synemin interacts with the lim domain protein zyxin and is essential for cell adhesion and migration

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Research Article

Synemin interacts with the LIM domain protein zyxin and isessential for cell adhesion and migration

Ning Suna, Ted W. Huiatta, Denise Paulinb, Zhenlin Lib, Richard M. Robsona,⁎

aMuscle Biology Group, Department of Biochemistry, Biophysics and Molecular Biology and of Animal Science, Iowa State University, Ames,3110 Molecular Biology Bldg, IA 50011-3260, USAbThe Université Pierre et Marie Curie-Paris 6, UMR7079 CNRS, 75005 Paris, France

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

⁎ Corresponding author. Fax: +1 515 294 0453E-mail address: [email protected] (R.MAbbreviations: IF(s), intermediate filament(

polyclonal antibody; mAb, monoclonal antibodactivation domain; FACS, Fluorescence activated

0014-4827/$ – see front matter © 2009 Elseviedoi:10.1016/j.yexcr.2009.10.015

A B S T R A C T

Article Chronology:

Received 24 July 2009Revised version received14 October 2009Accepted 16 October 2009Available online 21 October 2009

Synemin is a unique cytoplasmic intermediate filament protein for which there is limitedunderstanding of its exact cellular functions. The single human synemin gene encodes at least twosplice variants named α-synemin and β-synemin, with the larger α-synemin containing anadditional 312 amino acid insert within the C-terminal tail domain.We report herein that, by usingthe entire tail domain of the smaller β-synemin as the bait in a yeast two-hybrid screen of a humanskeletal muscle cDNA library, the LIM domain protein zyxin was identified as an interactionpartner for human synemin. The synemin binding site in human zyxin was subsequently mappedto the C-terminal three tandem LIM-domain repeats, whereas the binding site for zyxin within β-synemin is within the C-terminal 332 amino acid region (SNβTII) at the end of the long taildomain. Transient expression of SNβTII within mammalian cells markedly reduced zyxin proteinlevel, blocked localization of zyxin at focal adhesion sites and resulted in decreased cell adhesionand increased motility. Knockdown of synemin expression with siRNAs within mammalian cellsresulted in significantly compromised cell adhesion and cell motility. Our results suggest thatsynemin participates in focal adhesion dynamics and is essential for cell adhesion and migration.

© 2009 Elsevier Inc. All rights reserved.

Keywords:

Intermediate filamentSynemin

ZyxinCytoskeletonFocal adhesions

Introduction

Intermediate filaments (IFs) are ∼10 nm diameter, filamentouscytoskeletal polymers that provide crucial structural supportwithin mammalian cells [1,2]. In addition to the traditionalmechanical scaffolding role, recent studies have indicated thatIFs may have novel functions in many important cellular processessuch as cell adhesion/migration, signal transduction, targeting ofproteins and lipids and organization of subcellular organelles [3,4].The IFs, which are heterogeneous in nature, are comprised of cell-type-specific intermediate filament (IF) proteins [5-8]. More than

.. Robson).s); FA(s), focal adhesion(sy; SNT, synemin tail; SNβT,cell sorting

r Inc. All rights reserved.

67 IF genes encoding individual IF proteins have been identified inhumans, all of which comprise the IF protein superfamily [9].

Synemin is a very large, unique member of the IF proteinsuperfamily [10]. Themolecular structure of the syneminmoleculeis characteristic of an IF protein, with a central 312 amino acidconserved α-helical rod domain flanked by a very short, 10 aminoacid N-terminal head domain and a very long, ∼1000 amino acidC-terminal tail domain [11,12]. Although first discovered withinmuscle cells [13], synemin is also expressed in many non-musclecells and in some types of cancer cells. These include vertebrateerythrocytes [14,15], lens cells [16,17], normal and malignant

); GST, glutathione S-transferase; BSA, bovine serum albumin; pAb,β-synemin tail; SNαT, α-synemin tail; DB, DNA binding domain; AD,

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http://dx.doi.org/10.1016/j.yexcr.2009.10.015

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astrocytes [18-20], glia and neurons [21], hepatic stellate cells [22],human hepatoma cells [23], human malignant biliary epithelialcells [24] and cultured HeLa cells [25], which is a human cervixepithelial adenocarcinoma cell line. The wide expression profile ofsynemin, and particularly its expression in cancer cells, suggeststhat synemin has important, fundamental cellular functions.

Synemin is not able to self-assemble into homopolymericfilaments in vivo [11,18,26,27]. Association of synemin with themajor type III IF proteins, desmin and/or vimentin, via theirconserved α-helical rod domains, form heteropolymeric IFs withinmammalian cells [10]. Synemin also interacts with the non-IFproteins α-actinin [11,28], vinculin [28,29], talin [22,30], α-dystrobrevin [31], dystrophin/utrophin [32], plectin-1 [33] aswell as protein kinase A [34]. Thus, synemin appears capable oflinking the heteropolymeric synemin/desmin or synemin/vimen-tin IFs to several subcellular structures via interactions with non-IFprotein binding partners.

The single human synemin gene (gene name:Dmn) encodes twolarge splice variants named α-synemin (180 kDa by SDS–PAGE;172.7 kDa from sequence) and β-synemin (150 kDa by SDS–PAGE;140.1 kDa from sequence) [26]. The only difference betweenα- andβ-synemins is anadditional 312 aminoacid insert near the endof thelong C-terminal tail domain of α-synemin [26]. In previous studies,we have shown that the 312 amino acid insert within α-synemininteracts with human vinculin/metavinculin andwith talin, therebyconferring extra functions upon the larger α-synemin [29,30]. Incontrast, possible role(s) of the smallerβ-syneminwithin living cellshas remained unclear.

To identify other possible interacting proteins for synemin, weused the entire tail domain of human β-synemin (SNβT) as the baitin a yeast two-hybrid screen of a human skeletal muscle cDNAlibrary, and zyxin was identified as a binding partner of synemin.Zyxin is a LIM domain protein present primarily at sites of actin–membrane interactions [35-37]. Several lines of evidence suggestthat zyxin plays important roles in regulating cell adhesion andmigration, actin filament assembly and nucleus-cytoplasm commu-nications [36,38-42]. Our results suggest that, via direct interactionwith zyxin, synemin participates in actin cytoskeleton dynamicswithin living cells and is important for cell adhesion and migration.

Materials and methods

Generation of cDNA Constructs

The human α- and β-synemin cDNAs were provided by Dr. DenisePauin at the University of Paris and were described previously [26].For yeast two-hybrid assays, human synemin cDNA fragmentsencoding amino acid residues 321–579 (SNTIa), 580–920 (SNTIb),1153–1464 (SNTIII), 922–1253 (SNβTII), 922–1565 (SNαTII) andtheentire tail domain ofβ-synemin (SNβT, aminoacid residues 322–1253) (Fig. 2D)were amplified by PCR and introduced into the yeasttwo-hybrid bait vector pDEST32 (Invitrogen) by Gateway Recom-bination Technology using LR Clonase (Invitrogen). The humanzyxin cDNA fragment encoding amino acid residues 1–383 (Zyx 1-383) was PCR amplified from the Human 3-Frame Skeletal MusclecDNA Library (Invitrogen) using the primer pair 5′-GGGGA-CAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGCCCCCCG-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTTTAGAGTTCGTTGA-CAGCCACATTCTGCCTCTG-3′. The resulting cDNA fragment was

subsequently cloned into the yeast prey vector pDEST22 (Invitro-gen). The resulting plasmids were all sequenced in the DNASequencing and Synthesis Facility at Iowa State University andwere all confirmed to be in framewith theDNAbinding domain (DB,pDEST32 constructs) or the activation domain (AD, pDEST22constructs) of the Gal4 protein.

For expression of recombinant proteins, the cDNA fragmentsencoding SNTIa, SNTIb, SNTIII, SNβTII and SNβT were PCRamplified and subcloned into the pFLAG-ATS expression vector(Sigma) at the 5′ HindIII and 3′ BglII sites as FLAG-taggedexpression constructs. The cDNA fragments encoding full-lengthhuman zyxin (Zyx 1-572, amino acid residues 1–572), Zyx 1-383and the three zyxin LIM domains (Zyx 375-572, amino acidresidues 375–572) were PCR amplified from the Human 3-FrameSkeletal Muscle cDNA Library and were subcloned into the pGEX-4T2 GST expression vector (GE Healthcare) at 5′ BamHI and 3′ XhoIsites. Sequences of all the expression constructs were confirmed byautomated sequencing in the DNA Sequencing and SynthesisFacility at Iowa State University.

Yeast two-hybrid screening

Yeast two-hybrid screening was performed according to theProQuest Two-Hybrid System with Gateway Technology manual(Invitrogen). Briefly, 10 μg of both the pDEST32-SNβT plasmids andthe human 3-frame skeletal muscle cDNA library were co-trans-formed into the yeast strain MaV203 (Invitrogen), plated on to theSC-Leu-Trp-His plates containing 25 mM 3-aminotriazole (3-AT)and incubated for 72 h at 30 °C. Plates containing growing yeastcolonies were replica-cleaned and incubated for another 48 h at30 °C. Each of the resulting yeast colonies growing on the selectiveplateswas then streakedonto a freshSC-Leu-Trpplate and incubatedat 30 °C for 48 h. Plasmids were isolated from the positive yeastclones and sequenced using the sequencing primer pair for thepDEST22 vector. To confirm the positive interactions within theyeast two-hybrid system, retransformation assays were performed.The identified pDEST22 plasmids containing zyxin cDNA fragmentswere co-transformed with the pDEST32-SNβT plasmids intoMaV203 yeast cells. Five master plates were then created bystreaking the colonies onto SC-Leu-Trp plates along with the fiveyeast control strains (controls A–E) (Invitrogen) and the two self-activation control yeast strains (the “bait only” control containingDB-SNβT plus empty AD vectors and the “prey only” controlcontaining AD-Zyx 1-572 plus empty DB vectors). The master plateswere then patched ontoWhatman filter paper # 541 for X-gal assaysand were replica-plated onto plates of SC-Leu-Trp-Ura, SC-Leu-Trpcontaining 0.2% 5-fluoorotic acid (5-FA) and of SC-Leu-Trp-Hiscontaining 100 mM 3-AT for observation of growth phenotypes.

β-Galactosidase liquid culture assays

β-Galactosidase liquid culture assays using chlorophenol red-β-D-galactopyranoside (CPRG) (Roche Diagnostics) as the substratewere performed following instructions of the Clontech YeastProtocol Handbook [43]. The yeast control stains (controls A–E)used in the assays came from the ProQuest Two-Hybrid Systemwith Gateway Technology Kit (Invitrogen). These five controlsrepresented increasing strength of interactions in the β-Galacto-sidase liquid culture assays and served as the standards. Theexperiments were performed in triplicate and the calculated mean

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of β-galactosidase units±SD for each assay was plotted usingMicrosoft Excel.

Expression and purification of recombinant proteins

Recombinant proteins were all expressed in Escherichia coli BL21-Codon Plus (DE3) bacterial cells (Stratagene). FLAG-taggedsynemin fusion proteins were affinity purified using anti-FLAGM2-agarose affinity gel (Sigma). GST-tagged zyxin fusion proteinswere batch purified using Glutathione Agarose (Sigma).

Antibodies

The anti-synemin 2856 polyclonal antibody (pAb) and the anti-zyxin pAb B71 have been described previously [11,40]. Anti-vimentin monoclonal antibody (mAb) V9, horseradish peroxidase(HRP)-conjugated anti-FLAG M2 mAb, anti-α-tubulin mAb andanti-vinculin mAb hVIN-1 were purchased from Sigma. Anti-GFPpAb and HRP-conjugated anti-GST mAb were obtained from SantaCruz Biotechnology. Anti-human zyxin mAb, Alexafluor secondaryantibodies and Alexafluor-594 phalloidin were obtained fromInvitrogen. Anti-VASP mAb were obtained from BD Biosciences.

Co-immunoprecipitation (Co-IP), FLAGimmunoprecipitation and GST pull-down assays

For Co-IP assays, 1×107 HeLa cells were lysed with the MPERMammalian Protein Extraction Reagent (Pierce) and incubatedwith 5 μg anti-human zyxin mAb for 1 h at 4 °C. Fifty microliters of25% Protein A/G Plus Beads (Santa Cruz Biotechnology) was thenadded to the reaction and incubated for another 1 h at 4 °C. Thesame amount of HeLa cell lysates incubated with 5 μg anti-GAPDHmAb (Cell Signaling) plus 50 μl Protein A/G Plus Beads, or with50 μl beads alone, served as two negative controls. The reactionswere washed twice with 1× PBS and then eluted with 2× SDSsample buffer. The resulting eluates were subjected to SDS–PAGEand analyzed by Western blotting with anti-synemin 2856 pAband then reprobed with anti-human zyxin mAb.

FLAG immunoprecipitation assays were performed followinginstructions of the Anti-FLAG M2 Affinity Gel manual (Sigma).Briefly, 4 μg of each GST-tagged zyxin fusion proteinwas incubatedin 500 μl 1× PBS containing 4 μg purified FLAG-SNβT and 40 μl ofthe pre-cleaned anti-FLAGM2 beads for 3 h at 4 °C. The beads werethen centrifuged, washed twice with 1× PBS containing 0.1%Tween-20 (PBST) and eluted with 2× SDS sample buffer. Theresulting eluates were subjected to SDS–PAGE and analyzed byWestern blotting with HRP-conjugated anti-GST mAb and HRP-conjugated anti-FLAG M2 mAb.

GST pull-down assays were performed essentially as previouslydescribed [29].

Surface plasmon resonance

Surface plasmon resonance was performed using a Biacore T100(GE Healthcare) located in the Proteomics Facility at Iowa StateUniversity. Purified GST-Zyx 1-572 fusion protein was immobi-lized on the activated flowcell 2 of a CM5 sensor chip using the GSTCapture Kit (GE Healthcare) at an Rmax value of ∼100. GST aloneimmobilized on the activated flowcell 1 served as a reference inthe experiments. Increasing concentrations (from 250 to 3000 nM)

of purified FLAG-SNβT were then injected over the flowcells for anassociation phase of 180 sec at a flow rate of 10 μl/min andfollowed by a 100-s disassociation phase. The protein–proteininteractions were then detected in real time by the Biacore T100control software (GE Healthcare). The resulting sensorgrams, aswell as affinity of the interaction, were analyzed by the BiacoreT100 evaluation software (GE Healthcare) using the referencesubtracted (flowcell 2-1) data sets.

Cell culture, cell viability and proliferation assays

HeLa cells and NIH/3T3 cells were kindly provided by Dr. MaritNilsen-Hamilton and Dr. Janice E. Buss, respectively, at Iowa StateUniversity. A-10 cells, a rat vascular smooth muscle cell line, werepurchased from American Type Culture Collection (ATCC). Humanuterine smooth muscle cells (UTSMC) were obtained from Lonza.HeLa cells, NIH/3T3 cells and A-10 cells were maintained withDulbecco's modified Eagle's medium (DMEM) supplemented with10% fetal bovine serum (FBS) at 37 °C (95% air, 5% CO2). HumanUTSMC cells were maintained with Smooth Muscle Medium-2Bulletkit (Lonza) at 37 °C with 95% air and 5% CO2.

Cell viability was calculated with a Guava Viacount (GuavaTechnology) in the Cell and Hybridoma Facility at Iowa StateUniversity using trypan blue staining. Cell proliferation assayswere performed with the CyQUANT Cell Proliferation Assay Kit(Invitrogen) following the manufacturer's instructions.

Immunofluorescence, transfection studies and flowcytometry

Immunofluorescence staining of cultured cells was performedessentially as previously described [29]. For transfection studies,cells seeded on collagen-coated coverslips with optimal confluence(∼50% to ∼70%) were transfected with mammalian expressionconstructs using JetPEI TransfectionReagent (PolyplusTransfection).The cells were then immunostained with appropriate primary andAlexafluor secondary antibodies. Immunofluorescence microscopywas conducted using a LEICA DEMIRE2 inverted microscopeequipped with a CCD camera located in the Cell and HybridomaFacility at Iowa State University. Cells that expressed EGFP-SNβTIIand EGFP alonewere collected by fluorescence activated cell sorting(FACS) using a Beckman Coulter EPICS Altra Flow Cytometer in theCell and Hybridoma Facility at Iowa State University.

RNA interference and quantitative-PCR

The Hs_Dmn_4_HP siRNA (Syn-siRNA) (target sequence: CAC-GAGGGAGCAAGAAAGAAA) specific for both human α- and β-synemins and the Alexa Fluor 488 labeled negative control siRNA(NG siRNA) were obtained from Qiagen. HeLa cells cultured in 6-well plates (Corning COSTAR) were transfected with 5 nM of eachsiRNA using HiPerFect Transfection Reagent (Qiagen). Total RNAand cDNA of the siRNA-treated HeLa cells were then prepared 72 hpost-transfection using the RNeasy Mini Kit (Qiagen) and theQuantiTect Reverse Transcription Kit (Qiagen), respectively,following the manufacturer's instructions. Quantitative PCR tomeasure synemin mRNA levels was done with Hs_Dmn_1_SGQuantiTect Primer Assay (Qiagen) using a Stratagene Mx4000Multiplex Quantitative PCR System (Stratagene) in the DNASequencing and Synthesis Facility at Iowa State University. Protein


expression levels were analyzed by Western blotting usingappropriate antibodies 72 h post-transfection of the siRNAs.

Cell adhesion and migration assays

Cell adhesion assays were essentially performed as previouslydescribed [44] with modifications. Briefly, 1×105 cells wereseeded onto wells of a 12-well CellBIND Surface tissue culturemicroplate (Corning Life Sciences) for 1 h. Cells were then washedgently with 1× PBS for 5 times, detached from the plate with 0.25%Trypsin-EDTA and the numbers of cells were counted with GuavaViacount. The experiments were performed in triplicate. Theadhesion rate was calculated as the number of adherent cells/1×105. Data were plotted using Microsoft Excel. The 2-D woundhealing assay and the modified Boyden chamber cell migrationassay using non-coated transwell inserts (6.5 mm 24-well formatwith 8 μm pore size, Corning Life Sciences) were performedessentially as previously described [45]. For the modified Boydenchamber assay, 1×105 cells were seeded on the upper side of themembrane of the transwell inserts within serum-free DMEMmedia, while DMEM media containing 10% FBS was added at thebottom side. After incubation for 24–48 h at 37 °C, the cells werefixed with 2% formaldehyde in 1× PBS and stained with1.67 mmol/L CellTrace Calcein AM (Invitrogen). Cells on theupper side of the membranes were then removed by gentlyscraping with a wet cotton swab. The resulting membranes werethen cut out of the transwell inserts, mounted on glass slides withVectashield Mounting Medium (Vector Laboratories) and ob-served under the microscope. Numbers of cells from 5 differenthigh-power fields (HPF) (20× objective) on each membrane, from

ig. 1 – Zyxin was shown to interact with the β-synemin tailomain in the yeast two-hybrid screen of a human skeletaluscle cDNA library. (A) Schematic diagram showing domain

tructure of the human zyxin molecule as well as the zyxinegions from the nine positive clones resulting from the yeastwo-hybrid screening. Only 8 clones are listed because two ofhe nine positive clones were identical (AD-Zyx 322-572). Thelone containing the shortest zyxin region (AD-Zyx 375-572)ontains the C-terminal three tandem repeats of the LIMomains. (B) Results of the yeast two-hybrid retransformationssays. Each AD-Zyx construct was co-transformed again withB-SNβT into the MaV203 yeast strains and was tested with thehree reporter gene systems (LacZ, His3 and Ura3) of theroQuest yeast two-hybrid system. All of the co-transformantsontaining AD-Zyx construct plus DB-SNβT exhibitedhenotypes similar to the positive control, whereas the selfctivation controls of the bait only (DB-SNβT plus AD vector)nd the prey only (AD-Zyx 1-572 plus DB vector) showedegative phenotypes. (C) Co-immunoprecipitation of syneminith zyxin from HeLa cell lysates. 1 × 107 HeLa cell lysates werecubated with anti-human zyxin mAb immobilized on Protein/G Plus beads. The same amount of HeLa cell lysates incubatedith anti-GAPDHmAb immobilized on Protein A/G Plus Beads,r with the beads alone, served as two negative controls.recipitated synemin was detected with the anti-synemin 2856Ab. Syneminwas specifically precipitated alongwith zyxin butot precipitated by the immobilized anti-GAPDH mAb and therotein A/G Plus beads only, from HeLa cell lysates. Note that-synemin was highly expressed in the HeLa cells.

FdmsrttccdaDtPcpaanwinAwoPpnPα

triplicate experiments, were counted. Data (mean±SD) wereplotted with Microsoft Excel.

Results

Zyxin was identified as interacting with synemin by yeasttwo-hybrid screening using SNβT as the bait

The hypervariable N-terminal head and C-terminal tail domains ofIF proteins are often the regions mediating interactions with non-IF protein binding partners [4,46]. The large type VI IF proteinsynemin contains a very short, 10 amino acid head domain and avery long, ∼1000 amino acid tail domain. Thus, it is likely that thelong tail domain of synemin harbors most of the binding sites forany non-IF protein interaction partners. To identify novel proteinsinteracting with synemin, the entire tail domain (amino acidresidues 322–1253) of human β-synemin was used as the bait(DB-SNβT) in a yeast two-hybrid screen of a human skeletalmuscle cDNA library. A total of approximately 2×106 transfor-mants were screened and resulted in 15 positive colonies growingon SC-Leu-Trp-His plates containing 25 mM 3-aminotriazole. Nine


of these hosted prey plasmids containing cDNA fragmentsencoding various regions of human zyxin in frame with the Gal4activation domains. Importantly, these zyxin regions exactlyconstituted a series of N-terminal deletions of the full-lengthzyxin, with the smallest one (Zyx 375-572) representing the threetandem repeats of the LIM domains at the C-terminal end ofhuman zyxin (Fig. 1A).

To exclude the possibility that our results obtained from theyeast two-hybrid screening were false positives, the MaV203 yeastcells were co-transformed with each AD-Zyxin construct plus theDB-SNβT plasmid, and interactions were then tested using thethree reporter genes (LacZ, His3 and Ura3) of the ProQuest yeasttwo-hybrid system. The five control yeast strains (controls A–E,ProQuest yeast two-hybrid system), as well as the self-activationcontrols of the bait only (DB-SNβT plus AD vector) and the preyonly (AD-Zyx 1-572 plus DB vector) were also included. Similar tothe positive controls (controls B–D), the yeast strains containingthe several AD-Zyxin constructs plus DB-SNβT demonstratedstrong β-galactosidase activities, grew vigorously on the SC-Leu-Trp-Ura and SC-Leu-Trp-His + 100 mM 3-AT-selective plates andexhibited inhibited growth on SC-Leu-Trp-Ura plates containing0.2% 5-Fluoroorotic acid. In contrast, the negative control (DBvector plus AD vector) and the two self-activation controls (DB-SNβT plus AD vector and AD-Zyxin 1-572 plus DB vector) showedphenotypes contrary to the positive controls (Fig. 1B). Theseresults confirmed the interaction of SNβT with zyxin regionswithin the yeast two-hybrid system.

Interaction of synemin with zyxin was further illustrated byCO-IP assays using HeLa cell extracts. HeLa cells were selected forthe CO-IP assays because they express synemin [25] and arerelatively easy to culture. As shown in Fig. 1C, synemin wasspecifically precipitated along with zyxin from the HeLa celllysates by immobilized anti-human zyxin mAb. Lack of β-syneminin the lane of the CO-IP with the anti-human zyxin mAb may haveresulted from a very small amount of β-synemin precipitated andthereby not detected by Western blotting. In toto, this resultindicated an in vivo association of human synemin with zyxin.

Mapping the synemin binding site within human zyxin

To confirm the binding site of synemin in zyxin and to providequantitative estimates of the interactions, co-transformants of DB-SNβT plus each zyxin region in the AD vectors (AD-Zyx 1-572, AD-Zyx 82-572, AD-Zyx 197-572, AD-Zyx 375-572 and AD-Zyx 1-383)were analyzed for β-galactosidase activity with liquid cultureassays using CPRG. These AD-Zyxin constructs represented over-lapping regions that together include the full-length amino acidsequence of zyxin (Fig. 2A). The five control yeast strains (controlsA–E), with interaction strengths ranging from none (control A, DBvector plus AD vector) to very strong (control E, AD vector plus DB-full-length Gal4) were also included in the assay as standards toindicate relative interaction strengths. The co-transformant of DB-SNβT plus AD vector served as a negative control. As shown in Fig.2B, human zyxin regions Zyx 1-572, Zyx 82-572, Zyx 197-572 andZyx 375-572 all showed positive interactions with the SNβT incomparison to the positive, weak interaction of control B (DB-human RB+ AD-human E2F1). The zyxin region 1-383 did notshow any interaction with SNβT, demonstrating that the zyxin LIMdomains are required for the zyxin/synemin interaction. Inaddition, the interaction of the full-length zyxin (Zyx 1-572)

with SNβT displayed a relatively moderate interaction strength incomparison to control C (DB-Drosophila DP+AD-Drosophila E2F),which represented a moderate to strong interaction. Deleting theN-terminal 81 amino acids of zyxin (Zyx 82-572) resulted in aneven higher affinity for SNβT than that of the full-length zyxin (Zyx1-572). This may reflect an intramolecular interference caused bythe N-terminal 81 amino acids of zyxin in its interaction withhuman synemin. In comparison with the other zyxin regions, thezyxin LIM domains alone (Zyx 375-572) displayed a relativelyweak interaction with SNβT, indicating that the sequences inaddition to the LIM domains are required for the optimalinteraction with the SNβT.

Interactions of different zyxin regions with SNβT were alsoexamined in vitro by immunoprecipitation assays. The GST-taggedfull-length zyxin (GST-Zyx 1-572) and the zyxin LIM domains (GST-Zyx 375-572), but not theN-terminal 383 amino acids of zyxin (GST-Zyx 1-383), were specifically precipitated by FLAG-SNβT immobi-lized on anti-FLAGM2 resins (Fig. 2C). These results were consistentwith the results obtained from the yeast two-hybrid β-galactosidaseliquid culture assays (Fig. 2B) and further confirmed that the zyxinLIM domains contain the binding site for synemin.

Mapping the zyxin binding site within human synemin

To narrow the specific binding site for zyxin within humansynemin, SNβT was further divided into three consecutive regionsnamed SNTIa, SNTIb and SNβTII as diagrammed in Fig. 2D. The 312amino acid insert, which is present only within α-synemin(SNTIII), and the region including both SNβTII and SNTIII in theα-synemin tail (SNαTII) (Fig. 2D) were also included in themapping study to determinewhether they interact with zyxin. ThecDNAs encoding SNTIa, SNTIb, SNβTII, SNTIII and SNαTII wereintroduced into the yeast two-hybrid DB vectors and were co-transformed with AD-Zyx 82-572 plasmids into the MaV203 yeastcells. Interactions were examined using β-galactosidase liquidculture assays. Co-transformants of DB-SNβT plus AD-Zyx 82-572and of DB vector plus AD-Zyx 82-572 served as the positive controland the negative control, respectively. As shown in Fig. 2E, SNβTIIdisplayed a very strong interaction with zyxin as compared to thepositive control. SNTIa, SNTIb and SNTIII did not interact withzyxin. These results demonstrated that the binding site for zyxin insynemin is within the C-terminal 332 amino acids at the end of theβ-synemin tail domain. SNαTII showed a similar interactionstrength as the SNβT did (Fig. 2E), indicating that the presenceof the 312 amino acid insert within the α-synemin tail did notabolish the interaction. Thus, α-synemin also interacts with zyxin.

The interactions of synemin regions with zyxin were furthertested in vitro by GST pull-down assays. SNβTII was specificallyprecipitated by immobilized GST-Zyx 1-572, whereas SNTIa, SNTIband SNTIII were not (Fig. 2F). These results confirmed the dataobtained in the yeast two-hybridβ-galactosidase liquid culture assays.

Analysis of the interaction affinity

To provide a quantitative assessment of the interaction of SNβTwith zyxin, surface plasmon resonance (SPR) was conducted asdescribed in the Materials and methods section. The purified GST-tagged full-length zyxin (GST-Zyx 1-572) was used as the ligand inthe SPR analysis, whereas the purified recombinant FLAG-SNβTwas used as the analyte. Zyxin displayed positive interactions with

Fig. 3 – Biacore surface plasmon resonance analysis of the interaction between SNβT and zyxin. (A) Reference subtractedsensorgrams showing interaction of purified recombinant zyxin with SNβT. Purified GST-Zyx 1-572 was immobilized on a CM5sensor chip as the ligand. Increasing concentrations of FLAG-SNβT were injected as the analyte over the sensor chip with anassociation time of 180 s and a dissociation time of 100 s. From the bottom to the top sensorgrams represent 250, 500, 1000, 2000 and3000 nM, respectively. The red and black lines represent the real and curated sensorgrams, respectively. Note that the interactionreached saturation at high concentrations (2000 and 3000 nM) of FLAG-SNβT injected. RU denotes response units. (B) Affinity of theinteraction was calculated from results of two separate experiments. The Kd value for the interaction was estimated at 5.68×10−8

M. Concentrations are on the x axis and represent the concentration of FLAG-SNβT injected.


increasing concentrations of SNβT, as reflected by the sensorgrams(Fig. 3A). When the concentration of SNβT was high, theinteraction reached saturation. The Kd value of the interactionwas calculated as 5.68×10−8 M (Fig. 3B).

Effect of overexpression of SNβTII on zyxin localization

To examine the possible function of the interaction of syneminwith zyxin within a cellular context, HeLa cells were transfected

Fig. 2 –Mapping the binding sites within synemin and zyxin. (A–C) Sshowing the domain organization of the human zyxin molecule anZyx 1-383) used in the mapping studies. (B) Yeast two-hybrid β-gazyxin region in the AD vector with DB-SNβT. The five controls (conincluded as the standards of the interaction strength. The co-transfcontrol. The values οf β-galactosidase units represent the mean±Sof recombinant FLAG-SNβT with purified recombinant GST-taggedincubated in the reaction mixture containing purified FLAG-SNβT iBoth GST-tagged Zyx 1-572 and Zyx 375-572, but not GST-Zyx 1-383C-terminal 332 amino acid sequence (SNβTII) of the β-synemin taimolecules as well as each region used in the yeast two-hybrid β-gaβ-synemin (SNβT, a.a. residues 322–1253), the three sub-regions ofand SNTβII, a.a. residues 922–1253), the 312 amino acid insert presC-terminal region of α-synemin tail (SNαTII, a.a. residues 922–1565two-hybrid β-galactosidase liquid culture assays testing the interac82-572. The co-transformants of empty DB vector plus AD vector apositive controls, respectively. The values οf β-galactosidase unit rethat both SNTβII and SNTαII showed interaction with Zyx 82-572 inEach FLAG-tagged synemin fusion protein was incubated with puriprecipitated only FLAG-SNβTII but not FLAG-SNTIa, FLAG-SNTIb andnegative control.

with EGFP-tagged SNβTII plasmids. The subcellular locations ofEGFP-SNβTII and zyxin were then examined with epifluorescencemicroscopy. Cells with a moderate GFP expression level wereselected for analysis. In comparison with surrounding cells,transient expression of EGFP-SNβTII within the transfected HeLacells resulted in a markedly reduced zyxin localization at the FAsites (Figs. 4D–F). Expression of EGFP alone within the transfectedcells did not alter the localization pattern of the endogenous zyxin(Figs. 4A–C), indicating that zyxin localization to the focal

ynemin binds to the zyxin LIM domains. (A) Schematic diagramd regions (Zyx 1-572, Zyx 82-572, Zyx 197-572, Zyx 375-572 andlactosidase liquid culture assays testing the interaction of eachtrols A–E) from the ProQuest yeast two-hybrid system wereormant of empty DB vector plus AD vector served as a negativeD from triplicate experiments. (C) Immunoprecipitation assayszyxin regions. Each GST-tagged zyxin fusion protein wasmmobilized on pre-cleaned anti-FLAG M2 beads for 3 h at 4 °C., were precipitated by FLAG-SNβT. (D–F) Zyxin binds to thel domain. (D) Schematic showing human α- and β-syneminlactosidase liquid culture assays. The entire tail domain ofSNβT (SNTIa, a.a. residues 321–579; SNTIb, a.a. residues 580–920;ent only in α-synemin (SNTIII, a.a. residues 1153–1464) and the) were cloned into the yeast two-hybrid DB vectors. (E) Yeasttion of each synemin region in the DB vector with AD-Zyxnd of DB-SNβT plus AD-zyx 82-572 served as the negative andpresent the mean±SD from triplicates of the experiments. Notecomparison to the positive control. (F) GST pull-down assays.

fied GST-Zyx 1-572 in the GST pull-down assays. GST-Zyx 1-572FLAG-SNTIII. GST alone incubated with FLAG-SNβTII served as a

Fig. 5 – Transient expression of SNβTII led to a reduced zyxinprotein level within transfected HeLa cells. (A) Approximately10,000 cells were collected by flow cytometry, lysed andsubjected to SDS–PAGE and Western blotting. Wild-type HeLacells without transfection and EGFP-positive HeLa cells servedas the controls. Loss of zyxin, but not vinculin, was found inEGFP-SNβTII-positive HeLa cells. (B) RT-PCR showing similarmRNA levels of zyxin and vinculin in EGFP-SNβTII-positive,EGFP-positive and wild-type HeLa cells.


adhesions (FAs) was inhibited specifically by SNβTII. The loss ofzyxin at the FAs was not due to disruption of the normalarchitecture of the FAs themselves, as illustrated by the intactFAs labeled with vinculin within the EGFP-SNβTII transfected HeLacells (Figs. 4G–I), nor was it due to disruption of the normalarchitectures of other cytoskeletal components including vimentinIFs (Figs. 4M–O), actin cytoskeletons (Figs. 4P–R) and α-actinin(Supplementary Fig. 1). Loss of zyxin at FAs was specifically causedby transient expression of SNβTII only, but not by other regions ofthe synemin tail domain (SNTIa, SNTIb and SNTIII) (Supplemen-tary Fig. 1). These results in toto suggested that interaction ofSNβTII with zyxin was specific and inhibited the localization ofendogenous zyxin at the FA sites within the transfected cells.Previous studies have shown that positioning of vasodilator-activated phosphoprotein (VASP) at FA sites is dependent onappropriate zyxin localization [40,47,48]. We therefore examinedwhether loss of zyxin at FA sites by exogenous SNβTII would alsoblock VASP localization to the FAs. Indeed, the localization of VASPto the FAs was also markedly reduced within EGFP-SNβTII-expressing cells in comparison to surrounding cells (Figs. 4J–L),indicating that it was a result of loss of zyxin at the FA sites.

To examinewhether exogenous SNβTII would also lead to loss ofzyxin at the FA sites in othermammalian cellular contexts, A-10cells,a rat vascular smooth muscle cell line, and NIH/3T3 cells were alsotransfected with EGFP-SNβTII plasmids. Reduced zyxin localizationat the FA sites was also observed in the A-10 cells and NIH/3T3 cells(Supplementary Fig. 2) transfected with EGFP-SNβTII, suggesting ageneral mechanism underpinning this phenotype.

By using fluorescent activated cell sorting (FACS), EGFP-SNβTII-positive HeLa cells were collected, lysed and immediately

Fig. 4 – Transient expression of SNβTII in HeLa cells led to loss of zypAb in green and with each appropriate mAb or reagent to the corExpression of EGFP alone did not alter zyxin localization at the FAslocalization within the FAs at the edge of the transfected cells. Panexpressionwithin the transfected cells did not alter vinculin localizaVASP at the FA sites in EGFP-SNβTII transfected cells in comparisonpanel K. Asterisks in panels D–F and J–L indicate GFP-positive cellswas not disrupted by EGFP expression as indicated by labeling FAsphalloidin, respectively.

subjected to Western blot analysis. The results indicated that thezyxin, but not the vinculin, protein level within EGFP-SNβTII-expressing cells was markedly reduced (Fig. 5A), whereas thezyxin mRNA level was normal as indicated by RT-PCR whencompared with the zyxin mRNA level within wild-type HeLa cells(Fig. 5B). Thus, loss of zyxin at the FA sites resulted from a reducedzyxin protein level within the EGFP-SNβTII-positive cells.

Effect of overexpression of SNβTII on cell adhesion andmotility

Zyxin plays important roles in cell adhesion and cell motility.Knockdown of zyxin expression within mouse fibroblasts resultedin enhanced cell adhesion and increased motility [40]. Wetherefore examined whether overexpression of SNβTII, whichblocks zyxin localization to the FAs, will lead to similar pheno-types. FACS sorted EGFP-SNβTII-positive HeLa cells and EGFP-positive HeLa cells were used in the cell adhesion and migrationassays, in which the EGFP-positive HeLa cells served as controls.The EGFP-SNβTII-positive cells showed a similar viability (Fig. 6A)and proliferation ability (Fig. 6B) with the EGFP-positive cells afterseeded on a culture dish for 72 h after FACS sorting. In the celladhesion assays, the calculated adhesion rate of EGFP-SNβTII-positive cells was lower than that of the EGFP-positive cells andwas statistically significant (p<0.05) (Fig. 6C). To examine theeffect of overexpression of SNβTII in cell migration, 2-D woundhealing assays and modified Boyden chamber assays wereperformed using FACS sorted EGFP-SNβTII-positive HeLa cellsand EGFP-positive HeLa cells. In the 2-D wound healing assays,EGFP-SNβTII-positive cells closed the denuded area faster than theEGFP-positive cells (Fig. 6D). In the modified Boyden chamberassays, the EGFP-SNβTII-positive cells exhibited a statisticallysignificant (p<0.01) higher ability to migrate through the porousmembranes compared to the EGFP-positive cells (Fig. 6E). Theseresults indicated that overexpression of SNβTII decreased theadhesion but increased migration of the transfected HeLa cells.

Knockdown of synemin did not alter zyxin expression andlocalization within HeLa cells

To investigate whether loss of synemin would influence zyxinexpression and localization within cells, synemin-knockout mod-els were generated using siRNA specific to both human α- and β-synemins. Although it was previously shown that synemin isexpressed in cultured HeLa cells by mass spectrometry [25], wealso characterized and confirmed synemin expression in HeLa cellsby Western blotting (Fig. 7A), reverse transcriptase-PCR (Fig. 7B)and immunofluorescence microscopy (Fig. 8A). Western blottingshowed that, compared to the relatively equal and low amount of

xin at the FA sites. The cells were double labeled with anti-GFPresponding endogenous protein in red as indicated. (A–C). (D–F) Transient expression of EGFP-SNβTII blocked zyxinel F represents the boxed area in panel E. (G–I) EGFP-SNβTIItion at the FAs. (J–L) EGFP-SNβTII markedly reduced the level ofwith surrounding cells. Panel L represents the boxed area in

. The normal cytoskeletal architecture of the GFP-positive cells(G–I), IFs (M–O) and F-actin (P–R) with vinculin, vimentin and

Fig. 6 – Overexpression of SNβTII led to reduced cell adhesion and enhanced cell motility. (A) EGFP-SNβTII-positive HeLa cells hadsimilar viability with EGFP-positive HeLa cells after FACS collection. (B) Cell proliferation assays. Starting from 5000 cells seeded in96-well tissue culture dishes, EGFP-SNβTII-positive HeLa cells showed a similar proliferation rate with EGFP-positive HeLa cells within72 h of culture. (C) Cell adhesion assays comparing the adhesion rate of EGFP-SNβTII-positive HeLa cells and EGFP-positive HeLa cells.EGFP-SNβTII-positive cells exhibited a lower rate of adhesion to theCellBIND surfaces (⁎p=0.037). (D) Representative images ofwoundhealing assays showing that EGFP-SNβTII-positive HeLa cells almost closed the “wound” 24 h after wounding, whereas EGFP-positiveHeLa cells did not. (E) Modified Boyden chamber assays comparing the migration ability of EGFP-SNβTII-positive HeLa cells andEGFP-positive HeLa cells. FACS collected 1×105 cells were seeded on the upper side of the membrane and were allowed to migratedownward through the 8-μm pores for 24 h at 37 °C. EGFP-SNβTII-positive cells exhibited statistically significant higher ability oftraversing the membrane than did the EGFP-positive cells (⁎p=0.004). HPF, high-power field.


α- and β-synemins in both A-10 and UTSMC cells, α-synemin washighly expressed in HeLa cells and β-synemin expression was verylow (Fig. 7A). Reverse transcriptase-PCR using a specific primerpair that amplifies an ∼1 kb fragment from β-synemin mRNA andan ∼2-kb fragment from α-synemin mRNA, also showed that α-synemin was highly expressed in the HeLa cells (Fig. 7B). Syneminprimarily co-localized with vimentin IFs within the HeLa cells asindicated by double immunofluorescence labeling of the syneminand vimentin (Fig. 8A). Thus, these results in toto confirmed thesynemin expression within the cultured HeLa cells.

Transfection of the HeLa cells with the Hs_Dmn_4_HP siRNA(the siRNA specific for both human synemin isoforms, designatedas Syn-siRNA from here on) for 72 h (Syn-siRNA-HeLa cells)resulted in a significant decrease in both the synemin mRNA level(Fig. 7C) and protein level (Fig. 7D), with no obvious change inprotein levels of either zyxin or vinculin (Fig. 7D). Immunofluo-rescence staining of synemin within Syn-siRNA-HeLa cells indi-cated that synemin was absent in >98% of the cells (Fig. 8B).

Double immunofluorescence labeling of endogenous synemin andvimentin, or of synemin and zyxin, within Syn-siRNA-HeLa cellsshowed that both vimentin filaments and zyxin localizationremained intact (Fig. 8B). Other cytoskeletal architectures, suchas microfilaments and microtubules, were also normal (data notshown). These results indicated that zyxin expression andlocalization is not affected when synemin is absent within cells.

Synemin is essential for cell adhesion and migration

Intermediate filaments, such as vimentin IFs [3] and keratinfilaments [49,50], have been shown to play essential roles in celladhesion and migration. For example, Kleeberger et al. [45]recently reported that nestin, another large mammalian type VIIF protein that shares overall general molecular structure withsynemin, is important for the migration and metastasis of prostatecancer cells. We also examined whether synemin itself may play arole in cell adhesion and migration.

Fig. 7 – RNAi inhibition of synemin synthesis did not alter theprotein level of zyxin within HeLa cells. (A, B) Characterizationof synemin expression within HeLa cells. (A) Western blottingshowing that α-synemin is highly expressed in HeLa cells,whereas both α- and β-synemin are expressed at a relativelysimilar level in A-10 and UTSMC cells. (B) RT-PCR showing thatα-synemin mRNA is highly expressed within HeLa cells. (C, D)Knockdown of synemin expression within HeLa cells usingsiRNA. (C) Quantitative PCR use analysis of the synemin mRNAlevel within HeLa cells transfected with siRNAs. The syneminmRNA level was reduced by ∼83% with HS_Dmn_4_HP siRNA(Syn-siRNA) within HeLa cells 72 h post-transfection. (D)Western blotting showing synemin (Syn) knockdown withinHeLa cells treated with Syn-siRNA for 72 h. Wild-type HeLa cells(Wt), HeLa cells treated with transfection reagent only (mock)and HeLa cells transfected with Alexa Fluor 488 labelednegative control siRNA (NG siRNA) served as controls.Expression levels of α-tubulin (α-Tub) served as a householdprotein standard. Knockdown of synemin within HeLa cells didnot change the protein levels of either vinculin (Vin) or zyxin(Zyx).


In the cell adhesion assays, the calculated adhesion rate for theSyn-siRNA-HeLa cells was 29.9%, while the HeLa cells transfectedwith the fluorescent-negative control siRNA (Fl siRNA-HeLa cells)was 54.8% (Fig. 9A). Statistical analysis indicated that the Syn-siRNA-HeLa cells exhibited a significantly compromised ability toadhere to the CellBIND surface tissue culture microplates(p<0.05). The decreased adhesion rate of the Syn-siRNA-HeLacells was not a result of a decreased number of viable cells becausethe viability of the Syn-siRNA-HeLa cells was similar to the wild-type HeLa cells as measured by trypan blue staining using GuavaViacount (data not shown).

To examine the function of synemin in cell migration, 2-Dwound healing assays were performed using Syn-siRNA-HeLa cellsand the Alexa Fluor 488 labeled negative control siRNA (NG siRNA)-

treated HeLa cells (NG-siRNA-HeLa cells). Syn-siRNA-HeLa cellsexhibited a markedly compromised ability in closing the “wound”,whereas NG-siRNA-HeLa cells recovered most of the denuded areaof the plate 48 h after wounding (Fig. 9B). In the modified Boydenchamber assays, the NG-siRNA-HeLa cells exhibited a robust abilityto migrate through the porous membranes, whereas migration ofthe Syn-siRNA-HeLa cells through the membranes was markedlyinhibited (Figs. 9C and D). These results indicated that syneminpromoted the adhesion and migration of the HeLa cells.

Discussion

We have discovered that zyxin is an interaction partner for thelarge type VI IF protein synemin. Using the entire tail domain ofhuman β-synemin in yeast two-hybrid screening of the humanadult skeletal muscle total cDNA library yielded 15 positive clones.Nine of these 15 positive clones contained cDNA sequencesencoding the full-length, or truncated, human zyxin (Fig. 1A).Subsequent yeast two-hybrid retransformation assays (Fig. 1B), invivo co-immunoprecipitation assay (Fig. 1C) and in vitro protein–protein interaction assays (Figs. 2 and 3) indicated that theinteraction of synemin with zyxin is specific and did not resultfrom false positives in the yeast two-hybrid systems.

The binding site for zyxinwithin human β-syneminwasmappedto the C-terminal 332 amino acid sequence at the end of the taildomain (SNβTII, amino acid residues 922–1253) (Figs. 2E and F).These results represent the first evidence that β-synemin contains abinding site for a protein that localizes at actin–membraneinteractions and modulates cell–cell and cell–extracellular matrixadhesion. Our results herein also indicate that human α-synemin,which contains the332amino acid sequence (SNβTII), interactswithzyxin as well (Fig. 2E). We have previously shown that the 312amino acid insert present only within α-synemin (SNTIII) confersadditional functions upon α-synemin by interactions with vinculinand talin, both of which are cytoskeletal proteins present at actin–membrane interactions [29,51]. Notably, SNTIII itself did not bind tozyxin (Figs. 2E and F). However, its presence interfered with theinteraction of the SNβTII region with and thereby reduced theaffinity of, α-synemin for zyxin (Fig. 2E). Except for the 312 aminoacid additional insert present within its tail domain, α-synemin isidentical to and appears to share all of the potential functions of, thesmaller β-synemin. However, it is possible that differing ratios ofα-synemin to β-synemin modulate interactions with their specificproteinpartners and thereby fulfill specific functionswithindifferentcellular contexts. The latter is in concert with the fact that both α-and β-synemins are differentially expressed in the three types(skeletal, cardiac and smooth) of muscle cells [26,27] and that wehave shown in this study that HeLa cells, a human cervix epithelialadenocarcinomacell line, express a very high level ofα-synemin, buta very small amount of β-synemin (Figs. 7A and B).

Transient expression of SNβTII within mammalian cells led to amarkedly reduced zyxin protein level and the loss of zyxin at theFA sites, without disruption of other cellular architectures (Fig. 4,Supplementary Figs. 1 and 2). This phenotype was specific forSNβTII only, but not for other regions within the synemin taildomain (Supplementary Fig. 2), again indicating the specificityof the interaction. In addition, this may also reflect that, incells expressing exogenous SNβTII, zyxin was trapped withinthe cytoplasm by interaction with SNβTII and the resulting

Fig. 8 – Zyxin localizationwas not alteredwithin Syn-siRNA-treated HeLa cells. (A) Double labeling of synemin (green) and vimentin(red) of wild-type HeLa cells with anti-synemin 2856 pAb and anti-vimentin mAb V9. Synemin co-localized with vimentin infilamentous structures within wild-type HeLa cells. (B) Knockdown of synemin did not change the filamentous organization ofvimentin and the localization patterns of zyxin within Syn-siRNA-treated HeLa cells.


mislocalization of zyxin would subsequently lead to its degrada-tion. This interaction could also be transient within the cells andtherefore no obvious co-localizations of zyxin and synemin can beobserved by immunofluorescence. Zyxin has been shown able totravel between the nucleus and the FAs within cells and maytherefore mediate communications between these two subcellularcompartments [38,39]. Whether synemin and synemin-containingIFs are involved in regulating the translocation of zyxin betweenthe nucleus and the FAs remains to be elucidated.

Overexpression of SNβTII within HeLa cells also led to areduced cell adhesion and increased cell migration ability (Fig. 6).This phenotype of increased cell motility resembles the phenotypeof zyxin ablated mouse fibroblast cells shown by Hoffman et al.[40]. However, overexpression of SNβTII led to reduced celladhesion, which is contrary to the enhanced cell adhesion ofzyxin-null fibroblasts. It is possible that overexpression of SNβTIIdestabilizes the FAs to some extent, thereby resulting in reducedcell adhesion. In addition to zyxin, overexpression of SNβTII mayalso have effects on other potential interaction partners that

regulate cell adhesion/migration and lead to current phenotype. Intoto, these results imply an important and novel function of thelong synemin tail in regulating cell adhesion and cell motility.

The binding site for synemin within zyxin was mapped to the C-terminal region containing three tandem repeats of the LIMdomains(Figs. 2B and C). The LIM domains are modular protein-bindinginterfaces mediating protein–protein interactions [37,52,53]. And ithas been reported that the zyxin LIM domains contain binding sitesfor several other proteins, including cysteine-rich protein-1 (CRP-1)[52] and p130Cas [54]. Zyxin has also been implicated in serving asan adapter protein that recruits different partners to appropriatesubcellular compartments and promotes their activity [37]. Thezyxin LIM domains in particular appear able to direct zyxin and Ena/VASP family proteins to FAs [38]. Thus, synemin may be recruitedsimultaneously with Ena/VASP family proteins, CRP-1 and p130Casby the zyxin molecules to the FAs and thereby participate in actinremodeling and FA dynamics within cells.

We also report herein by the RNAi studies the functions forsynemin in promoting cell adhesion and cell motility (Fig. 9).

Fig. 9 – Synemin promotes cell adhesion and cellmotility. (A) Cell adhesion assays comparing the adhesion rate of the Syn-siRNA andNG siRNA transfected HeLa cells. Knockdown of synemin expression within HeLa cells resulted in compromised adhesion to theculture plates (⁎p=0.024). (B) Syn-siRNA transfected HeLa cells exhibited markedly compromised healing ability in the woundhealing assays 48 h after wounding. Representative photos from three independent experiments are shown. Bar, 100 μm. (C)Modified Boyden chamber cell migration assays comparing the relative abilities of the Syn-siRNA and NG siRNA transfected HeLacells in traversing the porous membrane. Many fewer Syn-siRNA transfected cells traversed the membrane after 48 h of migrationwhen compared with the NG siRNA transfected HeLa cells. Bar, 100 μm. (D) Quantitation of the number of cells that traversed theporous membranes in the modified Boyden chamber assays. HPF, high-power field.


Indeed, Pan et al. [55] reported in a recent study that synemincontributes to the migratory properties of astrocytoma cells byinfluencing the dynamics of the actin cytoskeleton. These func-tions are also partially reflected by the evidence that synemin isable to interact with several proteins present in adhesion sites,including vinculin, talin and zyxin. Both vinculin and talin havebeen shown to regulate cell adhesion and migration [56-58]. Andzyxin has also been shown to be important in regulating actinfilament assembly, with loss of zyxin within mice fibroblastsresulting in increased cell motility [40]. Differing ratios of α- andβ-synemin may also modulate interactions with zyxin, vinculinand talin and thereby help regulate cell adhesion and migration.Thus, a very high amount of α-synemin expressed within HeLacells could be beneficial for the invasiveness of these cancer cells.Indeed, another mammalian type VI IF protein, nestin, has beenshown to exhibit similar functions in enhancing prostate cancercell migration and metastasis [45].

The identification of the interaction of synemin isoforms withzyxin increases our understanding of the cellular functions ofsynemin. Synemin may link the synemin-containing heteropoly-

meric IFs to FA dynamics via its interactions with zyxin, vinculinand talin. As a result, synemin, like nestin, may play importantroles in cell adhesion and migration and possibly be involved incancer cell metastasis.

Acknowledgments

We thank Drs. Mary Beckerle and Laura Hoffman at the HuntsmanCancer Institute, University of Utah, for kindly providing us theanti-zyxin B71 pAb and for their critical reading of this manuscript.

This project was supported by National Research InitiativeCompetitive Grant no. 2003-35206-12823 from the USDA Coop-erative State Research, Education, and Extension Service. Thisjournal paper of the Iowa Agriculture and Home EconomicsExperiment Station, Ames, Iowa, Project No. 6616, was supportedby Hatch Act and State of Iowa funds. The costs of publication ofthis article were defrayed in part by the payment of page charges.The article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.


Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.yexcr.2009.10.015.

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synemin interacts with the lim domain protein zyxin and is essential for cell adhesion and migration

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