Kishida, M., Inoue, A.,
Asahara, T.,Yasui, W. and Kikuchi, A. (2006). Expression of Wnt-5a
is correlated withaggressiveness of gastric cancer by stimulating
cell migration and invasion.Cancer Res. 66, 10439-10448.
Lansbergen, G., Grigoriev, I., Mimori-Kiyosue, Y., Ohtsuka, T.,
Higa, S.,Kitajima, I., Demmers, J., Galjart, N., Houtsmuller, A.
B., Grosveld, F. et al.(2006). CLASPs attach microtubule plus ends
to the cell cortex through a complexwith LL5β. Dev. Cell 11,
21-32.
Matsumoto, S., Fumoto, K., Okamoto, T., Kaibuchi, K. and
Kikuchi, A. (2010).Binding of APC and dishevelled mediates
Wnt5a-regulated focal adhesiondynamics in migrating cells. EMBO J.
29, 1192-1204.
Maurer-Stroh, S., Koranda, M., Benetka, W., Schneider, G.,
Sirota, F. L. andEisenhaber, F. (2007). Towards complete sets of
farnesylated andgeranylgeranylated proteins. PLoS Comput. Biol. 3,
e66.
3128
RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3115-3129
doi:10.1242/jcs.185439
Journal
ofCe
llScience
http://jcs.biologists.org/lookup/doi/10.1242/jcs.185439.supplementalhttp://jcs.biologists.org/lookup/doi/10.1242/jcs.185439.supplementalhttp://jcs.biologists.org/lookup/doi/10.1242/jcs.185439.supplementalhttp://dx.doi.org/10.1038/nrm2369http://dx.doi.org/10.1038/nrm2369http://dx.doi.org/10.1016/S0092-8674(01)00288-4http://dx.doi.org/10.1016/S0092-8674(01)00288-4http://dx.doi.org/10.1016/S0092-8674(01)00288-4http://dx.doi.org/10.1016/S0092-8674(01)00288-4http://dx.doi.org/10.1016/S0092-8674(01)00288-4http://dx.doi.org/10.1016/j.ajhg.2008.10.003http://dx.doi.org/10.1016/j.ajhg.2008.10.003http://dx.doi.org/10.1016/j.ajhg.2008.10.003http://dx.doi.org/10.1016/j.ajhg.2008.10.003http://dx.doi.org/10.1016/j.ajhg.2008.10.003http://dx.doi.org/10.1016/S0925-4773(03)00095-9http://dx.doi.org/10.1016/S0925-4773(03)00095-9http://dx.doi.org/10.1016/S0925-4773(03)00095-9http://dx.doi.org/10.1016/j.ceb.2007.10.009http://dx.doi.org/10.1016/j.ceb.2007.10.009http://dx.doi.org/10.1242/dev.121384http://dx.doi.org/10.1242/dev.121384http://dx.doi.org/10.1242/dev.121384http://dx.doi.org/10.1038/ncomms7751http://dx.doi.org/10.1038/ncomms7751http://dx.doi.org/10.1038/ncomms7751http://dx.doi.org/10.1038/ncomms7751http://dx.doi.org/10.1038/nchembio.137http://dx.doi.org/10.1038/nchembio.137http://dx.doi.org/10.1038/nchembio.137http://dx.doi.org/10.1038/nchembio.137http://dx.doi.org/10.1371/journal.pgen.1005259http://dx.doi.org/10.1371/journal.pgen.1005259http://dx.doi.org/10.1371/journal.pgen.1005259http://dx.doi.org/10.1016/j.ceb.2015.06.004http://dx.doi.org/10.1016/j.ceb.2015.06.004http://dx.doi.org/10.1016/j.devcel.2016.04.011http://dx.doi.org/10.1016/j.devcel.2016.04.011http://dx.doi.org/10.1016/j.devcel.2016.04.011http://dx.doi.org/10.1016/j.devcel.2016.04.011http://dx.doi.org/10.1016/S0962-8924(99)01612-8http://dx.doi.org/10.1016/S0962-8924(99)01612-8http://dx.doi.org/10.1083/jcb.201408039http://dx.doi.org/10.1083/jcb.201408039http://dx.doi.org/10.1042/bj3510019http://dx.doi.org/10.1042/bj3510019http://dx.doi.org/10.1042/bj3510019http://dx.doi.org/10.1042/bj3510019http://dx.doi.org/10.1073/pnas.1403357111http://dx.doi.org/10.1073/pnas.1403357111http://dx.doi.org/10.1073/pnas.1403357111http://dx.doi.org/10.1073/pnas.1403357111http://dx.doi.org/10.1016/j.neulet.2009.09.050http://dx.doi.org/10.1016/j.neulet.2009.09.050http://dx.doi.org/10.1016/j.neulet.2009.09.050http://dx.doi.org/10.1146/annurev.cellbio.011209.122036http://dx.doi.org/10.1146/annurev.cellbio.011209.122036http://dx.doi.org/10.1146/annurev.cellbio.011209.122036http://dx.doi.org/10.1242/dev.054080http://dx.doi.org/10.1242/dev.054080http://dx.doi.org/10.1016/j.devcel.2011.06.011http://dx.doi.org/10.1016/j.devcel.2011.06.011http://dx.doi.org/10.1016/j.devcel.2011.06.011http://dx.doi.org/10.1101/gad.13.17.2315http://dx.doi.org/10.1101/gad.13.17.2315http://dx.doi.org/10.1101/gad.13.17.2315http://dx.doi.org/10.1101/gad.13.17.2315http://dx.doi.org/10.1038/35011068http://dx.doi.org/10.1038/35011068http://dx.doi.org/10.1038/35011068http://dx.doi.org/10.1038/35011068http://dx.doi.org/10.1093/jb/mvr023http://dx.doi.org/10.1093/jb/mvr023http://dx.doi.org/10.1093/jb/mvr023http://dx.doi.org/10.1093/jb/mvr023http://dx.doi.org/10.1038/nature01745http://dx.doi.org/10.1038/nature01745http://dx.doi.org/10.1038/ncb1607http://dx.doi.org/10.1038/ncb1607http://dx.doi.org/10.1038/ncb1607http://dx.doi.org/10.1038/ncb1271http://dx.doi.org/10.1038/ncb1271http://dx.doi.org/10.1038/ncb1271http://dx.doi.org/10.1083/jcb.146.5.1033http://dx.doi.org/10.1083/jcb.146.5.1033http://dx.doi.org/10.1083/jcb.146.5.1033http://dx.doi.org/10.1016/S0925-4773(03)00004-2http://dx.doi.org/10.1016/S0925-4773(03)00004-2http://dx.doi.org/10.1016/S0925-4773(03)00004-2http://dx.doi.org/10.1128/MCB.26.3.898-911.2006http://dx.doi.org/10.1128/MCB.26.3.898-911.2006http://dx.doi.org/10.1128/MCB.26.3.898-911.2006http://dx.doi.org/10.1158/0008-5472.CAN-06-2359http://dx.doi.org/10.1158/0008-5472.CAN-06-2359http://dx.doi.org/10.1158/0008-5472.CAN-06-2359http://dx.doi.org/10.1158/0008-5472.CAN-06-2359http://dx.doi.org/10.1016/j.devcel.2006.05.012http://dx.doi.org/10.1016/j.devcel.2006.05.012http://dx.doi.org/10.1016/j.devcel.2006.05.012http://dx.doi.org/10.1016/j.devcel.2006.05.012http://dx.doi.org/10.1038/emboj.2010.26http://dx.doi.org/10.1038/emboj.2010.26http://dx.doi.org/10.1038/emboj.2010.26http://dx.doi.org/10.1371/journal.pcbi.0030066http://dx.doi.org/10.1371/journal.pcbi.0030066http://dx.doi.org/10.1371/journal.pcbi.0030066
-
Narimatsu, M., Bose, R., Pye, M., Zhang, L., Miller, B., Ching,
P., Sakuma, R.,Luga, V., Roncari, L., Attisano, L. et al. (2009).
Regulation of planar cell polarityby Smurf ubiquitin ligases. Cell
137, 295-307.
Navarro, A., Anand-Apte, B. and Parat, M.-O. (2004). A role for
caveolae in cellmigration. FASEB J. 18, 1801-1811.
Okuda, H., Miyata, S., Mori, Y. and Tohyama, M. (2007). Mouse
Prickle1 andPrickle2 are expressed in postmitotic neurons and
promote neurite outgrowth.FEBS Lett. 581, 4754-4760.
Olofsson, J., Sharp, K. A., Matis, M., Cho, B. and Axelrod, J.
D. (2014). Prickle/spiny-legs isoforms control the polarity of the
apical microtubule network in planarcell polarity. Development 141,
2866-2874.
Pankov, R., Endo, Y., Even-Ram, S., Araki, M., Clark, K.,
Cukierman, E.,Matsumoto, K. and Yamada, K. M. (2005). A Rac switch
regulates randomversus directionally persistent cell migration. J.
Cell Biol. 170, 793-802.
Parsons, J. T. and Parsons, S. J. (1997). Src family protein
tyrosine kinases:cooperating with growth factor and adhesion
signaling pathways. Curr. Opin. CellBiol. 9, 187-192.
Parsons, J. T., Horwitz, A. R. and Schwartz, M. A. (2010). Cell
adhesion:integrating cytoskeletal dynamics and cellular
tension.Nat. Rev. Mol. Cell Biol. 11,633-643.
Peng, Y. and Axelrod, J. D. (2012). Asymmetric protein
localization in planar cellpolarity: mechanisms, puzzles, and
challenges. Curr. Top. Dev. Biol. 101, 33-53.
Rottner, K., Hall, A. and Small, J. V. (1999). Interplay between
Rac and Rho in thecontrol of substrate contact dynamics. Curr.
Biol. 9, 640-648.
Schuyler, S. C. and Pellman, D. (2001). Microtubule
“plus-end-tracking proteins”:the end is just the beginning. Cell
105, 421-424.
Simons, M. and Mlodzik, M. (2008). Planar cell polarity
signaling: from flydevelopment to human disease. Annu. Rev. Genet.
42, 517-540.
Singh, J. and Mlodzik, M. (2012). Planar cell polarity
signaling: coordinationof cellular orientation across tissues.
Wiley Interdiscip. Rev. Dev. Biol. 1,479-499.
Stehbens, S. and Wittmann, T. (2012). Targeting and transport:
how microtubulescontrol focal adhesion dynamics. J. Cell Biol. 198,
481-489.
Stehbens, S. J., Paszek, M., Pemble, H., Ettinger, A., Gierke,
S. and Wittmann,T. (2014). CLASPs link focal-adhesion-associated
microtubule capture tolocalized exocytosis and adhesion site
turnover. Nat. Cell Biol. 16, 561-573.
Streuli, C. H. and Akhtar, N. (2009). Signal co-operation
between integrins andother receptor systems. Biochem. J. 418,
491-506.
Strutt, D. I. (2002). The asymmetric subcellular localisation of
components of theplanar polarity pathway. Semin. Cell Dev. Biol.
13, 225-231.
Strutt, H., Thomas-MacArthur, V. and Strutt, D. (2013).
Strabismus promotesrecruitment and degradation of farnesylated
prickle in Drosophila melanogasterplanar polarity specification.
PLoS Genet. 9, e1003654.
Sweede, M., Ankem, G., Chutvirasakul, B., Azurmendi, H. F.,
Chbeir, S.,Watkins, J., Helm, R. F., Finkielstein, C. V. and
Capelluto, D. G. (2008).Structural and membrane binding properties
of the prickle PET domain.Biochemistry 47, 13524-13536.
Tao, H., Suzuki, M., Kiyonari, H., Abe, T., Sasaoka, T. and
Ueno, N. (2009).Mouse prickle1, the homolog of a PCP gene, is
essential for epiblast apical-basalpolarity. Proc. Natl. Acad. Sci.
USA 106, 14426-14431.
Tao, H., Manak, J. R., Sowers, L., Mei, X., Kiyonari, H., Abe,
T., Dahdaleh, N. S.,Yang, T., Wu, S., Chen, S. et al. (2011).
Mutations in prickle orthologs causeseizures in flies, mice, and
humans. Am. J. Hum. Genet. 88, 138-149.
Tissir, F. and Goffinet, A. M. (2006). Expression of planar cell
polarity genes duringdevelopment of the mouse CNS. Eur. J.
Neurosci. 23, 597-607.
Todaro, G. J. and Green, H. (1963). Quantitative studies of the
growth of mouseembryo cells in culture and their development into
established lines. J. Cell Biol.17, 299-313.
Tree, D. R. P., Shulman, J. M., Rousset, R., Scott, M. P., Gubb,
D. and Axelrod,J. D. (2002). Prickle mediates feedback
amplification to generate asymmetricplanar cell polarity signaling.
Cell 109, 371-381.
Veeman,M. T., Axelrod, J. D. andMoon, R. T. (2003a). A second
canon. Functionsand mechanisms of β-catenin-independent Wnt
signaling. Dev. Cell 5, 367-377.
Veeman,M. T., Slusarski, D. C., Kaykas, A., Louie, S. H.
andMoon, R. T. (2003b).Zebrafish prickle, a modulator of
noncanonical Wnt/Fz signaling, regulatesgastrulation movements.
Curr. Biol. 13, 680-685.
Webb, D. J., Parsons, J. T. and Horwitz, A. F. (2002). Adhesion
assembly,disassembly and turnover in migrating cells – over and
over and over again. Nat.Cell Biol. 4, E97-E100.
Weber, U., Pataki, C., Mihaly, J. and Mlodzik, M. (2008).
Combinatorial signalingby the Frizzled/PCP and Egfr pathways during
planar cell polarity establishment inthe Drosophila eye. Dev. Biol.
316, 110-123.
Xie, H., Pallero, M. A., Gupta, K., Chang, P., Ware, M. F.,
Witke, W., Kwiatkowski,D. J., Lauffenburger, D. A., Murphy-Ullrich,
J. E. and Wells, A. (1998). EGFreceptor regulation of cell
motility: EGF induces disassembly of focal adhesionsindependently
of the motility-associated PLCγ signaling pathway. J. Cell Sci.
111,615-624.
Yamamoto, H., Kitadai, Y., Yamamoto, H., Oue, N., Ohdan, H.,
Yasui, W. andKikuchi, A. (2009). Laminin γ2 mediates Wnt5a-induced
invasion of gastriccancer cells. Gastroenterology 137,
242-252.e6.
Yamana, N., Arakawa, Y., Nishino, T., Kurokawa, K., Tanji, M.,
Itoh, R. E.,Monypenny, J., Ishizaki, T., Bito, H., Nozaki, K. et
al. (2006). The Rho-mDia1pathway regulates cell polarity and focal
adhesion turnover in migrating cellsthrough mobilizing Apc and
c-Src. Mol. Cell. Biol. 26, 6844-6858.
Yang, Y. andMlodzik, M. (2015). Wnt-Frizzled/planar cell
polarity signaling: cellularorientation by facing the wind (Wnt).
Annu. Rev. Cell Dev. Biol. 31, 623-646.
Yang, T., Bassuk, A. G. and Fritzsch, B. (2013). Prickle1 stunts
limb growththrough alteration of cell polarity and gene
expression.Dev. Dyn. 242, 1293-1306.
Yokozaki, H. (2000). Molecular characteristics of eight gastric
cancer cell linesestablished in Japan. Pathol. Int. 50,
767-777.
Zallen, J. A. (2007). Planar polarity and tissue morphogenesis.
Cell 129,1051-1063.
Zhou, W., Parent, L. J., Wills, J. W. and Resh, M. D. (1994).
Identification ofa membrane-binding domain within the
amino-terminal region of humanimmunodeficiency virus type 1 Gag
protein which interacts with acidicphospholipids. J. Virol. 68,
2556-2569.
3129
RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3115-3129
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llScience
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