Rudhira/BCAS3 is a cytoskeletal protein that controls Cdc42 activation and directional cell migration during angiogenesis

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Research Article

Rudhira/BCAS3 is a cytoskeletal protein that controls Cdc42activation and directional cell migration during angiogenesis

Mamta Jaina, Ganesh P. Bhata, b, K. VijayRaghavanb, c, Maneesha S. Inamdara,⁎aJawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, IndiabNational Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, IndiacInStem, NCBS, Bangalore, India

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author at: Molecular Biology a560 064, India. Fax: +91 80 22082766.

E-mail address: [email protected] (M.S.Abbreviations: MTOC, Microtubule Organizin

0014-4827/$ – see front matter © 2012 Elseviedoi:10.1016/j.yexcr.2012.01.016

A B S T R A C T

Article Chronology:

Received 29 October 2011Revised version received 11 January 2012Accepted 15 January 2012Available online 25 January 2012

Cell migration is a common cellular process in angiogenesis and tumor metastasis. Rudhira/BCAS3(Breast Cancer Amplified Sequence 3) is a conserved protein expressed in the embryonicvasculature and malignant tumors. Here, we show for the first time that Rudhira plays an activerole in directional cell migration. Rudhira depletion in endothelial cells inhibits Matrigel-inducedtube formation and retards healing of wounded cell monolayers. We demonstrate that duringwound healing, Rudhira rapidly re-localizes and promotes Cdc42 activation and recruitment to theleading edge of migrating cells. Rudhira deficient cells show impaired downstream signaling ofCdc42 leading to dramatic changes in actin organization and classic cell polarity defects such as

loss of microtubule organizing center (MTOC) and Golgi re-orientation. Biochemical assays and co-localization studies show that Rudhira interacts withmicrotubules as well as intermediate filaments.Thus, Rudhira could control directional cell migration and angiogenesis by facilitating crosstalkbetween cytoskeletal elements.

© 2012 Elsevier Inc. All rights reserved.

Keywords:

RudhiraCell migrationCytoskeletonCdc42Polarity

Introduction

Embryonic development requires the formation of blood vessels,which occurs by vasculogenesis and angiogenesis. While there isabundant neo-angiogenesis and remodeling during embryonicdevelopment, adult endothelial cells, are quiescent for the mostpart [1]. Post-natal angiogenesis is a highly regulated process andseen only in special situations such as tissue repair and in the femalereproductive cycle. Loss of control on endothelial proliferation andmigration leads to aberrant angiogenesis. Tumors often depend onthe vasculature for their growth and spread [2] and overexpressmitogenic and angiogenic factors and their receptors. Mutations ingenes controlling angiogenesis are often associated with vascular

nd Genetics Unit, Jawaharla

Inamdar).g Centre; BCAS3, Breast Ca

r Inc. All rights reserved.

diseases or metastatic tumors [3]. The latter context is characterizedby aberrant expression of molecules controlling cell migration andcell polarity, two key events in angiogenesis aswell as tumormetas-tasis [4,5]. Here we investigated the mechanism by which Rudhira/BCAS3 affects these processes.

Rudhira is a predicted WD40 domain protein. During mousedevelopment Rudhira is expressed in erythropoietic and angiogenicprecursors but not in their differentiated progeny [6]. This transientexpression of Rudhira is also seen in normal adult angiogenesis [6].BCAS3, the human ortholog of Rudhira is 98% identical and thegene maps to a breakpoint of hematological neoplasms on chromo-some 17q23.1. BCAS3 is also expressed in vascular development inembryoid bodies in vitro [7] and was originally identified as a gene

l Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore

ncer Amplified Sequence 3; IF, Intermediate filament.

http://dx.doi.org/10.1016/j.yexcr.2012.01.016

mailto:[email protected]


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over-expressed in breast cancers [8]. It is reported to be a target ofmetastasis associated protein-1 (MTA-1) [9]. Further BCAS3 expres-sion is also upregulated in grade III and IV glioblastomas where it ismisexpressed in the tumor cells [7]. This suggests that a strict con-trol on Rudhira/BCAS3 expression in tissues is essential to preventaberrant angiogenesis as well as maintain normal cell phenotype.These studies implicate a role for Rudhira/BCAS3 in angiogenesisas well as tumor metastasis. As both normal and aberrant scenariosof Rudhira function require control of cell migration and cell polari-ty, we investigated whether Rudhira is required for these cellularprocesses.

Cell migration is generally induced in response to an appropri-ate chemotactic signal (reviewed in [5]) and requires a regulatedchange in the cytoskeleton and extracellular matrix. Actin re-organization during cell migration is regulated by the small RhoGTPase family of proteins represented by RhoA, Rac1 and Cdc42[10]. It is characterized by regulation of focal adhesions byRhoA, and lamellipodia or filopodia controlled by Rac1 andCdc42 respectively [11,12]. Rho GTPases also regulate microtu-bule and intermediate filament dynamics during cell migration[13,14]. Microtubule dynamics at the leading edge is regulatedby Rho GTPases where they help establish cell polarity byguanine-nucleotide exchange factor (GEF)-mediated activationof Cdc42 [15]. Although the direct mechanism by which RhoGTPases regulate intermediate filaments is not known, theymediate a change in phosphorylation status [14]. Rudhira beinga WD40 protein could interact with cytoskeletal elements tohelp regulate cell migration during angiogenesis and metastasis.Nevertheless, the molecular details of how Rudhira functionsare not known.

In this study, we modulated Rudhira in endothelial cells andinvestigated its effects on cell migration and angiogenesis. To elu-cidate mechanistic details of these effects, we assessed the effectof modulating Rudhira on actin re-organization, cell polarity andstudied its interaction with cytoskeletal elements.

Materials and methods

Antibodies and reagents

Rudhira monoclonal antibody was raised against custom designedpeptides (Imgenex Corporation, Bhubaneswar, India) and testedfor antigen specificity (see supplementary data Fig. S1). Primaryantibodies used were directed against: Vinculin, FLAG tag, γ-tubulin, vimentin, (SIGMA Chemical Co., USA); c-myc, β-tubulin(DSHB, Iowa); and Cdc42 (Cytoskeleton, Inc.). Phalloidin conju-gated with Alexa 546 or 488 (Molecular Probes) was used tolabel actin. Secondary antibodies used were goat anti-rat Alexa488, goat anti-mouse Alexa 568, and goat anti-mouse Alexa 633(all fromMolecular Probes). CytochalasinB, Nocodazole, Cyclohex-imide, LPA (Lysophosphatidic acid), Bradykinin and Wortmanninwere from SIGMA Chemical Co., USA. PDGF (Platelet-derivedgrowth factor) was from PeproTech Inc, USA.

Cell culture

Cell lines usedwere: Saphenous vein endothelial cells (SVECs) (fromKaustabh Rau, National Centre for Biological Sciences, Bangalore);NIH3T3, HEK293 and HeLa (American Type Culture Collection). All

cell lines were cultured in DMEM containing 10% FBS (Gibco-BRL,USA) and 2 mM Glutamax (Invitrogen, Carlsbad, USA).

Constructs and transfections

pCMV-Rudh-IRES2-EGFP was constructed by PCR amplification offull length Rudhira (931 amino acids) from cDNA clone (No.6430542M21, RIKEN BRC, Japan) and directionally cloned intoEcoRI-SalI sites of pIRES2-EGFP (Clontech). pCAG EGFP was akind gift from Peter W. Andrews (Centre for Stem Cell Biology,Sheffield, UK). To generate the bicistronic construct pCAGRu-d2AGFP, the upstream GFP in pCMV GFP(2A)GFP (kind gift fromHirofumi Suemori, Kyoto University, Japan) was first replacedwith the Rudhira ORF. Then, Rudh2A was PCR amplified from thegenerated pCMV-Rud2AGFP and ligated into XhoI-AgeI sites ofpCAG-EGFP. To generate RudhFL/FLAG (AA 1–928) the fragmentwas PCR amplified from full length Rudhira cDNA and cloned inpCMV-Tag2B (Stratagene). Raichu (1054X) plasmid was a kindgift from Michiyuki Matsuda, Kyoto University, Japan. mCherry-Actin construct was a kind gift from Klemens Rottner, Universityof Bonn, Bonn. The dominant negative mutant of Cdc42 (mycCdc42 DN) and activator mutant of RhoA (RhoA QL) were a gener-ous gift from Giorgio Scita (IFOM, Milan, Italy). HEK293 cells weretransfected using FuGENE 6 (Roche Molecular Biochemicals). NIH3T3 cells were microporated using a Neon™ Transfection system(Invitrogen) as per the manufacturer's protocols. Cells were usedfor experiments 36 h after transfection. Cell viability upon Rudhiraoverexpression was checked by staining with PKH26 (SIGMA) andEthD1 (Molecular Probes). Rudhira overexpression was quantifiedby measuring the total cell fluorescence in control and Rudhiraoverexpression cells stained with Rudhira antibody and plottedas a relative fold change.

In order to quantify the number of filopodia per cell after Rudhiraover-expression in NIH 3T3, single cell images were taken at 100×magnification and the cell protrusions were counted manually. Thenumber of protrusions marked by phalloidin was counted in afixed number of cells and was represented as number of filopodiaper cell in both control and Rudhira over-expression cells. Forthe quantitation of rescue phenotypes after co-expression ofRudh2AGFP with either Cdc42DN or RhoQL, a fixed number of GFPpositive cells were scored for either presence or absence of thecorresponding phenotype (stress fibers/focal adhesions in case ofRhoQL and filopodia in case of Cdc42DN). The data was then repre-sented as percentage of cells showing the phenotype.

siRNA transfection

SVECs were transfected with siRNA directed against rudhira(mouse BCAS3: SmartPool; Dharmacon, Lafayette, CO) using Lipo-fectamine 2000 (Invitrogen) following the manufacturer's recom-mendations. Firefly luciferase siRNA was included as a control fornon-specific effects. siRNA transfected cells were harvested after48 h for RNA extraction or after 72 h for protein extraction. Forall other experiments, cells were harvested after 72 h of transfec-tion. BCAS3 shRNAs FI325714, FI325715 (Origene, Rockville, MD)were transfected in SVECs and used for staining with Rudhiraand Phalloidin. Cells with depleted rudhira were identified byimmunostaining and phenotype was assessed by co-stainingwith respective marker antibodies (GM130, γ-tubulin). For quan-titative analysis, a fixed number of cells at the woundmargin were

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counted in both control and rudhira knockdown dishes and repre-sented as percentage of cells showing the phenotype. A thresholdwas set depending on the background in the dish and only cellsthat showed no or very weak staining were considered foranalyses. For quantitative analyses of Cdc42 recruitment, a fixednumber of cells showing Cdc42 recruitment was counted in thecontrol as well as rudhira knockdown cultures and relativeCdc42 recruitment was plotted by normalizing the control valueto 1.

RT PCR and western blotting

RNA was isolated from siRNA transfected cells using TRIzol reagent(Invitrogen, Carlsbad, CA). Reverse transcription was performedusing Superscript II (Invitrogen). Primer set used in PCR analyseswas against Rudhira ORF. For protein lysates, cells were pelleted,washed with PBS and lysed in a buffer containing 25 mM HEPES,150 mM NaCl, 1% NP-40, 10% glycerol, 10 mM MgCl2. 1 mM EDTA,1 mM Sodium orthovanadate and 10 μg/ml protease inhibitorcocktail (SIGMA Chemical Co., USA) on ice and clarified by centrifu-gation. Protein was estimated using Bradford reagent and 50 μg oflysate was loaded on SDS-PAGE. Gel was electroblotted on PVDFmembrane, probed with specific antibody and developed usingECL chemiluminescence (Thermo Scientific, Rockford, IL, USA).

In vitro angiogenesis assays

100 μl of Matrigel (Basement Membrane Matrix, BD Biosciences,USA) was coated onto a 96 well plate and incubated at 37 °C for30 min for gel formation. 72 h after transfection with siRNA,1.5×104 cells were plated onto Matrigel and incubated in 5%CO2 at 37 °C for 4 h. Endothelial tube formation was monitoredand imaged at 4× magnification. Average tube length was quanti-fied using Image J software (developed by National Institutes ofHealth, Bethesda, MD). 3D collagen tube formation assay wasperformed as described before [16]. Briefly, 15,000 SVECs wereplated onto 1.5 mg/ml Collagen (GIBCO Cat. No. A10483) 48 hafter siRNA transfection. After 24 h, cells were overlaid with1.5 mg/ml collagen, and tube formation was monitored up to48 h. Collagen plugs were fixed in 4% paraformaldehyde andsectioned for hematoxylin-eosin staining.

Wound healing and Transwell migration assay

SVECs were grown to confluence and transfected with siRNA. 48 hafter transfection, the cell monolayer was wounded using a 200 μlpipette tip, gently washed with PBS and incubated with freshmedia supplemented with 5% FBS. Cells were monitored using anOlympus IX70 inverted fluorescence microscope and imaged atdifferent time intervals using a Roper Coolsnap CCD camera andImage-Pro Plus software (Media Cybernetics). The rate of woundclosure was measured by calculating the distance covered permin for around 300 cells at each wound margin. For the trackingassay, control and knockdown cells were monitored in real timepost-wounding and 32 cells were selected randomly from boththe wound margins and analyzed for their directionality. HEK293cells were transfected with pIRES2-EGFP and Rudh IRES2-EGFPand used for wounding assay (see supplementary Fig. S2). Forthe transwell migration assay, Transwell inserts with 8 μm poresize (Costar) were coated with 10 μg/ml of porcine Fibronectin

(SIGMA Chemical Co., USA). 48 h after siRNA transfection, cellswere serum-starved for 12 h and plated onto the upper chamberof the Transwell filter. Serum was added to the lower chamberto serve as a chemoattractant. After 3 h, cells on the top of thefilter were removed using a cotton swab. Cells that had migratedto the bottom were fixed and stained with crystal violet. The dyewas extracted with 30% acetic acid and absorbance measuredspectrophotometrically at 540 nm.

Re-localization assay

Rudhira re-localization in migratory cells was studied by woundingSVEC monolayers. Cells were fixed at the initiation of the assay andat 6 h, then stained for Rudhira and imaged. For the time seriesexperiment, monolayers were scratched and fixed at various timepoints and stained with anti-Rudhira and anti-Cdc42 antibodies.For each time point, n≥600 cells were analyzed over three indepen-dent experiments.

Cytoskeleton-related experiments

To disrupt actin, cells were treated with 6 μM of CytochalasinB for2 h and then fixed for staining. For studying the effect of Rho GTPaseactivators, cells were serum starved for 16 h, inducedwith Lysopho-sphatidic acid (LPA) (400 ng/ml for 15 min), Platelet-derivedgrowth factor (human PDGF BB— 100 ng/ml for 10 min) or Bradyki-nin acetate (200 ng/ml for 15 min) and then fixed for staining.Microtubules were disrupted by treating cells with 10 μM Nocoda-zole for 12 h, fixed and stained. Intermediate filaments weredisrupted by treating cells with 10 μg/ml Cycloheximide for 4 h,fixed and stained. For detergent extraction, cells were treated withextraction buffer (100 mM PIPES, 1 mM MgCl2,1 mM EGTA, 1%Triton X-100, 0.4 M NaCl, 4% Polyethyleneglycol, 1 mg/ml DNAse)for 10 min at room temperature in order to preserve IFs, washed,fixed and stained. For co-immunoprecipitation assays, 1 mg ofHeLa cell lysate was incubated overnight with anti-tubulin or anti-vimentin antibody or isotype controls. Immune complexes werecaptured on Protein G-sepharose beads (SIGMA), washed threetimes in lysis buffer and analyzed by immunoblotting with anti-β-tubulin, anti-vimentin and anti-Rudhira antibody. For FLAG pulldown, 1 mg of lysate was incubated overnight with anti-FLAG anti-body along with sepharose beads, washed twice with lysis bufferand immunoblotted with β-tubulin or vimentin antibody.

FRET and PAK-PBD assay

HEK293T cells were co-transfected with Raichu 1054X and FLAGvector and Rudh-FLAG construct. 24 h post transfection, cellswere washed with PBS and lysed using 20 mM Tris pH7.5,100 mM NaCl, 5 mM MgCl2 and 0.5% Triton-X. The lysate wasused to acquire spectra on a fluorescent spectrometer (Hitachi,F-2500) with excitation wavelength of 433 nm. To confirm thepresence of intramolecular FRET, cell lysates were incubatedwith proteinase K (100 μg/ml) at 37 °C for 10 min before analyzingwith spectrofluorometer. For imaging based FRET, 48 h post trans-fection, cells were imaged on a Nikon Eclipse Ti microscope. Forimaging of Raichu probe, we used 434 nm for CFP excitation andtwo emission filters, 470±15 nm for CFP and 535±15 nm forYFP (Chroma). After background subtraction, the YFP emissionupon excitation in CFP channel was normalized to YFP emission


upon excitation in YFP channel and plotted as normalized YFPemission to compare between vector and Rudhira transfectedcells. For the photobleaching experiment, cells were illuminatedwithout ND filter for 10 s and CFP as well as YFP emission uponexcitation in CFP channel was captured. The increase in CFP wasnormalized to decrease in YFP and plotted as a comparisonbetween vector and Rudhira transfected cells. For the PAK-PBDassay, HEK293T cells were co-transfected with WTCdc42GFP(kind gift from Nagaraj Balasubramanian, University of Virginia,Charlottesville VA) and Rudh-FLAG construct. 48 h post transfec-tion, cell lysates were prepared and subjected to Western Blotting.700 μg of protein was incubated with PAK-1 PBD conjugatedagarose beads (Millipore) for 3 h at 4 °C, centrifuged, washed,resuspended in 30 μl of SDS loading dye and processed forWestern blotting with anti-Cdc42 antibody (Santa Cruz Biotech-nology). 50 μg of whole cell extract was used for probing thetotal Cdc42 content in both FLAG vector and Rudh-FLAG trans-fected lysates.

Fluorescence microscopy and live cell imaging

Cells to be imaged were plated onto glass coverslips. After experi-mentation, cells were fixed with 2% paraformaldehyde for 20 min,permeabilized with 0.1% TritonX-100 for 10 min and blocked with4% FBS for 1 h. After incubation with appropriate primary andsecondary antibodies, cells were imaged using a laser scanningconfocal microscope (Zeiss LSM510 Meta) with a 100X plan/1.3NAoil immersion objective. Images were processed using LSM 510Image software and are projections of a Z series unless otherwiseindicated. Brightfield (phase contrast) images were captured withan inverted microscope (IX70, Olympus) using 4X plan/0.1 NAobjective equipped with a cooled charge coupled device (CCD)camera (CoolSNAP; Roper Scientific, Inc). Time-lapse videos werecaptured using a motorized inverted microscope (IX81, Olympus)equipped with sample heater (37 °C), a CO2 incubation chamber(Tokai Hit) and an Olympus CCD camera (FVII with CellP software).All videoswere recorded at the frequency of one image every 10 min(for Matrigel assay) or 20 min (for cell migration assay). Videos inAVI format were edited with Image J (NIH). Images were adjustedfor brightness and contrast using Adobe Photoshop CS2 whererequired.

Statistical analyses

Statistical significance analysis was done using student's t-test orone-way ANOVA (analyses of variance) with Statistica software.P values<0.05 were considered significant.

Results

Rudhira ‘knockdown’ phenotypes suggest a role in endothelialtube formation

Rudhira is expressed in embryonic vasculature and hence we firsttested its role in angiogenesis. A characteristic feature ofendothelial cells is their ability to migrate in a directed fashion,coalesce and form tubes in response to a stimulus. We loweredrudhira expression in endothelial cells and tested their ability toform tubes on Matrigel, an extracellular matrix commonly used

to induce endothelial tube formation [17]. Knockdown of rudhirawith a SMARTpool of siRNA (see methods) showed a 70% reduc-tion in gene expression as seen by RT-PCR and Western blot anal-ysis (Figs. 1A and B). Rudhira knockdown was also achieved byusing two different gene-specific shRNA (Origene, Rockville, MD)in SVECs (Figs. S2A-H). Loss of rudhira significantly reduced tubeformation (~3.5 fold) in a time and dose-dependent mannerwhen compared with controls (Figs. 1C and D–K). Real timeanalyses of knockdown cells showed clumping and randommove-ment indicating a possible defect in their orientation, which couldaffect their ability to coalesce and form tubes (Figs. 1D–K andVideo S1). We further validated these results by a 3D collagentube formation assay [16] which showed reduced tube formationin rudhira depleted cells as compared to controls (Fig. S2I-N).These data show that Rudhira is required for endothelial tubeformation. Thus, Rudhira may regulate important events duringangiogenesis, such as cell migration.

Rudhira re-localizes towards the leading edge and promotescell migration in endothelial and other cell types

To test the role of Rudhira in cell migration, we first assessed thestatus of Rudhira in stationary and motile endothelial cells. Thecell-wounding assay generates a cell-free gap in a confluentmonolayer (schematically represented in Fig. 2A). This inducescells bordering the gap to polarize and move into the gap, eventu-ally filling it up [18]. In confluent stationary cells away from thegap, we found that Rudhira is expressed throughout the cytoplasmas seen in endothelial cell monolayers even after several hours ofwounding (Figs. 2B and C). However cells bordering the woundand migrating had drastically re-localized Rudhira towards theleading edge in the direction of movement (Figs. 2D–H). A timecourse analysis showed that Rudhira re-localized to the leadingedge within 15 min of wounding (Fig. 2E). Quantitative fluores-cence intensity analysis showed that Rudhira was re-localized in4±2% of motile cells within 30 min of wounding and 30±3%cells showed re-localization by 4 h (n≥600, see Materials andmethods). Thus we show that endogenous Rudhira is polarizedtowards the leading edge when cells are induced to move.

To investigate the functional significance of Rudhira re-localization in motile cells, we “knocked down” rudhira in endothe-lial cells and then tested their ability to heal wounded monolayers.Cell migration was greatly compromised in rudhira knockdowncells as analyzed by migration tracks. Rudhira-depleted cellsmoved much slower and randomly into the wound margin post-scratching as compared to controls (Figs. 2I–K). We observed a40% reduction in the migration rate (Figs. 2L–O, P and Video S2)and a greater reduction in directionality as compared to controls(Fig. 2Q) indicating that Rudhira affects both speed and directional-ity during migration. Using a transwell assay, we also observed asignificant reduction in individual cell migration (Figs. 2R–T).Conversely, over-expression of Rudhira in HEK293 cells, that havelow endogenous expression, showed an increase in migration rateas compared to vector controls (Supplementary Fig. S3A–C). Weruled out the possibility that wound healing on cell monolayers isdue to cell proliferation by treating the cells with mitomycin Cprior to the assay (Fig. S3C). Collectively, our results indicate thatRudhira promotes directional cell migration. To dissect out themechanism by which Rudhira functions we next investigated itseffects on the primary cytoskeletal components.

Fig. 1 – Tube formation is reduced in rudhira siRNA treated endothelial cells. (A, B) Semi-quantitative RT-PCR analysis to detectmRNA expression (A) and Western blot analysis to detect protein expression (B) of rudhira after transfection of endothelial cellswith siRNA as indicated. GAPDH RNA or protein levels served as controls. (C–K) Graph showing average tube length formed (C) andrepresentative images (D–K) of SVECs transfected with siRNA as indicated in the panels and assayed for tube formation onMatrigel.MT: mock-transfected; CT: control non-targeting siRNA (66 nM). Data represent the average of three independent experiments.Error bars indicate mean±SD. *: p<0.005, **: p<0.001 by one-way ANOVA compared with the control siRNA. Scale bar: 100 μm.


Rudhira co-localizes with both microtubules and intermediatefilaments

The distribution of Rudhira in stationary and migrating cellssuggested a role in cytoskeletal dynamics. The interplay betweencytoskeletal elements, namely actin, microtubules and intermedi-ate filaments is essential for cell migration [13,27]. Downstreameffectors of migratory stimuli lead mainly to changes in actinorganization and microtubule dynamics [25]. We investigatedthe possibility that Rudhira may interact with cytoskeletalelements.

Immunolocalization analyses showed that Rudhira co-localizessignificantly withmicrotubules (Figs. 3A–H) as well as intermediatefilaments (Figs. 3I–P) in stationary (Figs. 3A–C and I–K, quantified inD and L respectively) as well as motile cells (Figs. 3E–G and M–O,quantified in H and P respectively). Co-immunoprecipitaton assayswere next used to validate conclusions suggested by co-localization studies. For this, we chose HeLa cells as they have highlevel of endogenous Rudhira expression. We found that tubulin aswell as vimentin could independently co-precipitate Rudhira ascompared to isotype controls (Figs. 3Q and R) indicating thatRudhira interacts with both microtubules and intermediate

filaments in vivo. We confirmed our findings by a reverse Co-IPusing FLAG tagged Rudhira. We found that FLAG tag could pulldown both tubulin and vimentin in Rudhira transfected lysate ascompared to controls (Fig. 3S).

Next, we asked if Rudhira interacts with both the cytoskeletalcomponents, could Rudhira localization also depend on both micro-tubules and intermediate filaments. We extracted microtubuleswith detergent (Fig. S4A–C) or disrupted intermediate filamentswith cycloheximide (Fig. S4D–F) and examined the localization ofRudhira. In either case we saw that Rudhira maintained its filamen-tous architecture and localized primarily with the intact cytoskeletalelement — namely, Intermediate filaments in detergent extractedcells (Fig. S4C) and microtubules in cycloheximide treated cells(Fig. S4F). Interestingly, Rudhira collapses and forms perinuclearcables when cells are treated with Nocodazole, which depolymer-izes microtubules and aggregates intermediate filaments(Fig. S4G–I). Taken together, these results suggest that althoughRudhira interacts with microtubules and intermediate filaments,its architecture in the cell can bemaintainedwhen any one cytoskel-etal filament is intact. Whether this unique location allows Rudhirato mediate crosstalk between microtubules and intermediatefilaments needs further investigation.


Loss and gain of function experiments reveal a role for Rudhirain actin organization

Actin has an important and dynamic role during cell migration. Wealso checked whether Rudhira is associated with actin. Co-stainingfor Rudhira and actin showed no significant overlap in the twoexpression patterns (Fig. S5A–D). To test whether there is an indi-rect interaction of actin and Rudhira we disrupted or modulatedone and analyzed the effect on the other.

We first examinedwhether Rudhira affects actin localization. De-pletion of rudhira in migratory endothelial cells showed reducedfilopodial protrusions (Figs. 4E and F, compare to Figs. 4A and B,quantified in Fig. 4I). Knockdown of rudhira with two differentshRNAs also showed similar phenotypes (Fig. S6). However, we

did not observe significant changes in actin organization uponrudhira silencing in the stationary cells present distal to the woundmargin (Figs. 4G and H compare to Figs. 4C and D).

To test whether Rudhira alone can induce changes in actin, weexogenously expressed Rudhira in NIH 3T3 fibroblasts which havevery low endogenous levels of Rudhira. We used a bicistronicRudh2AGFP construct where GFP marks Rudhira-overexpressingcells (see Materials and methods). Cells with increased Rudhiraexpression (quantified in Fig. 4J) showed dramatic changes inactin organization (Figs. 4K–O). There was a significant reductionin stress fibers and increased filopodial extensions (Fig. 4O) ascompared to controls (Fig. 4N). In addition, staining for vinculin(Figs. 4P–S) revealed reduced focal adhesion complexes inRudhira over-expressing cells (Fig. 4S, arrowhead). Such changes

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Fig. 2 – Rudhira re-localizes towards the leading edge and promotes cell migration. (A) Schematic showing a wounded monolayerused for analysis with cells at the wound margin (red) and distal to the margin (green). (B–H) SVEC monolayers wounded, fixed atvarious time points post-scratch as indicated and stained for Rudhira (Gray). Nuclei were marked by DAPI (blue). Cells near(D–H) as well as distal (B, C) to the wound margin were imaged. Arrowheads indicate Rudhira relocalization towards the leadingedge. Cell boundary is marked by white dashed line and left woundmargin by solid white line. (I, J) Representativemigration tracksof SVECs treated with control (I) or rudhira siRNA (J) post scratching shown from both the wound margins (n=32). (K) Magnifiedview of migration track of a single cell treated with either control or rudhira siRNA. Note that for comparison, only a part of thetrack is shown in the control cell whereas the whole track is shown for rudhira siRNA treated cell. (L–O) Phase contrast images ofSVECs transfected with control or rudhira siRNA during wound healing assay. Wounded monolayers were imaged at 0 h (L, M) and10 h (N, O). (P) Graph representing the migration rate of SVECs transfected with control and rudhira siRNA. *: p<0.001 (Q) Averagepersistence of migration (directionality) calculated from tracks depicted in (I, J). Directionality was calculated by dividing netdistance traveled by total distance. **: p<0.001. (R–T) SVECs transfected with control or rudhira siRNAwere assayed for their abilityto migrate through transwell filters coated with fibronectin using serum as a chemoattractant. (R–S) representative brightfieldimages of migrated cells. (T) Graph showing the relative cell migration (quantified by absorbance after crystal violet staining). Datashown are mean±SD of at least three independent experiments *: p<0.001. Scale bar: (B–H) 100 μm; (L–O, R, S) 200 μm.

Fig. 3 – Rudhira is a cytoskeletal protein. Co-localization of Rudhira with tubulin (A–G) or vimentin (I–O) in stationary cells (A–Cand I–K) and migrating cells (E–G and M–O). D,H,L,P-Graphs representing percentage of colocalization between Rudhira andrespective cytoskeletal protein in stationary cells (D, L) and migratory cells (H, P). All Insets are single confocal section showing amagnified view of the respective boxed area. White solid line indicates left wound margin. Nuclei are marked by DAPI (blue). Scalebar: 100 μm. Error bar (D,H,L,P) indicates mean±SD of 20 cells from three independent experiments. (Q–R) Western blot showingco-IP of Rudhira with β-tubulin (Q) or vimentin (R) in Hela cells. IgG was used as an isotype control. 100 μg of whole cell lysate wasused as an input control. (S) Western blot showing the reverse Co-IP using FLAG tagged Rudhira to pull down both tubulin andvimentin. Vector transfected cells were used as a control. All the input controls in vector and Rudhira transfected cell lysates areshown.


image of Fig.�3


in actin organization are established hallmarks of a typical migra-tory cell [19]. Exogenously expressed Rudhira could induce thesesignatures even in stationary cells. Real time analyses in NIH3T3fibroblasts co-expressing either vector and mcherry-actin or bicis-tronic Rudh2AGFP and mCherry-Actin confirmed that the protru-sions in Rudhira over-expressing cells are indeed filopodia(Fig. 4T and Video S3). These results indicate that Rudhira can re-organize the actin cytoskeleton, thereby helping in cell migration.

Rudhira blocks RhoA activity and activates Cdc42 to facilitateactin re-organization

We next examined the effects of actin disruption or activation onRudhira. Depolymerization of the actin cytoskeleton with theactin inhibitor Cytochalasin B did not cause significant disruptionof Rudhira in the majority of cells (Fig. S5E-J). RhoA, Rac1 andCdc42 are the major small GTPases that affect actin cytoskeletal

image of Fig.�4


assembly and re-organization [12]. While RhoA promotes stressfiber and focal adhesion complex formation [20], Rac1 and Cdc42regulate the formation of lamellipodia and filopodia respectively[21,22]. LPA, PDGF and Bradykinin are the activators of RhoA,Rac1 and Cdc42 pathways respectively, and cause increased stressfibers, lamellipodia and filopodia respectively [23,24]. Cells trea-ted with either of these activators showed no change in Rudhiralocalization or expression (Fig. S5K–R). Collectively, these findingsindicate that Rudhira localization is not regulated by the actincytoskeleton and Rudhira has a regulatory role on actin reorgani-zation during cell migration.

As seen earlier, Rudhira causes a reduction in stress fiber andfocal adhesion complexes and increased filopodial extensions.Therefore we reasoned that Rudhira may act by blocking RhoA ac-tivity or by activating Cdc42. To test this we co-expressed eithervector (pCAG-EGFP) along with a constitutively active form ofRhoA (RhoQL) (Figs. 5A–D) or a dominant negative form ofCdc42 (Cdc42DN) (Figs. 5E and F) or Rudhira (pCAGRudh2AGFP)along with RhoQL (Figs. 5G–J) or Cdc42DN (Figs. 5K and L) inNIH3T3 fibroblasts and looked for rescue of the Rudhira over-expression phenotype. Cells co-transfected with Rudhira andRhoQL rescued the loss of stress fibers and focal adhesions(Figs. 5H and J, quantified in Figs. 5M and N respectively) as com-pared to cells overexpressing Rudhira alone (Figs. 4O and S re-spectively). On the other hand cells co-expressing Rudhira andCdc42DN showed significantly fewer filopodial extensions(Fig. 5L, quantified in Fig. 5O) compared to the cells expressingRudhira alone (Fig. 4O). Together, these results indicate thatRudhira-induced actin re-organization could regulate Cdc42 and/or RhoA. Since Cdc42 activation inhibits RhoA mediated pheno-types [19], we checked whether Rudhira over-expression canpromote Cdc42 activation.

Rudhira mediates Cdc42 activation and recruitment at theleading edge

Cdc42 regulates cell polarity, thereby mediating directional cellmigration. Cdc42 is activated and recruited to the leading edgeto determine the direction of cell migration [25,26]. Our analysisof Rudhira localization during cell migration suggested that itacts upstream of actin and the Rho GTPases. Further, Rudhiraalso localizes towards the leading edge in migrating cells. Wetherefore examined the spatio-temporal relationship between

Fig. 4 – Rudhira affects actin organization. (A–H) Representative imsubjected to the wounding assay and stained with Phalloidin (red) a(A, B, E, F) as well as distal to the woundmargin (C, D, G, H) were imDashed line in (E) marks a cell showing rudhira knockdown. (I) Gr(J, K) Graphs representing (J) fold change in Rudhira expression up(K) increased filopodia upon Rudhira over-expression, as comparedbar indicates mean±SD of a total of 18 cells (I), 40 cells (J) or 15 ccells transfected with pCAG EGFP (vector control) or pCAG Rudh2Astained with phalloidin (gray) (N, O) or anti-vinculin antibody (R,magnified view of filopodia seen in the boxed regions. Arrowheadadhesions. (A–H and L–S) Nuclei are marked by DAPI (blue). Scalechanges in actin dynamics at the membrane compared between ceRudhira with mCherry-Actin (lower panel).

Rudhira and Cdc42 re-localization to the leading edge (Fig. S7A–K). Immunostaining with antibodies directed against the respec-tive proteins revealed that Rudhira and Cdc42 are localized inspatially distinct regions at the leading edge of migratory cells(Fig. S7E and J). Further, a timed analysis showed that whileRudhira can be detected towards the leading edge 15 min postwounding (Fig. S7A–E), Cdc42 is not detected until 1 h at the lead-ing edge (Fig. S7F–J). In all cases Rudhira re-localization precededthat of Cdc42. This suggests that Rudhira may have a role in Cdc42activation and recruitment to the leading edge.

To check whether Rudhira mediates Cdc42 activation, we over-expressed Rudhira-FLAG in HEK293T cells and tested for Cdc42activation by biophysical and biochemical assays. FRET (Fluores-cence Resonance Energy Transfer) analysis of Cdc42 activation(Figs. 6A and B) showed that Rudhira over-expression significant-ly increased the FRET ratio (YFP/CFP) as compared to the vectorcontrol (Fig. 6B). Proteinase K treatment prior to the assayabolished the 527 nm peak (Fig. 6A) confirming the occurrenceof FRET from YFP to CFP in the absence of proteinase K. Weconfirmed these findings by imaging based FRET and found thatpre-bleaching, there is a significant increase in YFP emission inRudhira as compared to controls (Fig. 6C). Upon photobleachingYFP, we observed a significant increase in CFP emission in Rudhiraover-expressing cells whereas the CFP emission in control did notchange significantly (Fig. 6D, quantified in 6E). We also did a PAK-PBD ‘pull-down’ assay for a biochemical readout. Rudhira over-expression causes a dramatic increase in GTP-loaded Cdc42 ascompared to the control (Fig. 6F). This confirms Cdc42 activationupon Rudhira over-expression.

We further examined whether Rudhira is required for Cdc42recruitment at the leading edge and found that rudhira silencedcells show a dramatic reduction (~80%) in Cdc42 recruitment inthe migrating cells (Figs. 6G–J, quantified in 6K). This indicated arequirement for Rudhira in Cdc42 recruitment to the leading edge.Taken together, these results confirm that Rudhira is required forboth Cdc42 activation and recruitment.

Rudhira loss leads to defective Golgi positioning and inhibitsMTOC reorientation

MTOC and Golgi reorientation are the established hallmarks of apolarized cell (Etienne-Manneville, 2004). Rudhira re-localizationtowards the leading edge and its effect on actin organization

ages of SVECs transfected with control or rudhira siRNA,nd anti-Rudhira antibody (green). Cells near the woundmarginaged. Insets showmagnified view of filopodia in boxed regions.aph showing decreased filopodia upon rudhira silencing.on transfection with bicistronic pCAG Rudh2AGFP andto controls. *=p<0.0001 for (I, J) and p<0.005 for (K). Error

ells (K) from at least three independent experiments. (L–S) 3T3GFP (bicistronic Rudhira over-expression construct) wereS) (gray). GFP (green) marks the transfected cell. Inset showsin (S) indicates a GFP+cell showing reduction in focalbar: 100 μm. (T) Time lapse image series of a 3T3 cell showingll co-expressing vector with mcherry-actin (upper panel) and

Fig. 5 – Rudhira-induced actin re-organization is restored by RhoQL and Cdc42DN. Representative images of 3T3 cells transfectedwith pCAGEGFP+RhoQL (A–D), pCAGEGFP+Cdc42DN (E, F), Rudh2AGFP+RhoQL (G–J) or Rudh2AGFP+Cdc42DN (K, L) stainedwith Phalloidin (red) (B, F, H, L) or anti-vinculin antibody (red) (D, J). GFP marks the transfected cell. Arrow indicates stress fibers(H) or focal adhesions (J). Arrowhead points to cell lacking filopodia (L). Nuclei are marked by DAPI (blue). Scale bar: 100 μm.(M–O) Graphs representing restoration of stress fibers (M) and focal adhesions (N) upon co-transfection with RhoQL and rescue ofincreased filopodia phenotype upon co-transfection with Cdc42DN (O) as compared to Rudhira over-expression alone. *=p<0.01for (M, N) and p<0.001 for (O). Error bar indicates mean±SD of a total of 30 cells from three independent experiments.


strongly suggest that Rudhira is a mediator of cell polarity. So, wetested whether Rudhira can help in establishing cell polarity(Fig. 7). Rudhira depleted endothelial cells showed loss of MTOCreorientation (Figs. 7A–F and M) and a significant reduction inGolgi reorientation (Figs. 7G–L and N–O) towards the leadingedge. These findings show that Rudhira plays an important role inestablishing cell polarity preceding migration.

Discussion

We demonstrate that Rudhira is essential for endothelial cell tubeformation and migration. Rudhira expression can induce classicalmigratory phenotypes in stationary cells. It also interacts withmicrotubules and intermediate filaments. We show that Rudhiraactivates Cdc42 to affect actin organization. ThusRudhira interactionwith multiple components of the cytoskeleton could control cellpolarity and motility.

Depletion of rudhira from endothelial cells results in randomand retarded migration as seen in the Matrigel-induced tubeformation assay. Similar effects were reported for mutants orknockdown of several genes in the angiogenesis pathway[28,29]. This suggests that Rudhira contributes to angiogenesisby polarizing and inducing migration in endothelial cells. Thetransient expression of Rudhira during mouse angiogenesis inangiogenic precursors supports this view [6]. Rudhira may be

required only in the initial pathfinding/pioneer endothelial cells tomap the route for the rest of the angiogenic population. Howeverit must be noted that in human models of vascular developmentRudhira/BCAS3 expression is also seen in the mature endothelium[7]. This difference between human andmouse tissuemerits furtherinvestigation. It is interesting that over-expression of Rudhirainduces faster yet non random migration, suggesting a modulationof Rudhira functions by other factors.

The primary effect seen on Rudhira when stationary cells areinduced to migrate is the rapid re-localization of Rudhira towardsthe leading edge. This pattern of re-localization is similar to manyother cell polarity proteins like GSK3β, Par6 and Lkb1 [26,30–32].Cell polarization is primarily regulated by Cdc42 [25]. It involvesactivation and recruitment of Cdc42 at the leading edge andmany proteins are known to regulate this process [26,33]. Rudhiralocalizes towards the leading edge before Cdc42 suggesting that itcould regulate Cdc42 activation and recruitment to the leadingedge. However, the differential localization of Rudhira and Cdc42suggests that these events are mediated by other molecules.

Rudhira loss of function phenotypes are similar to those of otherregulators of cytoskeletal reorganization mediated by the RhoGTPase family of proteins [34–36]. Rudhira mediates both Cdc42activation and recruitment resulting in downstream signaling toeffect actin reorganization. Cdc42 is also responsible for establishingcell polarity, an essential process for directional cell migration [25].Rudhira depleted cells showed loss of cell orientation and random

image of Fig.�5

Fig. 6 – Rudhira mediates Cdc42 activation and recruitment. (A) FRET spectra on HEK293T lysates transfected with FLAG vector andRudh-FLAG construct. Emission readings were taken on a fluorescence spectrometer using excitation wavelength of 433 nm. Thelysate containing Rudhira over-expression construct was treated with proteinase K (denoted as Rudhira PK) to confirm thepresence of intramolecular FRET. (B) Quantification representing the FRET ratio as determined by ratio of YFP emission (527 nm)over CFP emission (475 nm). *=p<0.005. Error bar indicates mean±SD of a total of three independent experiments.(C) Quantification representing the normalized YFP emission (pre-bleach) compared across vector and Rudhira transfected cells*=p<0.005. Error bar indicates mean±SD of total of 34 cells analyzed. (D) Representative images showing CFP increase along withYFP decrease upon photobleaching compared across vector and Rudhira transfected cells. (E) Graph representing the normalizedCFP increase in Rudhira overexpression cells upon photobleaching as compared to controls. (F) Western blot analyses showingCdc42 activation upon Rudhira over-expression as compared to controls in a PAK-PBD assay. Immunoblots of total Cdc42 andRudhira over-expression on the whole lysate are shown. GAPDH levels serve as a loading control. Anti-FLAG antibody confirms thepresence of FLAG tag in the Rudhira over-expression construct. (G–J) Representative images of siRNA transfected cells in a woundhealing assay showing expression of Rudhira (green) and Cdc42 (gray). Nuclei are marked by DAPI (blue). Left wound margin isindicated by a white line. Arrowhead indicates Cdc42 recruitment at the leading edge. Cell showing rudhira depletion (I) and loss ofCdc42 (J) recruitment to the leading edge is marked by a dashed line. Scale bar: 100 μm. (K) Graphs showing reduction in Cdc42recruitment upon rudhira silencing as compared to controls. *: p<0.0001. Error bar indicates mean±SD of 136 cells from fiveindependent experiments.


image of Fig.�6

Fig. 7 – Rudhira regulates Golgi and MTOC reorientation. SVEC monolayers transfected with control siRNA (A–C and G–I) or rudhirasiRNA (D–F and J–L) were scratched, fixed and stained for Rudhira (green) along with γ-tubulin (red) (A–F) or GM130 (red) (G–L).Cell showing rudhira depletion (E, K) and loss of MTOC re-localization (D) or Golgi re-orientation (J) is marked by a dashed line.Arrows point to cell with normal re-orientation (A, G, J). Arrowheads point to abnormal re-orientation (D, J) in migrating cells.Nuclei are marked by DAPI (blue). Panels to the extreme right are merged images. Scale bar: 100 μm. (M, N) Graph showing effect ofrudhira depletion on MTOC (M) and Golgi reorientation (N). *: p<0.005, **: p<0.001 Error bar indicates mean±SD of 40 cells (M,N) from three independent experiments. (O) Schematic representation of direction of Golgi reorientation (green arrow) relative tothe wound margin (red line) in control and rudhira depleted cells.


movement in migration assays. Rudhira regulates both MTOC andGolgi reorientation, two established hallmarks of a polarized cell[25] demonstrating that it is an active player in establishing polarityfor directional cell migration. This is in agreement with previously

implied roles for Rudhira in angiogenesis and tumor metastasis,two processes where directed cell migration is essential.

Endothelial cell polarity regulated mainly by Cdc42, Rac1 andits downstream effectors also plays an essential role in the

image of Fig.�7


formation of vascular lumen [37,38] . Lumen formation is an es-sential step during vascular morphogenesis to establish bloodflow and proper functioning of the developing vascular network[39]. Rudhira can activate Cdc42 and hence establish endothelialcell polarity. Thus, our study provides new mechanistic insightsfor the process of lumen formation during vascular development.The in vivo significance of Rudhira phenotypes seen in cultureand its implications can now be tested in knockout mice andtumor metastasis models.

Rho GTPases are activated by guanine nucleotide exchangefactors (GEFs). Several Cdc42 specific GEFs are known [40,41], thatactivate Cdc42 at the leading edge. GEFs are characterized by a Dblhomology (DH) domain and a pleckstrin homology (PH) domain[42] or are homologous to DOCK proteins [43]. Rudhira does notbelong to either family of GEFs and it is a predicted WD40 domainprotein [6].WD40 domain containing proteins are known to interactwith multitude of proteins, so we expect that Cdc42 activation byRudhira would most likely occur by its interaction with a GEF or aGEF stimulator. These predictions are also based on the observationthat Rudhira does not co-localize with Cdc42 at any point of time.

The co-localization of Rudhira with bothmicrotubules and inter-mediate filaments suggests that Rudhira could mediate crosstalkbetween the twomajor cytoskeletal components. Several cytolinkerproteins like microtubule associated proteins (MAPs) [44,45], Inter-mediate filament associated proteins (IFAPs) [46] and molecularmotors [47,48] are known to cross-bridge the cytoskeletal elements.In addition, these proteins facilitate cell migration [49–56]. Sincemigration is a complex phenomenon that requires interplay of aplethora of molecules, detailed investigations of the potential roleof Rudhira as a cytolinker are required.

We propose a model for Rudhira function in directional cellmigration (Fig. 8). In stationary cells Rudhira co-localizes with cyto-skeletal elements such as microtubules and intermediate filaments

Fig. 8 – Proposed model for the role of Rudhira in directional cell mlines) and cytoskeletal components represented bymicrotubules (Mand actin (pink lines) at different stages of cell migration. Yellow l(A) Stationary adherent cell with reasonably uniform distributionActin is not represented here to avoid cluttering the figure. (B) In rand IF re-localize towards the wound margin (dashed line). This isedge (blue line). This induces actin reorganization and initiation oprotrusions. The MTOC and Golgi reorient (not shown). The cell is p(arrow).

(Fig. 8A). Upon wounding, Rudhira re-localizes towards the woundmargin along with re-orienting microtubules and intermediate fila-ments (Fig. 8B). Here Rudhira promotes activation and recruitmentof Cdc42 to the leading edge (Fig. 8B). Rudhira also polarizes thecell in the direction of the wound margin by causing the MTOCand Golgi to re-orient (not represented). The outcome is classic mi-gratory phenotypes in the actin cytoskeleton leading to directionalcell migration (Fig. 8C).

Cell migration is a primary event in several physiological andpathological processes likemorphogenesis, wound healing, immunesurveillance and tumor metastasis [57,58]. Mechanisms that controlthe interplay of the major cytoskeletal elements like actin, microtu-bules and intermediate filaments are not completely understood.Our data indicate that Rudhira is a mediator of these interactions.Rudhira is a potent inducer of cellmigration evenwhen exogenouslyexpressed in primarily non motile or slow moving cells. The gene islarge (over 750 kb inmouse and over 900 kb in human)with severalreported and predicted regulatory elements (www.ensembl.org;[9]). The requirement for multiple controls on expression is under-standable as Rudhira has been found to be mis-expressed in meta-static tumors. Our analysis provides insight into the potential roleof Rudhira in controlling cancer cell polarity and migration topromote metastasis. Thus, Rudhira could be a potential target forcontrol of vascular diseases and invasive tumors.

Conclusions

Rudhira is essential for directional migration of endothelial cells.Misexpression of Rudhira in non-endothelial cells promotesCdc42 activation thereby polarizing and mobilizing cells. HenceRudhira is a new component of the machinery by which endothe-lial cells and possibly tumor cells respond to migratory cues.

igration. Schematic showing localization of Rudhira (greenT) (red lines), intermediate filaments (IF) (orange dotted lines)ines represent co-localization of Rudhira with MT or IF.and co-localization of Rudhira with cytoskeletal components.esponse to a migratory stimulus such as a wound, Rudhira, MTfollowed by Cdc42 activation and recruitment to the leadingf cell polarization. (C) Actin reorganization results in filopodialolarized and initiates migration in the direction of the wound

http://www.ensembl.org

image of Fig.�8


Supplementary materials related to this article can be foundonline at doi:10.1016/j.yexcr.2012.01.016.

Acknowledgments

We thank Marc Kirschner and DSHB for antibodies, B.S. Suma forhelpwith confocalmicroscopy,M.R.S. Rao for use of live cell imagingmicroscope and Tejas Gupte for help with FRET experiments. Weare grateful to Nagaraj Balasubmaniam, Sivaraj Sivaramakrishnan,Elisabetta Dejana, Giorgio Scita and Ugo Cavallaro for discussions.This study was supported by a grant from the Department ofBiotechnology, Government of India, and intramural funds fromJNCASR.

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