The Bro1-domain-containing protein Myopic/HDPTP coordinates … · 2012-12-07 · Journal of Cell Science The Bro1-domain-containing protein Myopic/HDPTP coordinates with Rab4 to

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The Bro1-domain-containing protein Myopic/HDPTPcoordinates with Rab4 to regulate cell adhesionand migration

Dong-Yuan Chen1,*, Meng-Yen Li2,*, Shih-Yun Wu1, Yu-Ling Lin1, Sung-Po Tsai2, Pei-Lun Lai1, Yu-Tsen Lin1,Jean-Cheng Kuo3, Tzu-Ching Meng1,2,` and Guang-Chao Chen1,2,`

1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan2Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan3Institute of Biochemistry and Molecular Biology, National Yang Ming University, Taipei, Taiwan

*These authors contributed equally to this work`Authors for correspondence ([email protected]; [email protected])

Accepted 13 June 2012Journal of Cell Science 125, 4841–4852� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.108597

SummaryProtein tyrosine phosphatases (PTPs) are a group of tightly regulated enzymes that coordinate with protein tyrosine kinases to control protein

phosphorylation during various cellular processes. Using genetic analysis in Drosophila non-transmembrane PTPs, we identified one role thatMyopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays in cell adhesion. Depletionof Mop results in aberrant integrin distribution and border cell dissociation during Drosophila oogenesis. Interestingly, Mop phosphatase

activity is not required for its role in maintaining border cell cluster integrity. We further identified Rab4 GTPase as a Mop interactor in a yeasttwo-hybrid screen. Expression of the Rab4 dominant-negative mutant leads to border cell dissociation and suppression of Mop-induced wing-blade adhesion defects, suggesting a critical role of Rab4 in Mop-mediated signaling. In mammals, it has been shown that Rab4-dependentrecycling of integrins is necessary for cell adhesion and migration. We found that human HDPTP regulates the spatial distribution of Rab4 and

integrin trafficking. Depletion of HDPTP resulted in actin reorganization and increased cell motility. Together, our findings suggest anevolutionarily conserved function of HDPTP–Rab4 in the regulation of endocytic trafficking, cell adhesion and migration.

Key words: HDPTP, Myopic, Integrin, Rab4, Tyrosine phosphatase

IntroductionCell adhesion and cell migration are essential for the development

and coordinated function of multicellular organisms. Aberrant

regulation of these processes often results in the progression

of many diseases, including cancer invasion and metastasis.

Accumulating evidence has indicated that dynamic and reversible

protein tyrosine phosphorylation is essential for the regulation of

cell migration and cell adhesion (Zamir and Geiger, 2001; Burridge

et al., 2006; Huveneers and Danen, 2009). While many studies have

been devoted to the role of protein tyrosine kinases in these

processes, the function of protein tyrosine phosphatases (PTPs) in

cell adhesion and migration remains unclear.

The dynamic change of integrin-mediated focal adhesions plays

a critical role in cell adhesion and migration. Many focal adhesion

regulators such as focal adhesion kinase (FAK), Src, p130Cas and

paxillin are tyrosine phosphorylated (Panetti, 2002). The tyrosine

phosphorylation of these proteins affects focal adhesion dynamics.

Phosphorylation of tyrosine 397 in FAK promotes its association

with Src, and the activated FAK–Src complex subsequently

regulates focal adhesion dynamics by signaling downstream

targets (Mitra and Schlaepfer, 2006). Several PTPs have been

implicated in integrin signaling, cell adhesion and motility. One

study has shown that SHP-2 phosphatase influences FAK activity

(Yu et al., 1998). SHP-2 also promotes Src kinase activation by

inhibiting Csk (Zhang et al., 2004). Depletion of PTP-PEST has

been found to lead to the hyperphosphorylation of p130Cas, FAK

and paxillin, and a marked increase in focal adhesions (Angers-

Loustau et al., 1999). Moreover, PTP1B and PTPa, have also been

found to regulate Src phosphorylation and integrin-mediated

adhesion (Harder et al., 1998; Liang et al., 2005).

In Drosophila, a total of sixteen putative classical PTPs have been

identified (Andersen et al., 2005). Compared to mammalian PTPs,

Drosophila PTP family members are relatively simple, most

containing only one gene corresponding to each subtype (except

for DPTP10D and DPTP4E, which share similar domain structures).

Therefore, Drosophila can serve as an excellent model system for

the study of the physiological and developmental function of PTPs.

While much research has been devoted to the function of receptor

PTPs, the role of non-transmembrane PTPs (NT-PTPs) in

Drosophila development remains unknown. One of the most well

studied Drosophila NT-PTPs is Corkscrew (Csw). Csw is the

ortholog of human SHP-2 which has two SH2 domains at the N-

terminus and a PTP domain at the C-terminus. Csw functions as a

downstream effecter of Sevenless PTK and is essential for the

development of the R7 photoreceptor (Allard et al., 1996).

Phenotypic analysis showed that Csw can also act downstream of

many receptor tyrosine kinases, such as the Drosophila epidermal

growth factor receptor (DER) and the fibroblast growth factor

(Breathless) (Perkins et al., 1996; Feng, 1999). Protein tyrosine

phosphatase-ERK/Enhancer of Ras1 (PTP-ER) has been shown to

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function as a negative regulator downstream of Ras1 and tobe involved in RAS1/MAPK-mediated R7 photoreceptordifferentiation (Karim and Rubin, 1999). PTP61F, the Drosophila

ortholog of human PTP1B and TCPTP, has been reported to interact

with Dock, an adapter protein required for axon guidance (Clemenset al., 1996). PTP61F has recently been shown to coordinate withdAbl in regulating actin cytoskeleton organization via reversible

tyrosine phosphorylation of Abi and Kette (Huang et al., 2007; Kuet al., 2009). Moreover, dPtpmeg, a FERM and PDZ domain-containing NT-PTP, is reported to be involved in the formation of

neuronal circuits in the Drosophila brain (Whited et al., 2007),though its molecular function in this process is not known.

To explore the functional role of Drosophila NT-PTPs in celladhesion and migration, we performed genetic analyses to identifyNT-PTPs that could modulate border cell migration duringoogenesis. We found that Myopic (Mop), the Drosophila

homolog of the human His domain phosphotyrosine phosphatase(HDPTP), plays an important role in maintaining border cellcluster integrity. Depletion of Mop altered the normal distribution

of integrin receptor. While Mop has recently been reported toregulate EGFR and Toll receptor signaling (Miura et al., 2008;Huang et al., 2010), its molecular mechanism has remained

elusive. This study found that Mop interacts with Rab4 GTPase incontrolling integrin distribution and cell adhesion. We furtherdemonstrated that human HDPTP is essential for the intracellular

positioning of Rab4, integrin trafficking and cell migration. Thesefindings provide some insight into the mechanisms underlyingHDPTP in the regulation of cell adhesion and migration.

ResultsMolecular characterization of Drosophila NT-PTPsWe analyzed the eight Drosophila non-transmembrane PTPs (NT-

PTPs), including Ptp61F, dMeg2, CG7180, Myopic (Mop),dPtpmeg, dPez, PTP-ER and Csw, in Drosophila genome bymultiple sequence alignment of their PTP domain. Based on their

homology to the PTP domain of human PTP1B, 10 conservedmotifs of the PTP domain were aligned (Fig. 1A). Among them,two catalytic essential residues, aspartic acid within the WPD loop(motif 8) and the active site cysteine within the signature motif

HC(X)5R (motif 9), were identified. Interestingly, the criticalaspartic acid residue (D) in the WPD loop of Mop, dPez andCG7180 were replaced with lysine (K), glutamic acid (E) and serine

(S), respectively. Although the active site cysteine could be foundin all NT-PTPs, the overall signature motif of Mop showed muchgreater sequence divergence than all the other NT-PTPs.

We next measured the tyrosine phosphatase catalytic activitiesof Ptp61F, dMeg2, dPtpmeg, dPez and Mop using in vitro

phosphatase assays. The PTP domain of each NT-PTP wasexpressed in cultured Drosophila S2 cells, immunoprecipitated,and subjected to pNPP assay (Fig. 1B). PTP domain of dMeg2,dPtpmeg and Ptp61F showed detectable phosphatase activity by

pNPP assay (Fig. 1B). It appears that dPtpmeg has much higherPTP activity than that of dMeg2 and PTP61F (Fig. 1C).However, the PTP activity of dPez and Mop were barely

detectable by in vitro phosphatase assay (Fig. 1B). Our sequenceand biochemical analysis suggested that dPez and Mop may nothave tyrosine phosphatase enzyme activity.

Myopic is required to maintain border cell cluster integrity

To study the function of protein tyrosine phosphatases incollective cell migration, we used transgenic RNAi approach to

systematically knockdown each of the Drosophila NT-PTPs,including Ptp61F, dMeg2, CG7180, Myopic, dPtpmeg, dPez,

PTP-ER, and Csw, in migrating border cells during oogenesis.Border cells consist of a group of six to ten specialized migratory

cells that are derived from the follicular epithelium in thedeveloping egg chamber (Rørth, 2002; Montell, 2003). Thesecells adhere tightly to each other and migrate as a cluster toward

oocyte. Using the GAL4-UAS targeted expression system, double-stranded RNA (dsRNA) targeting each NT-PTP was expressed

under the control of the border cell-specific slbo–Gal4 driver. Wefound that RNAi-mediated downregulation of Drosophila NT-PTPs

did not have an obvious effect on border cell migration, except in thecase of dMeg2-RNAi, which resulted in a 23.3% (n5219) migrationdelay (Fig. 2A). We next examined whether the NT-PTPs were

involved in controlling border cell cluster integrity during collectivemigration. In control egg chambers (slbo–Gal4), border cells

(marked by GFP) are tightly associated to each other duringmigration (Fig. 2B,C). Strikingly, Mop-RNAi knockdown resultedin a marked increase in cluster dissociation (26.5%, n5276;

Fig. 2B). Western blot analysis showed that Mop-RNAi couldeffectively reduce the expression levels of Mop (supplementary

material Fig. S1A). Depletion of Mop caused the dissociation of oneor two cells from border cell cluster and retained in nurse cells by

stage 10; however, we did not observe obvious changes in the totalnumber of border cells, compared with the control (Fig. 2C;supplementary material Fig. S1B). These results together suggest

that Mop plays a role in cell adhesion, rather than cell invasiveness,during oogenesis. Moreover, the Mop-RNAi cluster dissociation

defect was rescued by co-expression of full-length Mop, Mop-DPTPand Mop-C/S mutant (supplementary material Fig. S2), suggesting

that the cell adhesion defect is PTP activity independent.Interestingly, overexpression of Mop with slbo–Gal4 disrupted thecluster integrity (30.7%, n5189; supplementary material Fig. S2),

indicating that a proper level of Mop expression is essential formaintaining border cell cluster integrity.

E-cadherin and integrin are adhesion-related molecules thathave been reported to play a role in maintaining border cell clusterintegrity (Llense and Martın-Blanco, 2008). Immunostaining

analysis of slbo–Gal4 controls revealed that both DE-cadherinand bPS-integrin are enriched at cell-cell contacts of border cells

(Fig. 3A,B). Depletion of Mop did not markedly affect the leveland distribution of DE-cadherin at cell-cell contacts (Fig. 3B).However, depletion of Mop resulted in significant changes in

integrin distribution (Fig. 3A). Most of the bPS-integrins appear toaggregate at the leading edges or cell periphery of migrating border

cells rather than in cell-cell contacts. Clonal analysis of mopmutants further confirmed our observation. In mop homozygous

mutant border cell clones (marked by the loss of GFP), bPS-integrins formed punctate aggregates in the peripheral region ofmop mutant cells (Fig. 3C). We also found a slight reduction and

diffuse localization of the endogenous DE-cadherin in mop mutantcells (Fig. 3C). Taken together, these findings show that Mop is

involved in the control of cell adhesion during border cell clustermigration, possibly through the control of integrin localization. We

next investigated the effect of Mop depletion in epithelial folliclecells surrounding the oocyte. DE-cadherin and bPS-integrin wereenriched at cell-cell contacts in control cells (GFP positive;

supplementary material Fig. S3A,C). In mop homozygous mutantfollicle cell clones (GFP negative), bPS-integrin and DE-cadherin

were diffusely distributed in the cell peripheral region(supplementary material Fig. S3A,C). A slight increase of

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DE-cadherin at cell-cell contacts was also observed in some mop

mutant clones (supplementary material Fig. S3C,D). These results

indicate that Mop is involved in regulating the distribution of

integrin and DE-cadherin in non-migrating follicle cells.

We next test whether misexpressing integrin in migrating border

cells may affect border cell cluster integrity. Strikingly, we found

that expression of myospheroid (mys, encoding bPS-integrin) with

slbo–Gal4 caused a marked increase in border cell dissociation, as

compared with controls (Fig. 3D,E). Immunostaining analysis

revealed that, unlike the control in which the bPS-integrin is

mainly localized at cell-cell junctions, misexpression of mys

resulted in an accumulation of bPS around the cell edges

(supplementary material Fig. S4). Moreover, genetic analysis

further showed that depletion of bPS-integrin (mys) could rescue

the Mop-RNAi-induced border cell dissociation phenotype

(Fig. 3E). These results together suggest that bPS-integrin

mislocalization may contribute to the cluster dissociation caused

by Mop depletion.

Integrin-related cell adhesion and migration is essential during

many developmental processes. During wing development, wing

imaginal discs fold into two layers of wing epithelia, dorsal and

ventral wing epithelia, which will later adhere to each other via an

integrin-mediated process (Bokel and Brown, 2002; Brower,

2003). Significantly, we found that ectopic expression of Mop

under the control of engrailed–Gal4 (en–Gal4) induced a blistered-

wing phenotype with high penetrance (90%, n5165; compare

supplementary material Fig. S5A,B). We further demonstrated that

the Mop-induced wing cell adhesion defect could be rescued by

specifically knocking down Mop at the same region (100%,

n5106; supplementary material Fig. S5C). These results indicate

that Mop plays a role in integrin-mediated cell adhesion.

Mop interacts with Rab4 GTPase

Recent studies have indicated that Mop plays a role in regulating

cell surface receptor signaling. Miura et al. found that Mop acts

as an endosomal protein in promoting EGF receptor signaling

Fig. 1. Sequence analysis and in vitro

phosphatase activity of Drosophila non-

transmembrane protein tyrosine phosphatases

(NT-PTPs). (A) Multiple sequence alignments of

NT-PTPs in the Drosophila genome. Amino acid

sequences of the conserved PTP domains of human

PTP1B and eight Drosophila NT-PTPs.

Identification of conserved motifs of PTP are based

on the alignments of human PTP domains

(Andersen et al., 2001). Amino acid residues of each

PTP are numbered at the left end and the right end

of each lane. Sequence alignment was generated by

Jalview (Waterhouse et al., 2009). Conserved motifs

are indicated by red bars, and sequences with

different shades of blue indicate the percentage

sequence identity of the consensus amino acids.

(B) In vitro phosphatase activity of Drosophila NT-

PTPs. HA-tagged PTP domain of Drosophila

dMeg2, dPtpmeg, dPez, Mop and Ptp61F was

immunoprecipitated and incubated in an assay

buffer containing p-nitrophenyl phosphate (pNPP).

The activity of PTPs was determined by measuring

the absorption spectra of hydrolyzed pNPP at

405 nm. Data are means 6 s.d. from triplicate

experiments. The level of immunoprecipitated HA-

containing PTPs were assayed by western blotting

with anti-HA antibody (bottom panel: a

representative immunoblot). (C) Relative tyrosine

phosphatase activity of NT-PTPs was quantified by

normalizing specific activity of each NT-PTP

(OD 405/relative level of immunoprecipitated PTP)

to that of dMeg2. Data are means 6 s.d. of

triplicate experiments.

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during photoreceptor differentiation (Miura et al., 2008).

Moreover, Huang et al. has reported that Mop is required for

Toll receptor innate immune signaling (Huang et al., 2010). They

found the Bro1 domain of Mop to be required for Mop to

function in Toll pathway activation, though the molecularmechanism remained unclear. We performed a yeast two-

hybrid screen of a Drosophila embryo cDNA library using theBro1 domain of Mop as bait (Fig. 4A). From a screen of about3.66107 transformants, we identified several positive clones.Sequence analysis revealed that one of these clones encoded the

Drosophila Rab4 GTPase (Fig. 4B). Rab4 has been implicated inthe regulation of membrane receptor recycling from earlyendosomes in mammalian cells (Roberts et al., 2001; Grant and

Donaldson, 2009). We then used GST pull down and co-immunoprecipitation assays to further examine the interactionbetween Mop and Rab4. GST–Mop–Bro1, but not GST,

efficiently interacted with Rab4 (Fig. 4C). To determinewhether full-length Mop interacts with Rab4, we transfectedHA-tagged full-length Mop (HA–Mop), Bro1 domain of Mop(HA–Mop–Bro1), and Myc-tagged Rab4 GTPase (Myc–Rab4)

into HEK293 cells, and Mop was immunoprecipitated with ananti-HA antibody. We found Rab4 to be co-immunoprecipitatedwith both full-length Mop and the Bro1 domain of Mop

(Fig. 4D). Since Rab4 has been found to localize at earlyendosome and recycling endosome (van der Sluijs et al., 1992;Sonnichsen et al., 2000), we investigated whether Mop was also

localized at these compartments. Immunofluorescence analysisshowed Mop to be distributed at enlarged vesicle-likecompartments and significantly colocalized with GFP–Rab4 in

Drosophila S2R+ cells (Fig. 4E). We further tested theinteraction between Rab4 and Mop by genetic analysis. Wingblistering defects induced by the overexpression of Mop could berescued by knocking down Rab4 or co-expressing GDP-bound

Rab4 (Rab4-SN; supplementary material Fig. S5D,E). RabGTPases cycle between the GTP-bound active form and theGDP-bound inactive form (Stenmark, 2009). To find out whether

Rab4 interacted with Mop in a guanine nucleotide-dependentmanner, we examined the interaction of Mop with bacteriallyexpressed GST–Rab4 in the presence or absence of nucleotide.

As shown in supplementary material Fig. S6, Mop associatedwith nucleotide-free, GDP-bound and GTPcS-bound forms ofRab4. These data together indicate that Rab4 interacts with Mopin a guanine nucleotide-independent manner.

Rab4 but not Rab5, Rab7 or Rab11 is involved inmaintaining border cell cluster integrity

The Rab family of small GTPases acts as molecular switches thatspatially and temporally regulate vesicle transport in the cell(Stenmark, 2009; Hutagalung and Novick, 2011). The binding of

Mop to Rab4 in our study raised the question of whether Mop alsointeracts with other Rab proteins. Because Mop has been shown tocolocalize with Rab5 on early endosomes (Miura et al., 2008), weinvestigated whether Mop and Rab5 also interacted with each other.

Co-immunoprecipitation assays revealed that Mop also interactedwith Rab5 but not with Rab7 and Rab11 (supplementary materialFig. S7). Because Rab GTPase-mediated endocytic trafficking has

been reported to play an important role in regulating border cellmigration (Assaker et al., 2010), we wanted to investigate whetherRab4, Rab5, Rab7 and Rab11 were involved in maintaining border

cell cluster integrity. Expression of the dominant-negative Rab5-S43N, Rab7-T22N or Rab11-S25N mutant in the migrating bordercells using slbo–Gal4 impaired border cell migration; however, none

of them affected the border cell cluster integrity (Fig. 5A,B).Strikingly, expression of the dominant negative GDP-bound Rab4-S22N mutant, but not Rab4-WT or the GTPase-deficient Rab4-Q67L

Fig. 2. Mop is required for maintaining border cell cluster integrity.

(A) Quantification of border cell migration in the indicated genotypes. RNAi-

mediated knockdown of Drosophila Mop, dMeg2, dPez, PTP-ER, Ptp61F,

dPtpmeg, CG7180 and CSW in border cells under the control of slbo–Gal4

(slbo-Gal4.UAS-mCD8-GFP). To define defects in border cell migration,

each stage 10 egg chamber was divided into three regions: 100% motility,

.50% motility and ,50% motility. Border cells were scored and the

percentages presented in histogram form. BC, border cell. (B) Quantification

of border cell dissociation in the indicated genotypes. Dissociation was

defined as the detachment of at least one border cell from the border cell

cluster and the detached cell(s) remained within the nurse cells of late stage 9

or stage 10 egg chambers. n.100. (C) Compared with control cells (slbo-

Gal4.mCD8-GFP), Mop knockdown in border cells (slbo-Gal4.UAS-

mCD8-GFP/UAS-MopRNAi) leads to greater dissociation. Border cells

dissociated from the cluster in the late stage 9 egg chamber are indicated by

arrowheads. Cell nuclei were stained with DAPI (blue) and F-actin was

stained with phalloidin (red). Scale bar: 20 mm.

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mutant, resulted in enhanced dissociation of border cells (Fig. 5B).

Moreover, unlike other Rab GTPases we tested, Rab4-S22N mutant

did not markedly affect border cell migration (more than 80% of

border cells migrated normally; Fig. 5A). We further investigated the

distribution of bPS-integrin in the control and Rab4-S22N expressing

border cells. Similar to depletion of Mop, the expression of dominant

negative Rab4 in border cells caused a redistribution of bPS-integrin

from cell-cell junction to cell peripheral in migrating border cells

(Fig. 5C). Taken together, our results further confirm the role that

Rab4 and Mop play in regulating border cell association and integrin

distribution during oogenesis.

Human HDPTP regulates early endosomal distribution

To find out whether Mop–Rab4-mediated integrin trafficking is

conserved in mammalian cells, we further investigated whether

the Mop human ortholog HDPTP would interact with Rab4A

GTPase. Co-immunoprecipitation assay on co-transfected

cells demonstrated that FLAG-tagged HDPTP interacted with

mammalian Rab4A (Fig. 6A), and immunofluorescence analysis

revealed that HDPTP and Rab4 colocalized with each other

(Fig. 6B), indicating that the interaction between HDPTP and

Rab4 GTPase is evolutionarily conserved. It has been shown that

HDPTP plays an essential role in the regulation of cargo sorting

and endosome morphogenesis (Doyotte et al., 2008). We

therefore investigated the effect of HDPTP depletion on Rab4

localization in HeLa cells (Fig. 6C,D). In control cells, GFP–

Rab4A and the early endosome antigen 1 (EEA1) were dispersed

throughout the cytoplasm (Fig. 6D). However, in the stable

HDPTP knockdown cells, GFP–Rab4A and EEA1-positive

endosomes were accumulated in the perinuclear region

(Fig. 6D). The shHDPTP-induced change in Rab4A distribution

could be reversed by the expression of wild-type HDPTP (data

not shown). We also noted that the localization of the late

endosomal marker Rab7 and the lysosomal marker LAMP-1

Fig. 3. Mop regulates the spatial distribution of adhesion

proteins. (A) bPS-integrin antibody staining of migrating border

cells in the control and slbo-Gal4.UAS-mCD8-GFP/UAS-

MopRNAi egg chambers. The bPS-integrins are enriched in cell-

cell contacts in control cells (top), whereas bPS-integrins appear

to aggregate at the cell periphery of Mop knockdown border cells

(bottom, arrowheads). (B) DE-cadherin antibody staining of

migrating border cells in the control and slbo-Gal4.UAS-mCD8-

GFP/UAS-MopRNAi egg chambers. DE-cadherin remains in cell-

cell contacts in Mop knockdown cells. (C) Redistribution and

aggregation of bPS-integrin in mopT612 homozygous mutant

border cell clones (marked by the absence of GFP). Nuclei were

stained with DAPI (blue). The cell margin is marked with white

dashed lines. (D) Compared with control cells (slbo-

Gal4.mCD8-GFP), misexpression of myospheroid (mys,

encoding bPS-integrin; slbo-Gal4.UAS-mCD8-GFP/UAS-mys)

leads to greater dissociation. Border cells dissociated from the

cluster in the late stage 9 egg chamber are indicated by an

arrowhead. Cell nuclei were stained with DAPI (blue) and F-

actin was stained with phalloidin (red). (E) Quantification of

border cell migration and dissociation in slbo-Gal4.UAS-

mCD8-GFP flies expressing MopRNAi, bPS-integrin (mys) or

indicated genotypes. Border cell migration and dissociation were

defined as in Fig. 2. n.100. Scale bars: 10 mm (A–C) and

20 mm (D).

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appeared to be normal when HDPTP was depleted (data not

shown). These results together indicate that HDPTP may play a

role in regulating the proper distribution of Rab4A in endosomal

compartments.

HDPTP depletion affects integrin recycling

Because mop mutation affects Drosophila bPS-integrin

distribution during oogenesis, we checked whether knocking

down HDPTP would have any influence on integrin receptor

distribution. As shown in Fig. 7A, b1 integrin was dispersed over

the cell surface and in the peripheral region of control cells.

Significantly, knocking down HDPTP resulted in an increasedaccumulation of b1 integrins to the perinuclear region of HeLa

cells (Fig. 7A). We then investigated the role of HDPTP ininternalization and recycling of b1 integrin in HeLa cells.Quantitative internalization assay by flow cytometry revealedthat the amount of b1 integrin endocytosed in HDPTP knockdown

cells was slightly reduced compared to control cells (Fig. 7B). Wefurther analyzed the effect of HDPTP knockdown on the recyclingof integrins from endosomes to the plasma membrane. To monitor

recycling of integrins from Rab4-mediated early endosomes, cellsurface integrins were biotin-labeled and allowed to beendocytosed at 22 C for 15 min. After we removed the integrins

remaining on the cell surface, we chased cells at 37 C for varioustime periods to permit recycling of internalized integrins. In shLuccells, ,60% of the internalized a5b1 recycled to the membraneafter 5 min (Fig. 7C). Depletion of HDPTP significantly reduced

the amount of a5b1 recycled to the plasma membrane (Fig. 7C).We then investigated the effect of knocking down HDPTP on therecycling of integrin from the perinuclear recycling compartments.

Cells were surfaced labeled and integrins were internalized at 37 Cfor 30 min. The recycling rate was slightly reduced in HDPTPknockdown cells (Fig. 7D). Taken together, our results indicate

that HDPTP plays an important role in the recycling of a5b1integrin from early endosomes.

HDPTP regulates actin structure and cell migration

Integrins are adhesion receptors that connect the extracellularmatrix (ECM) and the actin cytoskeleton to control cellmovement (DeMali et al., 2003). Upon binding to ECM

molecules, integrins induce the formation of focal complexesor focal adhesions which are found at the ends of actin stressfibers (Carragher and Frame, 2004; Romer et al., 2006). Since

HDPTP plays an essential role for integrin distribution andtrafficking, we asked whether HDPTP would be important for theorganization of focal adhesions and actin cytoskeleton. The

MDA-MB231 breast cancer cells stably expressing shHDPTP (orshLuc) were stained with anti-paxillin antibody and phalloidin tovisualize focal adhesion and actin structure, respectively.Interestingly, in contrast to the prominent actin stress fibers in

the control cells, the F-actin was organized into short and thinfilaments in the HDPTP-depleted cells (Fig. 8A). Notably,knocking down HDPTP also resulted in a redistribution of

paxillin from large focal complexes/adhesions at the cellperiphery to small punctate staining at the leading edge and inthe cytoplasm (Fig. 8A). These results suggest that HDPTP is

required for the actin cytoskeleton organization and focaladhesion formation. We next wanted to investigate what effectHDPTP depletion would have on cell motility using the in vitro

wound-healing and Transwell migration assays. Control and

shHDPTP MDA-MB231 cells were grown to confluence and thenwounded by scratching in the presence of mitomycin-C, aninhibitor of cell proliferation. We found that shHDPTP cells

migrated across the wound in greater numbers than that of controlcells over 24 hours (Fig. 8B). Time-lapse analysis revealed thatHDPTP knockdown dramatically increased cell migration speed

in MDA-MB231 cells (supplementary material Fig. S8A,B). Inthe Transwell assay, compared with control cells, there was amarked increase in the number of HDPTP knockdown cells that

migrated across the Transwell membrane to the underside of theinserts (Fig. 8C). Together, our findings indicate that HDPTPdepletion altered the assembly of focal adhesions and the

Fig. 4. Identification of Rab4 as a Mop-binding protein. (A) Schematic

presentation of the domain structures of Mop. The positions of the Bro1

domain and the protein tyrosine phosphatase (PTP) domain are indicated.

(B) The Bro1 domain of Mop interacts with Rab4 in the yeast two-hybrid

assay. The yeast transformants were patched for 2 days at 30 C to test for

growth ability on selective media. (C) Lysates from HEK293 cells expressing

HA-tagged Mop–Bro1 (amino acids 1–410) were incubated with either GST

or GST–Rab4 immobilized on glutathione beads. The pull-down products and

input Mop–Bro1 were analyzed by western blots with the HA antibody. Equal

inputs of GST and GST–Rab4 in pull-down reactions were validated by

Coomassie blue staining. (D) HEK293 cells transfected with HA-Mop, HA-

Mop-Bro1 and Myc-Rab4 were used for immunoprecipitation with anti-HA

antibody. The immunoprecipitates and total cell lysates (TCL) were analyzed

by western blotting with antibodies as indicated. (E) Coexpression of HA–

Mop and GFP–Rab4 in Drosophila S2R+ cells. 48 h after transfection, cells

were fixed, permeabilized and processed for staining with antibodies against

HA to visualize colocalization of Mop (Red) and Rab4–GFP (green) in S2R+

cells. Scale bar: 5 mm.

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organization of actin cytoskeleton within the cells, which may

account for the increased motility of these cells.

DiscussionAccumulating evidence has indicated that vesicular trafficking

regulates the distribution of plasma membrane content as well as

the localization of cytoskeletal proteins during cell adhesion and

migration. Drosophila border cells migrate as a cluster of strongly

adherent cells during the development of the egg chamber. During

this process, JNK signaling and endocytosis-mediated spatial

distribution of receptor tyrosine kinases play a critical role (Llense

and Martın-Blanco, 2008; Assaker et al., 2010), though

mechanisms involved in this process have remained elusive. In

this study we identified Mop, the Drosophila homolog of human

HDPTP, as a regulator of integrin trafficking. Mop is essential for

proper integrin localization and for maintaining border cell

integrity during oogenesis. We further demonstrated that Mop

and HDPTP interacts with Rab4 GTPase in both Drosophila and

mammals. Rab4 has been shown to regulate integrin recycling and

cell migration (Roberts et al., 2001; White et al., 2007). Our

findings indicate that Mop/HDPTP-mediated endocytic trafficking

plays an essential role in integrin-mediated cell adhesion and

migration.

Mop has been predicted as a nontransmembrane PTP (Andersen

et al., 2005). However, amino acid sequence analysis revealed that

Mop displays several differences from conserved PTP motifs

within the phosphatase domain. For example, the catalytic

essential aspartic acid (D) within motif 8 (WPDXGXP) is

replaced by a lysine residue (K). Although the active site

cysteine (C) in the catalytic motif 9 (VHCSAGXGR[T/S]G)

could be found, the overall signature motif of Mop was much more

divergent compared to other PTPs. Moreover, we could not detect

Mop tyrosine phosphatase activity using in vitro phosphatase

assays. These results suggest that Drosophila Mop may not be

enzymatic active. Alternatively, Mop may exhibit weak

phosphatase activity which can not be detected using either

pNPP or in gel phosphatase assay. A recent study by Lin et al.

indicated that human PTPN23/HDPTP exhibits relatively low

activity that is comparable with the specific activity of PTP1B

D181E mutant (Lin et al., 2011). We also found that expression of

Mop-C/S mutant, in which the catalytic cysteine in the active site is

replaced by serine, or Mop phosphatase domain deletion mutant

rescued the Mop-RNAi-induced border cell dissociation defects as

effectively as the wild-type Mop, indicating that the putative

tyrosine phosphatase activity is not essential for maintaining

border cell cluster integrity.

In addition to having a C-terminus phosphatase domain, Mop

has a sequence similar to that of yeast Bro1 at the N-terminus.

The Bro1 domain consists of a folded core of about 370 residues

and has been found in many proteins, including Bro1, Alix, and

Fig. 5. Dominant-negative Rab4 induces border cell dissociation.

(A,B) Quantification of border cell migration (A) and dissociation (B) in

slbo-Gal4.UAS-mCD8-GFP flies expressing MopRNAi or the indicated

Rab mutant genes. Ectopic expression of a dominant-negative form of

Rab5, Rab7 and Rab11 impairs border cell migration but not clustering.

Ectopic expression of a dominant-negative form of Rab4 leads to

dissociation of border cells. n.100. (C) Representative images showing the

distribution of bPS-integrin (yellow) and DE-cadherin (red) in border cells

of slbo-Gal4.UAS-Rab4SN, UAS-mCD8-GFP egg chamber. Nuclei were

stained with DAPI (blue). Scale bar: 10 mm.

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Rim20, known to regulate endosome trafficking (Kim et al., 2005).

The Bro1 domain has been shown to bind with multivesicular body

components (ESCRT-III proteins) such as yeast Snf7 and

mammalian CHMP4B for targeting Bro1-domain-containing

proteins to endosomes (Ichioka et al., 2007). Interestingly,

mutational analysis revealed that CHMP4B binding is not

required for HDPTP function, suggesting that the Bro1 domain

may have other functions (Doyotte et al., 2008). Here we identified

Rab4 as an interactor with Mop and HDPTP through the Bro1

domain. The Rab GTPases cycle between the GTP-bound active

and the GDP-bound inactive forms and are necessary for efficient

membrane vesicle transport between different subcellular

compartments (Stenmark, 2009). Among them, Rab4 and Rab11

have been implicated in controlling membrane trafficking through

the endocytic recycling pathways (Grant and Donaldson, 2009).

Several lines of evidence suggest that Rab4 is involved in Mop-

mediated integrin distribution. First, we found that expression of

dominant-negative Rab4S22N or depletion of Rab4 in migrating

border cells resulted in integrin redistribution and border cell

dissociation. Second, genetic analysis revealed that Rab4S22N and

Rab4-RNAi suppressed Mop-induced wing blistering phenotypes.

Third, in mammalian cells, Rab4 regulated integrin recycling from

early endosomes and was required for cell adhesion and spreading

(Roberts et al., 2001). We found that downregulation of HDPTP

disrupted integrin distribution, focal adhesion formation, actin

organization, and enhanced cancer cell motility. Moreover, since

Mop interacts with Rab4 in a nucleotide-independent manner, Mop

may act as an adaptor rather than an effector of Rab4 during

endosomal trafficking processes.

In mammals, Rab4 has been shown to regulate fast integrin

recycling and persistent cell migration (Roberts et al., 2001;

White et al., 2007). We also noticed that dominant-negative Rab4

caused integrin redistribution to the perinuclear region. However,

we found that expression of HDPTP could not rescue the

dominant-negative Rab4-induced integrin redistribution (data not

shown). This is consistent with our genetic data that Rab4 acts

downstream of Mop (supplementary material Fig. S5). We

propose that Mop/HDPTP may act as an adaptor to keep Rab4 in

proper endosomal domains. Depletion of HDPTP resulted in

Rab4 mislocalization, changes in the integrin dynamics and

increases in cell migration. Indeed, we found that misexpression

of bPS-integrin resulted in a marked increase in border cell

cluster dissociation and bPS-integrin knockdown suppressed

MopRNAi-induced border cell dissociation phenotype. Mop/

HDPTP is likely to function via its association with Rab4 on

early endosomes to regulate integrin sorting and recycling.

Integrins activate multiple signaling pathways involved in

regulating cell proliferation, survival and migration (Giancotti

and Ruoslahti, 1999). One major signaling event stimulated by

integrins is mediated by the FAK and Src tyrosine kinases (Mitra

Fig. 6. Human HDPTP interacts with Rab4A. (A) HEK293 cells

transfected with FLAG-tagged human HDPTP and GFP-Rab4A were

used for immunoprecipitation with anti-FLAG antibody. The

immunoprecipitates and cell lysates were analyzed by western

blotting with antibodies as indicated. (B) HeLa cells grown on

coverslips were co-transfected with FLAG-tagged HDPTP and GFP-

Rab4A. The cells were then fixed and stained with DAPI (blue) and

anti-FLAG antibody (red). (C) Western blot analysis of HDPTP

expression in HeLa cells stably transfected with HDPTP shRNA.

(D) HeLa cells stably expressing shLuc and shHDPTP#G were

transiently transfected with GFP-Rab4A and immunostained with

anti-EEA1 (red) antibody. Depletion of HDPTP results in

redistribution of EEA1 and Rab4 when compared with controls.

Scale bar: 10 mm.

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Fig. 7. Depletion of HDPTP disrupts integrin

recycling. (A) HeLa cells stably expressing shLuc and

shHDPTP were cultured on fibronectin-coated

coverslips and immunostained for b1 integrin (green)

and EEA1 (red). b1 integrins were distributed

throughout the control cells, but were more concentrated

at the perinuclear region of HDPTP knockdown cells.

Scale bar: 10 mm. (B) Cells in A were labeled at 4 C

with anti-a5 integrin antibody without permeabilizing

the cells. Cells were then incubated at 37 C for 3, 6 and

12 min for endocytosis of integrins. The rate of integrin

internalization was determined by flow cytometry as

described in Materials and Methods. (C,D) Recycling of

a5 integrin in control (shLuc) and HDPTP knockdown

(shHDPTP) HeLa Cells. Cells were surface labeled with

NHS-SS-Biotin, and internalization was allowed to

proceed for 15 min at 22 C (C) or for 30 min at 37 C

(D), and biotin was removed from receptors remaining at

the cell surface. Cells were then incubated with 10%

FBS/DMEM at 37 C for 5 and 10 min to allow

recycling to the plasma membrane. Integrin-

biotinylation was measured by capture-ELISA. The

proportion of integrin recycled to the plasma membrane

is expressed as the percentage of the pool of integrin

labeled during the internalization period (data are means

6 s.e.m. of at least nine replications). P-values were

calculated using Student’s t-test, *P,0.05; **P,0.01.

Fig. 8. Depletion of HDPTP increases cell migration.

(A) MDA-MB231 breast cancer cells stably expressing shLuc

and shHDPTP were immunostained with anti-paxillin (green)

antibody and TRITC–phalloidin (red) to visualize focal

adhesions and F-actin structures, respectively. Scale bar:

10 mm. (B) Cells as in A were cultured to a confluent

monolayer, wounded, and incubated in growth medium

containing 10 mg/ml mitomycin. Cell migration was observed

with the light microscope at the indicated time points. The

percentage of wound closure at 24 h after wounding is

presented below the images. (C) Cell migration was also

assessed using an in vitro Transwell migration assay. Cells as in

A were plated onto the upper well of a Transwell Boyden

chamber and allowed to migrate for 24 h. Cells that migrated

through the filter were stained with Crystal Violet and

quantified using a microplate reader. Data are means 6 s.e.m.

(n53). **P,0.01.

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and Schlaepfer, 2006; Harburger and Calderwood, 2009). FAKand Src are crucial regulators of cell adhesion and motility whichthey regulate by controlling the formation and turnover of focal

adhesions (Mitra et al., 2005). Several reports have showninteractions between HDPTP and FAK–Src complex. HDPTP hasbeen proposed to act as a molecular bridge between FAK and Src

in regulating endothelial and bladder carcinoma cell motility(Mariotti et al., 2009b; Mariotti et al., 2009a). Moreover, Lin et al.identified PTPN23/HDPTP as a negative regulator of cell

invasion in mammary epithelial cells (Lin et al., 2011). Inthose studies, FAK and Src were found to be substrates ofHDPTP, suggesting that loss of HDPTP may increase FAK–Srcactivity to promote cell motility. Intriguingly, in Drosophila, we

showed that Mop regulates the border cell association in a PTPdomain-independent manner. Recent studies in Drosophila andmammalian cells also found that the phosphatase activity of Mop

and HDPTP is not required for its biological function in receptorsignaling and tumor suppression (Miura et al., 2008; Gingraset al., 2009; Huang et al., 2010). Our findings on Mop/HDPTP–

Rab4 interaction and the regulation of integrin recycling providenew insights into the role of Mop/HDPTP in the regulation of celladhesion and migration, and they are not mutually exclusive fromprevious findings.

Materials and MethodsDrosophila strains and genetics

Flies were raised at 25 C following standard procedures unless otherwise noted. Thefollowing Drosophila strains were used: mopT612 and UAS-Mop-C/S (Miura et al.,2008), UAS-mys and UAS-mysRNAi (Bhandari et al., 2006). The UAS-Moptransgene was generated by subcloning the Mop from SD03094 into the pUASTvector. The UAS-MopDPTP mutant (amino acids 1–1496), in which C-terminusPTP domain of Mop was deleted, was obtained by PCR and subcloned into thepUAST vector. UAS-Rab4RNAi (v106651), UAS-FAKRNAi (v108608), UAS-CSWRNAi (v21756), and UAS-CG7180RNAi (v34369) were obtained from theVienna Drosophila RNAi Center. The mop mutant clones were generated using theFlp/FRT-mediated mitotic recombination system (Xu and Rubin, 1993). Fortransgenic RNAi lines target sequences were amplified from PCR and inserted asinverted repeats with pRISE vector as the destination vector (Kondo et al., 2006). Toconstruct an inducible RNA interference allele of NT-PTPs, we used the followingspecific primers: Mop-RNAi (forward: 59-caccTTATCGCGAGAGTTCCAGAAA-39 and reverse: 59-CTTAACGCTCTGCATCCTTTG-39), dPez-RNAi (forward: 59-caccAACACATCAGCTTCCACATCC-39 and reverse: 59-ACTCGAGACGCTGA-TGACTGT-39), dPtpmeg-RNAi (forward: 59-caccCGTTCAGGTGTCAAAAG-TGGT-39 and reverse: 59-TCCATTACGGTCGTTAGCATC-39), PTP-ER-RNAi(forward: 59-caccGAATCGGAACTGTCAAT-39 and reverse: 59-TCCACTGTT-CATTAGGTTGCC-39), dMeg2-RNAi (forward: 59-CACCGCATGATCTG-GGAACAACATT-39 and reverse: 59-TACACGTTGTTTTCTCGTCCC-39). Otherstocks were obtained from the Bloomington Stock Center.

Plasmids and antibodies

Human GFP tagged Rab4A-WT and Rab4A-S22N were gifts from J. Norman (TheBeatson Institute). Drosophila Rab4-WT, Rab4-Q67L and Rab4-S22N were kindlyprovided by M. Scott (Stanford University). Drosophila Rab5, Rab7 and Rab11 cDNAswere gifts from M. Gonzales-Gaitan (University of Geneva). Human HDPTP cDNAwas obtained from Open Biosystems (Thermo). The lentiviral shRNA clones used toknockdown human HDPTP were obtained from the National RNAi Core Facility ofAcademia Sinica. The targeted sequences for these clones are HDPTP shRNA #A:(Clone ID: TRCN0000080843) 59-GCTCAGGCAAAGATGATTATA-39, HDPTPshRNA #E: (Clone ID: TRCN0000003047) 59-CCGCCAGATCCTTACGCTCAA-39,and HDPTP shRNA #G: (Clone ID: TRCN0000003049) 59-GACAACGACTTC-ATTTACCAT-39. Luciferase shRNA was used as a control. Lentiviral production andinfection were performed as previously described (Tang et al., 2011). Antibodies usedfor the study were: anti-Mop (Abcam), anti-bPS (CF.6G-11, DSHB) anti-DE-cadherin(DCAD2, DSHB), anti-HDPTP (Proteintech), anti-a5 and anti-b1 (BD Biosciences),anti-paxillin (BD Biosciences), anti-EEA1 (Cell Signaling), anti-LAMP1 (Abcam).

Cell culture, immunoprecipitation and immunoblotting

Drosophila S2 cells were cultured at 25 C in Schneider’s Drosophila medium(Invitrogen) containing 10% fetal bovine serum (FBS) and 16 penicillin/streptomycin antibiotics (Invitrogen). Mammalian cells were cultured at 37 C inDMEM (Invitrogen) medium supplemented with 10% FBS and antibiotics. For

immunoprecipitations, HEK293T cells that were transiently transfected with theindicated plasmids were scraped from dishes in lysis buffer [50 mM Tris-HClpH 7.4, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM EDTA,10 mM NaF, 2 mM Na3VO4, 1 mM PMSF, and protease inhibitor cocktail(Roche)]. Cell lysates were immunoprecipitated with anti-Myc or anti-HAantibody and protein-G–Sepharose beads (GE Healthcare) at 4 C for 2 h. Thesebeads were washed three times with the lysis buffer. After resolution by SDS-PAGE,the immunoprecipitates were subjected to western blot analysis.

Immunofluorescence

For border cell migration experiment, ovaries were dissected in Schneider’smedium with 10% FBS, fixed with 4% paraformaldehyde in PBS for 15 min atroom temperature. After a brief washing with PBS, egg chambers werepermeabilized with 0.3% Triton X-100/PBS for 10 min. Samples were thenblocked with 5% normal goat serum in PBST (PBS + 0.1% Triton X-100) for 1 h,and incubated with primary antibodies overnight at 4 C. On the following day,samples were washed three times with PBST and incubated with fluorescent-labeled secondary antibodies for 2 h at room temperature. Egg chambers weremounted in anti-fade reagent containing 0.1 M n-propyl gallate (pH 7.4) and 90%glycerol in PBS. For immunostaining, cells grown on coverslips were fixed with4% paraformaldehyde and permeabilized with 0.1% Triton X-1000/PBS. Theywere then incubated with primary antibodies for at least 1 h, followed byincubation with secondary antibodies. F-actin was stained using TRITC-conjugated phalloidin (Sigma) and nucleus was stained using DAPI (1 mg/ml).Samples were visualized under an epifluorescence microscope (Olympus BX51) ora confocal laser scanning microscope (Zeiss LSM510).

pNPP phosphatase assay

The para-nitrophenyl phosphate (pNPP) phosphatase assays were carried out asdescribed previously (Montalibet et al., 2005). Briefly, S2 cells transientlyexpressing HA-tagged PTP domain of PTP61F (amino acids 1–310), dMEG2(amino acids 505–815), dPTPMEG (amino acids 497–856), dPEZ (amino acids 957–1252) and Mop (amino acids 1497–1803) were lysed and immunoprecipitated withanti-HA antibody. The immunocomplexes were incubated with 20 mM pNPP(Sigma) in pNPP assay buffer (50 mM Hepes, pH 7.5, 1% NP-40, 10 mM DTT) at37 C in the dark for 20 min. The absorbance of the product, pNPP, was measured at405 nm with a spectrophotometer.

Yeast two-hybrid screen

The DNA fragment encoding the Bro1 domain of Mop (amino acids 1–434) wascloned to pGBKT7 (Clontech) to generate a fusion to the Gal4 DNA-bindingdomain (pGBKT7-Bro1). The yeast strain AH109 expressing the pGBKT7-Bro1was transformed with a Drosophila embryonic cDNA library fused to the Gal4activation domain (Clontech). The double transformants were plated onto selectivemedium lacking adenine, histidine, leucine, tryptophan and further assayed for b-galactosidase activity by a colony lift filter assay. The positive clones were isolatedand sequenced.

Flow cytometry analysis to quantify integrin internalization

To study the internalization of the integrins, cells were cultured in 10 cm plateuntil they formed monolayers and the cells were washed twice with PBS and thendetached with the Trypsin-EDTA buffer. Cell surface integrins were labeled withanti-a5 antibody (BD Biosciences) for 20 minutes at 4 C and washed twice in coldPBS. We then allowed endocytosis to occur at 37 C for various intervals (0, 3, 6and 12 minutes). Cells were subsequently fixed with 4% paraformaldehyde for20 minutes at 4 C and washed with cold PBS. Washed cells were labeled withAPC-conjugated secondary antibody (BD Biosciences) for 20 minutes at 4 C tothe primary antibody coated on the cell surface. Antibodies conjugated cells werewashed twice with PBS before flow cytometry analysis (BD FACSCanto II).

Integrin recycling assay

Integrin recycling assay was performed as described previously (Roberts et al.,2001). Surface-labeled cells were incubated with serum-free DMEM at 22 C for15 min or 37 C for 30 min to allow integrin internalization. Biotinylated surfaceproteins were removed by incubation of 20 mM 2-mercaptoethanesulfonate(Mesna, Sigma) in 50 mM Tris (pH 8.6) and 100 mM NaCl for 30 min at 4 C.Recycling of internalized biotin-labeled integrin was stimulated by 10% FBScontaining DMEM at 37 C. At indicated time, medium was aspirated and cellswere washed twice with cold PBS. Surface protein biotinylation was removed by asecond reduction with MesNa incubation, followed by IAA incubation, cell lysis asdescribed above. The percentage of recycled integrin was calculated from controlcells maintained on ice. Biotinylated integrins were detected by capture ELISA asdescribed (Roberts et al., 2001). In brief, Nunc Maxisorp 96-well plates werecoated with 2.5 mg/ml anti-integrin antibodies in 0.05 M Na2CO3 (pH 9.6) at 4 Covernight and blocked for 1 h at room temperature with 5% BSA in PBScontaining 0.1% Tween-20 (PBST). Integrins were captured by overnightincubation of cell lysates with equivalent concentration at 4 C. After extensive

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washing with PBST, color was developed with TMB substrate (Sigma) andstopped with 1 N HCl. The absorbance was measured at 450 nm using amicroplate reader (Tecan).

In vitro wound-healing and Transwell migration assay

MDA-MB-231 breast cancer cells expressing HDPTP siRNA (shHDPTP) orluciferase-knockdown control (shLuc) were plated onto six-well culture plates inDMEM containing 10% FBS. After 24 h, the cell monolayers were woundedmanually by scratching with a pipette tip, and rinsed with PBS. Fresh DMEM with1% FBS and 10 mg/ml mitomycin C was added to follow healing for 24 h. Cellswere photographed at 0 h, 6 h and 24 h after wounding using a phase contrastmicroscopy and an Olympus IX70 camera. For time-lapse microscopy and celltracking analysis, cells were observed with a 206 phase-contrast objective andimages were collected every 10 min using a Cool SNAP CCD camera. Cell trackswere analyzed with ImageJ software. For Transwell migration assays, 16105 cellswere seeded into and grown in low serum medium (1% FBS) on the top ofTranswell chambers (8 mm pores, Millipore). The lower chamber was filled withmedium containing 10% FBS. Mitomycin C (Sigma) was added to inhibit cellproliferation. After 24 h, the migrated cells on the lower surface of membranewere fixed and stained with 0.5% Crystal Violet (Sigma). The absorbance wasmeasured at 570 nm using a microplate reader (Tecan).

AcknowledgementsWe thank M. Gonzales-Gaitan, M. Grotewiel, Y. Kageyama, J.Norman, L-M Pai, H.-W. Pi, M. Scott, J. Treisman, the BloomingtonStock Center, Vienna Drosophila RNAi Center, Fly Core Taiwan andthe Developmental Studies Hybridoma Bank (DSHB) for reagents.We thank S.Y. Tsai for the yeast two-hybrid screen and C.-C. Hung forthe confocal microscopy assistance.

FundingThis work was supported by the National Science Council of Taiwan[grant numbers NSC98-2311-B-001-019-MY3 to T.-C.M., NSC99-2311-B-001-017-MY3 to G.-C.C.], and by Academia Sinica.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.108597/-/DC1

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