INTRODUCTION

Proteins of the α-actinin superfamily crosslink actin filamentsinto tight bundles or meshworks of looser arrangement, andalso connect them to the plasma membrane (Matsudaira, 1991;Matsudaira, 1994; Puius et al., 1998). The common feature ofthese proteins is the presence of the N-terminal actin-bindingdomain (ABD), a protein module of about 250 amino acids;otherwise they have diverse organizations (Puius et al., 1998).The ABDs of the α-actinin-related proteins are typicallyfollowed by rod domains, which are composed of a number ofrepeats that often mediate the protein dimerization, as well asby diverse C-terminal structural motifs. The rod domainrepeats can have an immunoglobulin-like fold, as in the caseof the filamin family (Fucini et al., 1997; McCoy et al., 1999),or double helical coiled coil motifs, as in case of cortexillins(Faix et al., 1996). The spectrin family (α-actinin, β-spectrin,dystrophin, utrophin) is characterized by rod domainscomposed of triple-helical coiled coil repeats (Pascual et al.,1997). Dystrophin and utrophin do not assemble as dimers,despite the structural similarity of their rod domain repeatswith the those of β-spectrin. They also do not crosslink actinfilaments (Winder et al., 1995b), but rather connect them tomembrane-associated proteins, providing a link between theactin cytoskeleton and the extracellular matrix via thetransmembrane dystroglycan complex (Matsumura et al.,1992; Tinsley et al., 1994; James et al., 1996). Likewise,dimerization and actin crosslinking activity has not been shownfor plectin, dystonin and MACF (ACF7, trabeculin) andkakapo. The Drosophilaprotein, kakapo (Gregory and Brown,

1998; Prokop et al., 1998; Strumpf and Volk, 1998), and itsmammalian homologue, MACF (Karakesisoglou et al., 2000;Leung et al., 1999), have been identified as linkers betweenintegrins, actin filaments and microtubules. Plectin and dystonin,the most versatile cytolinkers, connect the microfilament systemto intermediate filaments and microtubules (Fuchs and Yang,1999; Leung et al., 1999; Wiche, 1998; Yang et al., 1999). Asan exception, the proteins of the fimbrin (plastin) family, whichlack rod domains, possess two ABDs, and therefore do notrequire dimerization to crosslink actin filaments into tightbundles (Matsudaira, 1994).

Actin binding is the most conserved property of the proteinsof the α-actinin superfamily. The actin-binding region of α-actinin, which became a prototype of the ABDs of the othermembers, consists of two subdomains (calponin homology(CH) domains) in tandem, which have probably arisen by geneduplication of a single copy (Castresana and Saraste, 1995;Matsudaira, 1991). A single CH domain is found in, amongothers, calponin, Vav, IQGAPs and SM22-α. According tophylogenetic analysis the CH domains of different proteinswere divided into three major groups (Banuelos et al., 1998;Stradal et al., 1998). The first two groups include N-terminal(CH1) and C-terminal (CH2) CH domains of the α-actininrelated proteins. This indicates that the N-terminal CH domainsof proteins with an ABD are more similar to each other thanto the C-terminal CH domains of the same proteins. The thirdgroup is formed by the CH domains of the proteins that containsingle CH domains.

The three-dimensional structures of the CH2 domain ofhuman β-spectrin (Djinovic-Carugo et al., 1999), and of

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We have identified and cloned a novel 42-kDa proteintermed α-parvin, which has a single α-actinin-like actin-binding domain. Unlike other members of the α-actininsuperfamily, which are large multidomain proteins, α-parvin lacks a rod domain or any other C-terminalstructural modules and therefore represents the smallestknown protein of the superfamily. We demonstrate thatmouseα-parvin is widely expressed as two mRNA speciesgenerated by alternative use of two polyadenylation signals.We analyzed the actin-binding properties of mouse α-parvin and determined the Kd with muscle F-actin to be8.4±2.1 µM. The GFP-tagged α-parvin co-localizes withactin filaments at membrane ruffles, focal contacts andtensin-rich fibers in the central area of fibroblasts. Domain

analysis identifies the second calponin homology domain ofparvin as a module sufficient for targeting the focalcontacts. In man and mouse, a closely related paralogue β-parvin and a more distant relative γ-parvin have also beenidentified and cloned. The availability of the genomicsequences of different organisms enabled us to recognizeclosely related parvin-like proteins in flies and worms, butnot in yeast and Dictyostelium. Phylogenetic analysis of α-parvin and its para- and orthologues suggests, that theparvins represent a new family of α-actinin-relatedproteins that mediate cell-matrix adhesion.

Key words: Actin cytoskeleton, Calponin homology, Focal contacts,Cell adhesion, Parvin

SUMMARY

Parvin, a 42 kDa focal adhesion protein, related to theα-actinin superfamilyThorsten M. Olski, Angelika A. Noegel* and Elena KorenbaumInstitute for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany*Author for correspondence (e-mail: noegel@uni-koeln.de)

Accepted 27 November 2000Journal of Cell Science 114, 525-538 © The Company of Biologists Ltd

RESEARCH ARTICLE

526

fimbrin (Goldsmith et al., 1997), utrophin (Moores et al.,2000) and dystrophin ABDs (Norwood et al., 2000) have beensolved. Although the primary structure of the proteins variessignificantly, the crystal structures revealed that the core CHdomain structure is preserved in all molecules, with three α-helices forming a triple helical bundle and the fourth one lyingperpendicular to them (Norwood et al., 2000). Despite thestructural similarity, CH domains of different groups seem tohave distinct functions. The N-terminal CH domain (CH1)alone is able to bind actin, although with lower affinity thanthe entire ABD (Way et al., 1992; Winder et al., 1995a). TheC-terminal CH (CH2) domain alone has week affinity for actin,but may contribute to stabilizing the overall binding of thecomplete ABD (Banuelos et al., 1998; Way et al., 1992;Winder et al., 1995a). Since the single CH domain of calponinfails to bind F-actin in in vitro sedimentation assays and failsto target stress fibers or membrane ruffles of transfectedfibroblasts (Gimona and Mital, 1998), the function of suchdomains remains unclear. Thus, structurally equivalent singleand tandem CH domains seem to have different properties withrespect to interaction with filamentous actin. Therefore, notonly the duplication, but also the alteration of amino acidcomposition of CH domains, results in a distinct modulecapable of interacting with filamentous actin.

In addition to interacting with F-actin, ABDs can haveadditional functions like binding to G-actin (Andrä et al.,1998), PIP2 (Fukami et al., 1996), the cytoplasmic domain ofhemidesmosomal integrin β4 subunit (Geerts et al., 1999),vimentin (Correia et al., 1999) and extracellular regulatedkinase (ERK). Thus, the ABD, originally considered as anactin-binding unit, may harbor multiple binding sites thatsupport versatile interactions.

Being the components of the subplasmalemmalcytoskeleton, many proteins of the α-actinin superfamilyprovide physical linkage of actin filaments to the integral andperipheral proteins of cell membranes (Puius et al., 1998).Several proteins like α-actinin (Blanchard et al., 1989), plastins(Arpin et al., 1994; Bretscher and Weber, 1980), filamin(ABP280) (Pavalko et al., 1989), plectin (Seifert et al., 1992),dystrophin and utrophin (Belkin and Burridge, 1995; Kramarcyand Sealock, 1990) have been identified at actin-rich focalcontacts, specific cytoskeleton-plasma membrane molecularcomplexes, recruited to the clustered integrins upon interactionwith extracellular matrix (ECM) (Bershadsky and Geiger,1999; Burridge and Chrzanowska-Wodnicka, 1996).Localization of these proteins to focal adhesions was shown tobe mediated via direct interaction of α-actinin and filaminswith β-integrins (Loo et al., 1998; Otey et al., 1990; Sampathet al., 1998), and dystrophin and utrophin with dystroglycan(Belkin and Smalheiser, 1996).

Considering the importance of proteins of the α-actininsuperfamily in organizing the subplasmalemmal cytoskeleton,we have screened the expressed sequenced tag (EST) databasesearching for new proteins with ABD domains. We report thecloning and characterization of α-parvin as an actin-bindingprotein localized to cell-matrix adhesions via its C-terminal CHdomain. Together with the closely related paralogue β-parvinand the more distant γ-parvin, identified in human and mice,as well as parvin-like proteins found in Caenorhabditis elegansand Drosophila melanogaster, α-parvin forms a novel familyof small α-actinin related proteins.

MATERIALS AND METHODS

Cloning of parvinsFor α-parvin, two overlapping EST-clones (GenBank AccessionNumbers AA691868 and AA791745) were identified by BLASTsearch (http://www.ncbi.nlm.nih.gov/BLAST/) as encoding a novelprotein homologous to the actin-binding domain of α-actinin. TheEST clones were obtained from the Resource Center of the GermanHuman Genome and sequenced. The 5′-end was extended by rapidamplification of cDNA ends (RACE) PCR with adaptor-ligated mousebrain and skeletal muscle Marathon-Ready cDNA (Clontech,Heidelberg, Germany) as a template, according to the manufacturer’sprotocols. The whole sequence was verified by RT-PCR ofoverlapping fragments (Fig. 1). The PCR products were cloned intopGEM-TEasy vector (Promega, Madison, WI) or pCR2.1-vector(Invitrogen, Carlsbad, CA) for sequencing. The cDNA sequence of α-parvin has been deposited in GenBank (Accession NumberAF237774).

EST clones for the human α-, β- and γ-parvin as well as for themouse β- and γ-parvin were identified by NCBI Xblastn program(Altschul et al., 1997) using the mouse α-parvin peptide sequence.The obtained EST sequences were used for contig assembly. ThecDNAs of human and mouse parvin were cloned by reversetranscribed (RT)-PCR, with total RNA prepared by phenol/chloroform extraction (Sambrook et al., 1989) as a template. RNAfrom human embryonic kidney (293) cell line was used for amplifyinghuman α- and β-parvins, RNA from the human T-cell line (Jurkat) forhuman γ-parvin, and from mouse heart for mouse β-parvin. Mouseembryonic cDNA (Clontech) served as template for cloning mouse γ-parvin. (The cDNA sequences have been deposited in GenBank underthe Accession Numbers AF237771 for human α-parvin, AF237769for human β-parvin, AF237772 for human γ-parvin, AF237770 formouse β-parvin and AF312712 for mouse γ-parvin.) Reversetranscription of total RNA was carried out using the M-MLV-reversetranscriptase (Promega). The resultant cDNA was amplified with HighFidelity Expand Taq-polymerase (Boehringer, Mannheim, Germany).DNA sequencing was performed with the dideoxy nucleotidetermination method using a DNA sequencer (ABIprism 377 DNAsequencer, Perkin-Elmer, Norwalk, CT) at the service facilities of theCenter for Molecular Medicine Cologne.

Northern blot and RT-PCR analysisFor the analysis of the mRNA transcription pattern, multiple tissuenorthern blots (Clontech) from mouse embryo and adult mice werehybridized with cDNA probes 1 or 2 (Fig. 1), according to themanufacturers protocols. Hybridization with a β-actin cDNA wasused as a control. Probes were 32P-labeled by random priming usingthe Prime-It kit (Stratagene, La Jolla, CA) according to theinstructions of the manufacturer. Trancription of α-parvin in mousetestis and myoblasts was analyzed by reverse transcriptase (RT)-PCRanalysis. Mouse testis Marathon cDNA (Clontech) was used as atemplate to amplify the fragments marked as probes 3 and 4 (Fig. 1).Total RNA isolated from C2F3 myoblasts on differentiation days 0,2, 4 and 6 was reverse transcribed and used as a template to amplifythe probe 3.

Plasmid construction and cell transfectionThroughout the plasmid preparations, standard molecular biologicalmethods (Sambrook et al., 1989) were applied. For bacterialexpression of recombinant α-parvin, cDNA was amplified usingprimers with add-on sequences for a SphI and a PstI restriction site,which allowed cloning into the pQE-30 vector (Qiagen, Hilden,Germany). Plasmids encoding α-parvin or its deletion derivativesfused to the C or N terminus of EGFP were generated by amplifyingthe full-length or truncated parvin by PCR, using forward and reverseprimers with extensions for restriction sites for KpnI and SmaI,respectively. The amplified products were cloned into KpnI/SmaI-cut

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527Parvin: actin-binding protein at focal contacts

pEGFP-N3 and pEGFP-C1 vectors (Clontech). The DNA fragmentsencoding parvin fused to GFP were then excised from the vector asEcoRI-NotI fragments and cloned into a retroviral vector pBMN-Zfrom which the lacZ gene had been removed. The pBMN-Z-vectorcontaining the parvin-EGFP fragments was used for transfection ofthe packaging cell line, Phoenix-ECO or Phoenix-AMPHO, asdescribed previously (Clemen et al., 1999). Infection efficiency wasnearly 100% and cells were stably producing the entire GFP-parvinor the truncated parvin deletion mutants.

Protein purifications and actin-binding assayThe recombinant His6-tagged parvin was expressed in Escherichiacoli M15[pREP4]. E. coli cells transfected with the pQE-30 plasmidencoding the 6xHis-α-parvin were grown at 37°C to an A600 of 1.6.Cells were lysed by freezing-thawing in a lysis buffer (50 mMNaHPO4, 300 mM NaCl, 10 mM imidazole, pH 8) in the presence ofbenzamidine, aprotinine, leupeptine, pepstatin A, PMSF andlysozyme (Sigma, Deisenhofen, Germany) for 30 minutes. Aftersonication and centrifugation at 4°C for 30 minutes at 15,000 rpm theclarified supernatant was incubated with Ni-agarose (Qiagen) at 4°Covernight and then loaded onto a column. Lysis buffer in the columnwas gradually exchanged against 10 mM imidazole, pH 8. Afterwashing with 20 mM imidazole, parvin was eluted with 150 mMimidazole buffer at pH 8. The protein concentration was determinedby the method of Bradford (Bradford, 1976).

Rabbit skeletal muscle actin was purified from acetone-driedmuscle powder, as described (Spudich and Watt, 1971), and stored inG-buffer (5 mM Tris-HCl, 0.5 mM ATP, 0.1 mM CaCl2, 0.5 mM DTT,pH 7.6) drop-frozen in liquid nitrogen. Concentration of G-actinsolutions was determined spectrophotometrically at 290 nm, using anabsorption coefficient of 0.63 mg ml−1 cm−1 (Houk and Ue, 1974).

For F-actin co-sedimentation assay, protein samples werecentrifuged at 100,000 g for 30 minutes immediately before use.Polymerization of 10 µM actin was initiated by mixing withpolymerization buffer (100 mM imidazole, 20 mM MgCl2, 2 mMCaCl2, pH 7.4) in the absence or presence of various concentrationsof α-parvin. After 1 hour of incubation at room temperature, themixture was centrifuged at 120,000 g for 1 hour at 4°C.Corresponding amounts of the supernatant and the pellet weresubjected to SDS-polyacrylamide gel electrophoresis followed byprotein staining with Coomassie Brilliant Blue. Protein bands werequantified with a scanning densitometer 300A (Molecular Dynamics,Sunnyvale, CA) and used to determine the amount of α-parvin boundto F-actin.

Cell culture and immunofluorescence microscopyC3H/10T1/2 fibroblasts and mouse myoblast cell line C2F3 werepropagated and differentiation of C2F3 was induced as describedpreviously (Clemen et al., 1999). Rat vascular smooth muscle cells(A7r5), human epidermal cells (A431) and primary human foreskinfibroblasts (HFF) were propagated in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% fetal calf serum, penicillinand streptomycin. For the propagation of human embryonic kidneycells (293) pyruvate was added to the medium. Human T lymphocytes(Jurkat) were maintained in RPMI 1640 containing 10% fetal calfserum and 2 mM L-glutamine.

Monoclonal antibodies against recombinant His-tagged mouse α-parvin were produced as described (Schleicher et al., 1984), exceptthat the ImmunEasy Mouse Adjuvant (Qiagen) was used. Culturemedium from hybridoma cells producing antibody against α-parvinwas taken for immunolabelling.

For immunofluorescence, C3H/10T1/2 fibroblasts were allowed toattach onto glass coverslips for 1-16 hours, rinsed with PBS, fixed in3% paraformaldehyde and permeabilized with 0.1% Triton X-100 inPBS for 5 minutes. Alternatively, cells were fixed with methanol at–20°C for 10 minutes. After preblocking in PBG (0.5% BSA, 0.045%fish gelatine, both from Sigma, in PBS at pH 7.4) supplemented with

10% goat serum, the cells were incubated with the primary antibody,washed with PBS and incubated with the secondary Cy3-conjugatedantibody. For the detection of F-actin, the cells were treated withTRITC-phalloidin. The mouse anti-vinculin, anti-talin, anti-α-actinin,TRITC-phalloidin and the secondary Cy3-conjugated antibodies werepurchased from Sigma. Mouse anti-tensin antibodies were purchasedfrom Transduction Laboratories (Lexington, KY). For mitochondriastaining fibroblasts were stained with MitoTracker-Red dye(Molecular Probes, Eugene, OR) according to the manufacturersprotocol. Specimens were mounted in Gelvatol/DABCO (Sigma) andexamined under a confocal laser-scanning microscope TCS-SP(Leica) or a fluorescence microscope DMR (Leica, Solms, Germany)equipped with cooled charged-coupled device (CCD) camera (PCO,Kehlheim, Germany).

RESULTS

Cloning and sequence analysis of mouse α-parvinand parvin-related proteinsIn a search for proteins with homology to the ABD of α-actininin the mouse EST database, we have identified two overlappingcDNA clones encoding a novel protein, that shares low, butstatistically significant, similarity with several known familymembers. Sequence analysis of cDNAs extended by RACE-PCR revealed two transcripts (4324 and 1591 bp) sharing a 132bp 5′-untranslated region (UTR) and one single open readingframe (ORF) extending over 1119 bp. These two transcriptsarise as a consequence of two different polyadenylation signals(AATAAA) at positions 1567 and 4299 bp, which result in 3′-UTRs of different length. In addition, a splicing variant thatlacks a large stretch in the 3′-UTR, namely bases 2519-3964,was identified by RT-PCR. Further analysis of the ESTdatabase identified another variant, W18337, which spliced outa 3′-UTR stretch from 1414 to 4242 bp (Fig. 1). The α-parvinORF encodes a 372-amino acid protein with a calculatedmolecular mass of 42,330.

The mouse α-parvin protein sequence was used to searchhuman and mouse EST databases. The obtained ESTsequences were assembled into three mouse and three humancDNA contigs that corresponded to α-parvin and its twoparalogues, designated as β- and γ-parvins, for the closely andthe more distantly related proteins, respectively. The humanand mouse β-parvin cDNA encoded a 365 amino acid proteinswith 74% identity and 85% similarity to mouse α-parvin (Fig.2A). The cDNAs of human and mouse γ-parvins coded forproteins of 331 amino acids that show 42% identity and67% similarity to paralogous α-parvins. C. elegansand D.melanogasterwere also found to harbor parvin-like proteins.Parvin-related protein of C. elegansshared 48% identity and66% similarity to mouse α-parvin (Fig. 2A). The parvin-likeCG12533 predicted gene product in D. melanogaster, a peptideof 692 amino acids, showed homology to parvin in the C-terminal 357 amino acids, starting with Met 326. Given that noDrosophilaEST was found to connect the cDNA upstream anddownstream of the ATG for Met 326, it might well be that theCG12533 predicted gene product was a result of erroneousjoining of two independent genes. Therefore, only the C-terminal 357 amino acid peptide sequence of the Drosophilagene was used in the phylogenetic analysis.

The parvins are composed of a single ABD preceded by aN-terminal stretch. The overall primary structure of parvins is

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very conserved with the N-terminal domain being the mostvariable region of the molecule (Fig. 2A) varying from 82amino acids in γ- to 125 amino acids in β- and 133 amino acidsin α-parvins. The N-terminal domains of α- and β-parvincontain two putative nuclear localization signals (NLSs) atpositions corresponding to residues 20 and 39 of α-parvin (Fig.2A), and three potential SH3-binding sites (consensus PxxP,where x is any amino acid but cysteine (Cohen et al., 1995)).The first and the second CH domains forming the ABDs shareabout 20% identity and are separated by a linker of about 60residues (Fig. 2A). The linker region between the two CHdomains is longer than in other related proteins and displayssome similarity to the parts of β-spectrin and plectin thatprecede their respective CH1 domains. The sequences of C.elegans and D. melanogaster parvin-like proteins displaystronger homology to mammalian α- and β-parvin than to γ-parvin.

Phylogenetic analysis of CH domainsA phylogenetic analysis of the CH domains (Fig. 2B) of parvinand various members of the α-actinin superfamily shows thatCH1 and CH2 of parvins create their own branch divergingfrom a point between the CH1 domains of ABD-containingproteins and single CH domains. The CH1 and CH2 domainsof the other α-actinin-related proteins cluster separately,reflecting the higher degree of similarity between CH1 domainsof different proteins compared with CH1 and CH2 domainsfrom the same protein, in agreement with previous findings(Stradal et al., 1998, Banuelos et al., 1998). The CH domainsof proteins containing a single copy of CH form the separatebranch with an exception of smoothelin, which, albeitcontaining only a single CH domain (van der Loop et al., 1996),shows a higher degree of similarity to the CH2 domains ofproteins with two CH domains in tandem. Surprisingly, unlikethe other α-actinin-related proteins, the C-terminal CH domains

of parvins shows higher degree of homology to their own CH1than to the CH2 domains of other ABD-containing proteins(Fig. 2D). The CH1 of α-actinin shares 24% identity and 50%similarity with the second CH domain, but only 17% identityand 36% similarity with the first CH domain of α-parvin. Thestronger similarity of the α-parvin CH domains to each otherthan to any other type of CH domain suggests that the parvinsrepresent a separate family of proteins within the α-actininsuperfamily. This is also confirmed by the sequence analysis ofthe whole ABDs that represent the major families of the α-actinin superfamily, together with parvins (Fig. 2C). In theevolutionary tree based on the alignment of the entire actin-binding regions, the parvins group separately from the rest ofthe proteins of the α-actinin superfamily. They are positionedcloser to the ABDs of fimbrin (Fig. 2C), probably becausefimbrins, as parvins, harbor relatively long, although different,linkers between their CH domains. The phylogenetic tree of theABDs shows two major branches in the parvin family. One

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0 2 3 4

1587-2493

1

2468-4092

CH1

1- 684

kb

115-1592

950-4324

EST clone AA 791745

EST clone AA 691868

653-1067

115-805probe 1

653-3577

probe 2

probe 3

2012-4092probe 4

CH2

EST clone W18337

* *

splicing variant amplified by RT-PCR

Fig. 1.Full-length cDNA of mouse α-parvin. Thick lines representRT-PCR and RACE-PCR clones. The ORF is boxed. The noncodingpart of the cDNA is represented by the thin line. Polayadenylationsignals giving rise to two transcripts are marked by asterisks. The CHdomains are shown as empty boxes. Probe 1 and probe 2 were usedfor Northern blotting (see Fig. 4A,B,G), probes 3 and 4 wereamplified in RT-PCR (see Fig. 4D,E).

Fig. 2. (A) Alignment of the members of the parvin family, mouseand human α-, β- and γ-parvin, as well as the parvin homologues inD. melanogasterand C. elegans. The amino acids identical in morethan 66% of sequences are shaded in black with white letters. Thesequence motif search identified two putative nuclear localizationsignals (marked by double lines above the sequences) and twocalponin homology domains (thick lines above the sequences). Thesequence data are deposited in GenBank/EMBL/DDBJ underAccession Numbers AF237774 (mouse α-parvin), AF237771(human α-parvin), AF237770 (mouse β-parvin), AF237769 (humanβ-parvin), AF312712 (mouse γ-parvin) and AF237772 (human γ-parvin). The Accession Numbers for D. melanogasterand C. elegansparvin-like proteins are AAF 49016.1 and AAC 48090, respectively.(B) Phylogenetic tree of the CH domains based on calculations fromthe ClustalW alignment of these domains. Note the phylogeneticgroups of the CH1 domains, CH2 domains and the ones of proteinscontaining single CH domains. The CH1 and CH2 domains ofparvins form a separate branch emerging between CH1 domains anddomains of single-CH-domain-containing proteins. α-, β- and γ-parvin refer to mouse proteins. Accession Numbers of the sequences:human filamin, AF184126; human dystrophin, P11532;Dictyosteliumcortexillin I, L49527; human calponin, P51911;human β-spectrin, M96803; Drosophilakakapo, AJ011924;Dictyosteliuminteraptin, AF057019; chicken fimbrin, A37097;mouse α-actinin, P12814; mouse smoothelin, AJ010305; humanutrophin, NM_013070; mouse plectin, AF188012; mouse MACF,AF150755; mouse dystonin, AF252549; human Vav 15498; humansmooth muscle protein sm22-α, Q01995; human IQGAP, P46940.(C) Phylogenetic tree of the entire ABDs composed of tandem CH1and CH2 domain. The ABDs of parvins (mm, M. musculus; hs, H.sapiens; dm, D. melanogaster; ce, C. elegans) form a group distinctfrom the ABDs of the other α-actinin-related proteins. The ABDs ofparvins appear to be closer related to the fimbrin ABDs than to theother ABDs, probably due to the length of the linker between the CHdomains. The tree branches representing ABDs of human and mouseα-parvin are not distinguishable, owing to insignificant a-single-residue difference between mouse and human ABDs. (D) Alignmentof CH domains of mouse α-parvin (parvin/1, residues 96-202 andparvin/2, residues 263-369) with CH domains of calponin (residues29-131), IQGAP (residues 45-159) and N-terminal CH domains ofα-actinin (residues 32-135), β-spectrin (residues 55-163), filamin(residues 17-122), dystrophin (residues 16-119) and plectin (residues43-145). The amino acids identical in more than 66% of sequencesare shaded in black with white letters. Phylogentic analysis ofsequences was carried out using ClustalW version 4.2 (Thompson etal., 1994).

529Parvin: actin-binding protein at focal contacts

branch includes mammalian γ-parvins and the second containsthe rest of the proteins with Drosophila and Caenorabditisparvins positioned closer to mammalian α-parvins. The branchof mouse α-parvin is undistinguishable from its humancounterpart, owing to the highly conserved ABDs bearing asingle substitution (Fig. 2C). This suggests that α-parvinrepresents the evolutionary most conserved family member.

Tissue distribution of α-parvinA northern blot containing purified polyA+ RNA from various

tissues was hybridized separately with probe 1, specific forboth transcripts, or probe 2, specific for the 4.4-kb transcript(see Fig. 1A). Northern blot analysis with probe 1 recognizedtwo mRNA species that were 1.6 and 4.4 kb in length (Fig.3B), the sizes of which correlate well with the cDNAsequences. According to the sequence analysis, these twotranscripts may have arisen as a consequence of the twodifferent polyadenylation signals. Two mRNA species showedvarying levels of expression in different mouse tissues. The1.6-kb mRNA was generally expressed at lower levels than the

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4.4-kb mRNA, however its tissue distribution correlated withthe larger transcript (Fig. 3B). Hybridization of the same blotwith the probe 2, specific for the longer mRNA, recognizedonly the 4.4-kb transcript (Fig. 3A). An especially high levelof the 4.4-kb mRNA was detected in kidney and heart, weakersignals were observed in brain, lung and liver. As no signal wasdetected in mouse testis by Northern blot, we attempted toassess the expression of the α-parvin gene by means of RT-PCR. The fragments named as probe 3, corresponding to theORF, and probe 4, corresponding to the 3′-UTR of the 4.4-kbtranscript (Fig. 1), were amplified from testis cDNA, revealingthe presence of both α-parvin mRNA species (Fig. 3D). Thisindicates that the amount of mRNA in testis was below thedetection level for northern blots. The fragment correspondingto the probe 3 was also amplified using cDNA of differentiatingmyoblasts as a template, where we detected transcripts at alltime points (Fig. 3E).

To analyze the developmental regulation of α-parvin, wehave hybridized probe 1 to a northern blot containing RNAfrom 7-, 11-, 15- and 17-day-old mouse embryos. Both α-parvin mRNA species were expressed throughout mousedevelopment. Again, the level of expression of the 4.4-kbtranscript was significantly higher in comparison with the levelof the shorter mRNA (Fig. 3G).

Interaction of α-parvin with filamentous actin in vitroAll proteins with ABDs composed of two CH domains knownto date are able to bind filamentous actin. Considering that theABD of α-parvin differs from the other ABDs of the other α-

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Fig. 3.Expression of α-parvin mRNA in mouse tissues and celllines. Northern blot analysis of the α-parvin mRNA from mouseadult tissues (A-C) and from mouse embryonic tissues (G,H).Migration of RNA standards is indicated on the right. The blots werehybridized with the radiolabelled α-parvin probe 1, encompassing aregion common to both 1.6- and 4.4-kb mRNA species (A,G), withthe probe 2 specific for 1.6-kb transcript (B) and with β-actin cDNAas a control (C,H). RT-PCR analysis of mouse testis (D) anddifferentiated myoblasts at days 0, 2, 4 and 6 (E,F). Fragmentsamplified are as follows: (D) lane 1 and (F) lanes 1-4,glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe as acontrol; (D) lane 2, probe 4 specific for 4.4-kb α-parvin transcript;(D) lane 3 and (E) lanes 1-4, probe 3 specific for both 1.4- and 4.4-kbtranscripts. Migration of DNA markers is indicated on the right.

Fig. 4. Analysis of actin-binding properties of α-parvin. (A) Co-sedimentation of His6-α-parvin with F-actin by ultracentrifugation.S, supernatant; P, pellet. Positions of α-parvin and actin are indicatedon the left. (B) Estimation of the Kd of α-parvin binding to actinfilaments. Various amounts of His6-α-parvin were mixed with F-actin followed by ultracentrifugation. Amounts of bound and freeHis6-α-parvin were measured by densitometry of the supernatantand pellet fractions. The data were obtained from four independentexperiments comprising 15 samples in total. The Kd value wascalculated to be 8.4±2.1 µM.

531Parvin: actin-binding protein at focal contacts

actinin-related proteins in its ‘reverse’ order of the CH domainsand a longer linker between them, we were interested toexplore whether this moderate similarity was sufficient toconfer actin-binding properties to α-parvin. We tested thepotential F-actin-binding properties of the full-length His-tagged recombinant α-parvin in a high-speed co-sedimentationassays with F-actin and found that α-parvin co-sedimentedwith F-actin (Fig. 4A, lane 4), whereas in control experimentsmost of the His-tagged α-parvin was found in the supernatantfraction after ultracentrifugation (Fig. 4A, lane 1). Smallamounts of parvin, however, significantly less than in thepresence of actin, were also seen in the pellet, probably causedby precipitation (Fig. 4A, lane 2). The actin-binding affinitywas estimated by quantifying the results of four independentsedimentation assays (15 samples in total) with varyingamounts of parvin (Fig. 4B). The Kd value for the interactionof the recombinant His-tagged α-parvin with actin wascalculated to be 8.4±2.1 µM, which is in the same range asreported for the ABDs of other members of this superfamily.

Subcellular localization of α-parvinTo determine the subcellular localization of parvin, weemployed GFP tagged α-parvin, because of the lack ofantibodies distinguishing between parvin isoforms. Overall,expression of GFP tagged α-parvin in C3H/10T1/2 fibroblasts,even in the strongly expressing cells, did not lead to changesin actin cytoskeleton and focal contacts organization whencompared with untransfected fibroblasts upon visualization

with rhodamine-phalloidin and anti-vinculin immunolabelling,respectively. Cells transfected with the constructs coding forGFP fused to the N terminus of α-parvin showed identicaldistribution patterns to the cells with GFP at the C terminus.Therefore, data shown throughout are obtained with GFP fusedto the C terminus of α-parvin. The expression of GFP-α-parvinfusion protein was verified by western blotting, using an anti-GFP monoclonal antibody (data not shown).

In well-spread fibroblasts, GFP-α-parvin preferentiallyappeared at the focal adhesions (Fig. 5A,B). In addition to that,GFP-parvin was localized to a distinct set of fibers in thecentral area of the cell running nearly parallel to one another(Fig. 5B). In fibroblasts forming contacts with severalneighboring cells, these fibers may run in various directions(Fig. 5A). GFP-parvin was also observed at membrane ruffles,which were well seen in spreading fibroblasts (Fig. 5C).

To find out whether the distribution of the GFP-α-parvinobserved in the C3H/10T1/2 fibroblasts reflects the generalproperties of the protein, or is limited to this cell type, wetransfected several other cell lines, including mouse myoblasts(C2F3), rat vascular smooth muscle cells (A7r5), humanepidermal carcinoma cells (A431) and primary human foreskinfibroblasts (HFF). GFP-α-parvin was associated with the cell-substrate structures (focal contacts) in all cell types studied. Inepithelial cells (A431) GFP-α-parvin also accumulated at somecell-cell adhesions in addition to the focal contacts and nuclei(Fig. 5D), which implies involvement of α-parvin in generalcell-adhesion events.

Fig. 5.Subcellular localization ofα-parvin in transfectedC3H/10T1/2 mouse fibroblastsand epidermal cells A431. (A,B) In well-spread fibroblasts,GFP-α-parvin is localized at focaladhesions (arrowheads), fibrillarstructures in the central area of thecell (empty arrowheads) andnuclei. (C) In spreadingfibroblasts, GFP-α-parvin isdetected at lamellar ruffles (arrow)and maturing focal adhesions(arrowheads). (D) In humankeratinocytes, GFP-α-parvin wasassociated with focal contacts(arrowheads), with some cell-cellcontacts (arrows), but not with all(asterisk), and stronglyaccumulated in nuclei. Scale bars:20 µm.

532

To confirm the presence of GFP-parvin in focal adhesionsand to explore the relationship between focal contacts andparvin in more detail, we labeled the transfected fibroblastswith antibodies against the focal contact proteins vinculin, talinand α-actinin. Notably, GFP-α-parvin was not alwaysassociated with focal contacts positive for vinculin. This canbe observed particularly well during fibroblast spreading,which involves formation of focal adhesions at the cellperiphery (Fig. 6). In some spreading cells, fixed within 30-90minutes after plating, GFP-α-parvin was not recruited tonascent or to the well-developed focal adhesions, as observedby the intensity of anti-vinculin labeling (Fig. 6A). Instead,GFP-α-parvin was distributed diffusely throughout thecytoplasm with enrichment at lamellipodial ruffling. At a laterstage of cell spreading, GFP-α-parvin can be seen at the cellmembrane and also weekly incorporated at vinculin-positivefocal adhesions (Fig. 6B). When the cell membrane wasdepleted of GFP-α-parvin, the GFP-α-parvin fluorescencebecame stronger at the focal contacts (Fig. 6C). In addition, itlocalized to the central fibers, which were weakly labeled withvinculin (Fig. 6C). In contrast, in fibroblasts, which wereallowed to spread overnight, the GFP-α-parvin fluorescenceoverlapped significantly with the anti-vinculin pattern (Fig.7D,F). Occasionally, GFP-α-parvin labeling at focal contactswas found to be more elongated towards the cell center whencompared with the anti-vinculin staining. The anti-vinculinlabeling of the α-parvin-positive central fibers appeared tobe weaker than the GFP-α-parvin fluorescence. These

observations suggest that α-parvin was not necessary for theformation of focal adhesions, as it was not associated withnascent newly formed adhesion complexes.

To investigate the nature of the GFP-α-parvin-rich fiberslocated in the central cell area we stained the transfectedfibroblasts with TRITC-phalloidin to label F-actin. In somecells GFP-α-parvin fluorescence was extended from focalcontacts towards the center of the cell, showing partial co-localization with stress fibers (Fig. 7A-C). Additionally, GFP-α-parvin fluorescence revealed that not all filaments appear tobe associated with F-actin. This was even more obvious inspecimen immunolabeled for α-actinin (Fig. 7H,I), whichlocalizes to focal contacts and actin stress fibers. We comparedthe GFP-α-parvin distribution with the anti-α-actinin patternand found that GFP-α-parvin co-localized with α-actinin infocal contacts. As GFP-α-parvin extended from the focaladhesions, it followed the stress fibers marked by the typicalfor α-actinin striated pattern. However, not all GFP-α-parvin-fibers in the center of the cell appear to overlap with anti-α-actinin staining. Thus, the data imply that association of α-parvin with central fibers was not mediated by actin. Next, weexamined whether GFP-α-parvin was also localized to focalcontacts stained by anti-talin antibodies and found that parvinco-localized completely with talin in focal adhesions as wellas with fibers in the central area of the fibroblasts (Fig. 7K,L).The resemblance of the α-parvin-rich central fibers to tensin-associated fibrillar adhesions, a distinct type of adhesionsinvolved in matrix remodeling (Katz et al., 2000; Zamir et al.,

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Fig. 6. The distribution of α-parvin (A-C) compared with vinculin (D-F) during cell spreading and focal contact formation. C3H/10T1/2 mousefibroblasts stably expressing GFP-α-parvin were immunostained with anti-vinculin antibodies. In cells fixed within 30-90 minutes of plating, α-parvin is attracted to lamellipodial ruffles (arrows), but not to nascent focal adhesions (A,D). The recruitment of α-parvin to focal contacts isincreased upon their maturation (arrowheads) (B,C,E,F). In addition, α-parvin is localized to the central fibers, which are weakly labeled withanti-vinculin (open arrowheads). Scale bars: 20 µm.

533Parvin: actin-binding protein at focal contacts

2000), led us to examine the co-distribution of GFP-α-parvinand tensin. Anti-tensin antibodies were hardly detected in thefocal contacts, but strongly labeled the central α-parvin fibers(Fig. 7N,O). Thus, α-parvin appears to be associated withvarious types of cell-matrixadhesion structures, includingmature focal contacts and fibrillaradhesion-like structures.

In addition, GFP-α-parvin wasstrongly enriched in nuclei ofsome cells (Figs 7G,J), but not inothers (Fig. 7A,D). This differenceappears to correlate with theexpression level of α-parvin in thetransfected cells. Cells expressinglower level of parvin did notaccumulate it in their nuclei,although it was clearly associatedwith the focal-contact-likestructures (Fig. 7A,D). Theappearance of GFP-α-parvin in thenucleus could be due to thepresence of the two nuclearlocalization signals identified inthe N-terminal part of α-parvin.Nuclear staining was also observedin control cells expressing GFPalone, probably as a result ofdiffusion of the 28-kD GFPthrough the nuclear pores.Although the molecular mass ofGFP-α-parvin fusion protein (71kDa) is higher than the generallyaccepted threshold of 30 kDa forproteins that can passively diffusethrough the nuclear pores, weemployed methanol fixation toexclude unspecific nuclear

targeting. Indeed, after this fixation, no GFP fluorescence wasobserved in control cells expressing GFP alone. The GFPfusion protein remained, in contrast, associated withintranuclear structures (data not shown).

Fig. 7. Confocal images of GFP-α-parvin in well-spread cells.C3H/10T1/2 fibroblasts stablyexpressing GFP-α-parvin werestained with TRITC-phalloidin (A-C) and immunolabelled forvinculin (D-F), α-actinin (G-I), talin(J-L) and tensin (M-O). Fluorescenceof GFP-α-parvin in focal contacts(arrows) overlaps with actin (C), vinculin (F), α-actinin (I), andtalin (L), but not tensin (O) pattern.Note, that α-parvin-rich fibers in thecentral area of the cell (arrowheads)do not co-localize with actin stressfibers visualized with TRITC-phalloidin (C) and anti-α-actinin (L).Images show GFP-α-parvin(A,D,G,J,M), the co-staining alone(B,E,H,K,N) or the composite imagesgenerated by superimposition of greenand red signals with overlappingregions appearing yellow (C,F,I,L,O).Scale bars: 20 µm.

534

To compare the distribution of the endogenous parvin withthe GFP-fused protein we employed the monoclonal antibodiesraised against mouse α-parvin. The immunolabelingoverlapped with the GFP-α-parvin pattern in the focal contacts(Fig. 8A-C) and the nuclei (not shown). The staining ofendogenous parvin in untransfected fibroblasts with the anti-parvin antibodies resulted in a pattern similar to the one ofthe GFP-α-parvin-expressing cells (Fig. 8D). The fiber-likestructures, also recognized by the antibodies, could result fromthe cross-reaction with β- and γ-parvin.

Domain analysis of α-parvinTo further characterize the function of α-parvin and to map thedomains responsible for targeting different cell compartments,a set of overlapping fragments of parvin fused to GFP wasgenerated and prepared for retroviral transfection intoC3H/10T1/2 fibroblasts. A schematic representation of thedomain structure with the expressed fragments is shown in Fig.9. None of the deletion mutants affected the apparent actinfilament organization and focal contactdistribution as monitored by labeling withTRITC-phalloidin and anti-vinculin,respectively. Deletion of the second CHdomain (GFP-α-parvin1-273) abolished its

localization to focal adhesions and centrally located fibers.GFP fluorescence in cells transfected with this construct wasdiffuse throughout the cytoplasm in fully spread cells (Fig.10A) and could not be found at focal contacts visualized withanti-vinculin antibody (Fig. 10B,D). However, the truncatedprotein was enriched at cell ruffles in moving or spreading cells(Fig. 10C).

In contrast, GFP-α-parvin257-372, composed solely of thesecond CH domain, was targeted to focal adhesions and centralfibrillar structures in fully spread fibroblasts (Fig. 10E,F). Inmigrating cells, GFP-α-parvin257-372 showed a distributionsimilar to the full-length construct as it was attracted to theleading edges of migrating cells (Fig. 10G,H). The distributionof this GFP-α-parvin257-372was similar to the staining patternof vinculin, although the fluorescence signal at the focalcontacts seen with this construct appeared to be generallyweaker than the one with the full-length GFP-α-parvin. Thisimplies that the second CH domain is responsible for theattraction of parvin to the fibrillar adhesions and thus couldrepresent a module interacting with molecular complexesmediating cell-matrix adhesion. The first CH domain of α-parvin appears to reinforce or stabilize the binding of thesecond CH domain, as the focal contact fluorescence of thelatter was weaker then the one of full-length GFP-α-parvin.

Transfection with the construct coding for GFP-α-parvin1-93, composed of the most N-terminal part of parvin,resulted in weak diffuse staining in the cytoplasm afterparaformaldehyde-Triton X-100 fixation, and fluorescence wascompletely abolished in methanol fixed cells. Yet, the GFP-α-parvin1-93 fluorescence was clearly seen in the nuclei of theintact unpermeabilized cells. Thus, these observations indicatethat the predicted NLSs found in the N-terminal part of α-parvin may be responsible for the import to the nucleus, butnot sufficient to confer targeting of parvin to its nuclear bindingpartner. All constructs of GFP-α-parvin and its fragments, withan exception of GFP-α-parvin188-372and GFP-α-parvin188-273

(see below), were found to localize to nuclei, with the full-length GFP-α-parvin resulting in the strongest nuclearfluorescence. Taken together, these data may indicate that thesites involved in association with nuclear targets are located inthe C-terminal two thirds of α-parvin, whereas the NLSs arepositioned in the N-terminal region.

An unexpected fluorescence pattern was observed with GFP-α-parvin188-372, which was attracted to reticular structuresidentified as mitochondria by co-staining with MitoTracker-Red marker, whereas the localization to focal or fibrillaradhesions, to lamellae and to the nuclei was completely

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Fig. 8.Partial co-localization of GFP-α-parvin and anti-parvinimmunostaining. Note, that GFP-α-parvin (A) co-localizes with theanti-parvin monoclonal antibody (B) at focal contacts (arrows) of thetransfected cells. (C) Merging of the A,B. (D) The immunostainingof the endogenous parvin in untransfected cells shows a patternsimilar to that of GFP-α-parvin-expressing cells (see B). All framesare confocal images. Scale bars: 10 µm.

adhesions fibers lamellae nuclei mitochondria focal central

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Fig. 9. Construction of truncated α-parvins. Adiagram of α-parvin domains and theirrepresentation in the respective truncationconstructs is presented on the left. The aminoacids comprised by each construct are given. Thetable on the right indicates the subcellularlocalizations observed in fibroblasts expressingthe constructs.

535Parvin: actin-binding protein at focal contacts

abolished (Fig. 10I,J). Further truncation of this constructyielded GFP-α-parvin188-273and identified the amino acids 188through 273 of α-parvin as being sufficient for targeting tomitochondria. Since no mitochondria targeting was observedwith the full-length GFP-α-parvin, it is possible that theassociation of GFP-α-parvin188-372 and GFP-α-parvin188-273

with mitochondria was an artefact caused by exposure ofresidues mediating mitochondrial targeting.

DISCUSSION

We have identified and characterized α-parvin, a novel 42-kDactin-binding protein associated with focal adhesions.Sequence analysis of α-parvin revealed a pair of CH domainsforming an ABD, which is characteristic for a large α-actinin/β-spectrin superfamily of proteins. In contrast to otherknown proteins of this superfamily, the C-terminally locatedABD of α-parvin is not followed by any of the protein motifstypical for the other members. Since α-parvin appears to be thesmallest member of the α-actinin-superfamily known so far, itwas given a name derived from the Latin term ‘parvus’ for‘small’.

In view of the differences between amino acid sequences ofthe CH domains of parvin and other α-actinin-related proteins,it was interesting to examine actin-binding properties of α-parvin. For this we used His-tagged bacterially expressed α-parvin. His-tagged proteins or their actin-binding domainswere used previously for estimating actin-binding properties,although these data should be evaluated with caution, since atleast in one case the direct comparison of nonfusion versus His-tagged protein (cortexillin fragment) showed that the bindingto actin was enhanced, probably due to electrostaticinteractions of the positively charged residues of the tag withthe negatively charged actin (Stock et al., 1999). Yet, theaffinities of His-tagged ABD of dystrophin to α- and γ-actin,estimated to 13.7 and 10.6 µM, respectively (Renley et al.,1998) were highly consistent with the data obtained with thesame dystrophin fragment expressed without the tag and β-actin (13 µM) (Winder et al., 1995a). The affinity of His-α-parvin to F-actin was determined to be 8.4 µM, which iscomparable with the Kd value of 4.7 µM determined for α-actinin (Way et al., 1992). The regions of the primarysequences of conventional ABDs involved in direct interactionwith actin filaments in dystrophin (Corrado et al., 1994;Fabbrizio et al., 1993; Levine et al., 1992), α-actinin(Hemmings et al., 1992), ABP-120 (Bresnick et al., 1990) andβ-spectrin (Karinch et al., 1990) were mapped to three shortstretches designated as actin-binding sites (ABSs). ABS1 andABS2 located in the CH1 domain of α-actinin-related proteinsare conserved in the C-terminal CH domain of parvin, whichshows higher overall homology to CH1 (Fig. 2D). It was alsoshown that the CH1 domains of α-actinin (Way et al., 1992)

Fig. 10. GFP-tagged parvin domains were expressed in C3H/10T1/2fibroblasts, and the localization of each domain was observed byGFP fluorescence (A,C,E,G,I) and indirect immunofluorescentstaining for vinculin (B,D,F,H). GFP-α-parvin1-273, encompassingthe N-terminal stretch, the first CH domain and the linker, wasdiffusely distributed over the cell with some enrichment at membraneruffles (A-D). The GFP fused to the second CH domain (GFP-α-parvin257-372), in addition to membrane ruffles (G), was also attractedto focal contacts and the central fibers (E-H), although weaker thanthe full length GFP-α-parvin. Arrowheads indicate the location offocal contacts, the arrows mark the location of membrane ruffles.Open arrowheads point at mitochondria-associated structures in cellsexpressing GFP-α-parvin188-372(I,J). Mitochondria were stainedusing the MitoTracker-Red probe (J). Scale bar: 20 µm.

536

and dystrophin (Winder et al., 1995a) harboring ABS1 andABS2, are sufficient for binding to F-actin, although theaffinity of interaction was lower than the one determined forthe whole ABDs. It could therefore well be, in contrast to theother members of the α-actinin superfamily, that the C-terminal CH domain represents the major actin-binding regionin parvins. The structural analysis suggest that linkers betweenCH domains are not directly involved in the interaction withactin filaments, but confer conformational flexibility to ABDsand play a role in arranging the CH domains to form an actin-binding surface (Norwood et al., 2000). Therefore anexclusively long linker region of parvin might not interferewith actin interaction and may be involved in positioning ofthe CH domains upon association with microfilaments. Thus,we demonstrated that the ABD of α-parvin is a functionalmodule capable of binding to filamentous actin in spite of therelatively low similarity to the conventional ABDs of α-actinin-related proteins.

In vivo α-parvin fused to GFP interacted with the site whereactin filaments are associated with cell-matrix adhesioncomplexes. Focal contacts or focal adhesions representmolecular complexes at the points of the closest appositionbetween the cell membrane and extracellular substrate thatmediates integrin-dependent cell-matrix adhesion. Theassembly and maturation of focal adhesions is a complexprocess (Bershadsky and Geiger, 1999; Burridge andChrzanowska-Wodnicka, 1996), where the order of appearanceand topology is not clear. In contrast to fimbrin, a protein withtwo ABDs of the α-actinin type that is involved in early stagesof attachment (Correia et al., 1999), α-parvin does not seem tobe involved in the maturation of the focal contacts (Fig. 6).GFP α-parvin appears at already formed adhesions and is alsoassociated with the net of fibers in the central area of the cell.There is a growing body of evidence that focal adhesions arediverse and highly motile structures, which move in anactomyosin-dependent fashion, and that this movement isresponsible for generation of different types of adhesioncomplexes (Katz et al., 2000; Smilenov et al., 1999; Zamir etal., 2000). The central fibers, positive for anti-tensin, maycorrespond to recently described tensin-rich fibrillar adhesions,whose molecular composition is distinct from classical focalcontacts and which were shown to be involved in matrixremodeling (Katz et al., 2000; Zamir et al., 1999). Fibrillaradhesions, which are elongated structures associated withfibronectin fibrils, contain little or no paxillin, vinculin, talinand focal-adhesion kinase, and are assembled at proximalmargins of focal contacts and translocated towards the cellcenter in an actomyosin-dependent manner (Zamir et al.,2000). The location of parvin not only at classical focalcontacts but also at tensin-rich central fibers poses theimplication of α-parvin in matrix remodeling and in turnoverof adhesion complexes, although the exact molecularmechanism remains to be examined. Our results also indicatethat the CH2 domain of α-parvin is sufficient for formingcomplexes with the proteins of focal contacts, since it wasrecruited to cell-adhesion sites when the domain alone wasexpressed in a fibroblast cell line. The CH2 domain of α-parvinalso harbors the conserved regions ABS1 and ABS2, which areinvolved in F-actin binding (see above). This observationsuggests that parvin may be complexed with proteins of focalcontacts by virtue of binding F-actin, and that it associates with

actin filaments in an adhesion-dependent manner. Although theC-terminal CH domain of parvin was responsible for both focalcontacts and fibrillar adhesions, the mechanism that targetedα-parvin to the central tensin-rich fibers may differ from theassociation with focal adhesions, given that the central fiberswere not often co-localized with actin filaments.

Actin seems to be an accepted nuclear component, and anincreasing number of actin-binding proteins have been reportedto reside in the nucleus or to translocate to the nucleus underregulatory control (reviewed by Rando et al., 2000). Theobserved nuclear targeting of GFP-α-parvin is in agreementwith the two putative nuclear localization signals, which wereidentified in the N-terminal part of α-parvin. Strong nuclearlocalization of GFP-α-parvin, which is too large to diffusepassively into the nucleus, suggests that the identified NLSsare functional. Analysis of the distribution of the truncated α-parvin shows that the fragments of α-parvin lacking both NLSsare also observed in nuclei, although not to the same extent asthe full-length GFP-α-parvin. The N-terminal α-parvinfragment encompassing the first 93 residues harboring bothpredicted NLSs is detected in nuclei of the intact cells, but notafter fixation and permeabilization. A likely explanation forthis could be that the GFP-α-parvin1-93 contains the NLSsresponsible for the transport to the nucleus, but lacks the CHdomains that may encompass the sites responsible for theinteraction with intranuclear targets.

Our data identify α-parvin as a novel component of focaladhesions recruited to the sites of cell-matrix adhesion via itssecond CH domain. Given that this CH domain of mouse α-parvin shares 84% identity and 94% similarity with paralogousβ-parvin, and 50% identity and 71% similarity with γ-parvin,we speculate that parvins constitute a novel emerging familyof actin-binding proteins involved in cell-matrix adhesion.Although the exact biological function of parvins remains to bedetermined, parvins probably represent structural componentsof a link between actin filaments and transmembrane complexesassociated with extracellular matrix.

This work was supported by a grant from the Center for MolecularMedicine Cologne. T.O. is a member of the Graduiertenkolleg‘Molekularbiologische Grundlagen pathophysiologischer Vorgänge’.We thank Dr M. Aumailley and Dr M. Schleicher for helpfuldiscussions and sharing of reagents and Dr F. Rivero for criticalreading of the manuscript.

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Note added in proofThe protein identical to mouse α-parvin was identified aspaxillin-binding protein and named actopaxin by Turner et al.(Turner (2000) J. Cell Sci. 113, 4139-4140).

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