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 (akikuchi@molbiobc.med.osaka-u.ac.jp)

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

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

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

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RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3115-3129 doi:10.1242/jcs.185439

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