Research Article 1663 Characterisation of IRTKS, a novel IRSp53/MIM family regulator with distinct filament bundling properties

Thomas H. Millard*, John Dawson and Laura M. Machesky‡ School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK *Present address: University of Bristol, Dept. of Biochemistry, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK ‡Author for correspondence (e-mail: [email protected])

Accepted 12 March 2007 Journal of Cell Science 120, 1663-1672 Published by The Company of Biologists 2007 doi:10.1242/jcs.001776

Summary IRSp53 is a scaffold that contains an IRSp53/MIM resembles a WASP-homology 2 (WH2) motif. Addition of homology domain (IMD) that bundles actin filaments and the Ct extension to IRSp53 causes an apparent shortening interacts with the small GTPase Rac. IRSp53 also binds to of bundles induced by the IMD in vitro, and in cultured the small GTPase Cdc42 and to Scar/WAVE and cells, suggesting that the Ct extension of IRTKS modulates Mena/VASP to regulate the actin . We the organising activity of the IMD. Lastly, we could not have characterised a novel IMD-containing protein, insulin detect actin monomer sequestration by the Ct extension of receptor tyrosine kinase substrate (IRTKS), which has IRTKS as would be expected with a conventional WH2 widespread tissue distribution, is a substrate for the insulin motif, but it did interact with actin filaments. receptor and binds Rac. Unlike IRSp53, IRTKS does not interact with Cdc42. Expression of IRTKS induces clusters Supplementary material available online at of short actin bundles rather than filopodia-like http://jcs.biologists.org/cgi/content/full/120/9/1663/DC1 protrusions. This difference may be attributable to a short carboxyl-terminal (Ct) extension present on IRTKS, which Key words: GTPase, Actin, Cytoskeleton, Filopodia, Motility

Introduction of the signals that control the formation of filopodia.

Journal of Cell Science The actin cytoskeleton is central to the movement, morphology Activation or overexpression of the GTPase Cdc42 induces and adhesion of eukaryotic cells (Pollard and Borisy, 2003). filopodia in some cell types, but the key downstream effectors Actin filaments are used in a diverse array of cellular contexts of Cdc42-induced filopodia are not known (Lamarche et al., and numerous actin binding proteins and regulators control the 1996; Nobes and Hall, 1995). There is increasing evidence that polymerisation and arrangement of filaments. In motile cells, Ena/VASP proteins are involved in filopodia formation, acting polymerisation of actin at the leading edge drives protrusion of by preventing filament capping, resulting in the production of the membrane. Protrusive actin structures include lamellipodia, long parallel filaments (Bear et al., 2002; Lebrand et al., 2004; which are broad, flat sheet-like projections, and filopodia, Mejillano et al., 2004). Diaphanous related (DRF) which are thin, needle-like projections (Small et al., 2002). proteins are also key controllers of filopodia formation Lamellipodia consist of a dense network of short, branched (Pellegrin and Mellor, 2005; Schirenbeck et al., 2005a; actin filaments, and our understanding of how these structures Schirenbeck et al., 2006; Schirenbeck et al., 2005b). In are formed have seen a dramatic increase in recent years addition, numerous actin bundling and crosslinking proteins (Svitkina and Borisy, 1999). Central to lamellipodia formation control the arrangement of actin filament protrusions (Revenu is the Arp2/3 complex, which nucleates actin filaments while et al., 2004; Vignjevic et al., 2006). Bundling is a crucial bound to the side of existing filaments, resulting in a branched determinant of the mechanical properties of actin protrusions filament network (Mullins et al., 1998). Signals are transduced (Gardel et al., 2004; Xu et al., 1998). Different actin structures from extracellular stimuli to the Arp2/3 complex by a pathway contain different complements of bundling proteins, for including the small GTPase Rac and Scar (WAVE) proteins instance fascin is found within filopodia and filamin A within (Bompard and Caron, 2004; Machesky and Insall, 1998). lamellipodia (Flanagan et al., 2001; Kureishy et al., 2002). Filaments in lamellipodia are short due to the presence of Like the proteins that control actin assembly, bundling proteins capping protein, which binds to the barbed end of growing are subject to regulation and it is becoming apparent that in filaments shortly after nucleation, preventing further growth order to generate specific actin structures, actin assembly and (Pollard and Borisy, 2003). In contrast to lamellipodia, bundling must be co-ordinately controlled (Revenu et al., filopodia consist of a combination of long, unbranched 2004; Vignjevic et al., 2006). filaments arranged in tight, parallel bundles (Svitkina et al., Insulin receptor substrate of 53 kDa (IRSp53) was originally 2003) and shorter unbundled filaments near the tips (Medalia identified as a phosphorylation substrate for the insulin et al., 2007). We currently do not have a clear understanding receptor, however, subsequently research has focused on its 1664 Journal of Cell Science 120 (9)

role in regulating the actin cytoskeleton (Yeh et al., 1996). isoform 2 or IRSp53-S, NM_017450). This sequence has Several groups have observed that overexpression of IRSp53 previously been identified by Yamagishi et al. who referred to results in the formation of filopodia-like protrusions, and it as IRTKS in sequence alignments (Yamagishi et al., 2004). IRSp53 localises to the tips of both filopodia and lamellipodia Homologous sequences have also been identified for several (Govind et al., 2001; Krugmann et al., 2001; Nakagawa et al., other species, including mouse (NP_080109, 87% identity), 2003; Yamagishi et al., 2004). IRSp53 is a modular protein chicken (XP_414749, 70% identity) and zebrafish containing several protein interaction domains, including an (AAH68330, 52% identity). The human IRTKS gene is located SH3 domain, which binds to several important actin regulators on chromosome 7 at q21.3–q22.1, whereas IRSp53 is on including Scar2/WAVE2 and the Ena/VASP protein, Mena chromosome 17 at q25. (Krugmann et al., 2001; Miki et al., 2000). IRSp53 also Alignment of human IRTKS with IRSp53 demonstrates that possesses distinct binding sites for the two Rho family the region corresponding to the IMD of IRSp53 is well GTPases, Rac and Cdc42, leading to the proposal that IRSp53 conserved, as are the SH3 domain and WW domain interaction functions as an adaptor, transducing signals from these motif (Fig. 1). Outside of these regions conservation is low, GTPases to cytoskeletal regulating proteins (Krugmann et al., with IRTKS notably lacking the partial CRIB domain which 2001; Miki et al., 2000). However, recent work has suggested mediates binding of Cdc42 to IRSp53. The C terminus of that IRSp53 also possesses effector functions (Yamagishi et al., IRTKS has no similarity to that of the well-characterised short 2004). The N-terminal 250 amino acids of IRSp53 consist of (S) splice variant of IRSp53, however, it does have homology a domain conserved in five mammalian proteins including the to the C-termini of two other IRSp53 splice variants, the L previously identified actin regulator missing in metastasis-B (long) and M (medium) forms (Fig. 1B) (Miyahara et al., 2003; (MIM-B), hence the name IRSp53/MIM homology domain Yamagishi et al., 2004). Note that unless otherwise stated, (IMD). The crystal structure of the IRSp53 IMD reveals it to IRSp53 refers to the S splice variant in this manuscript. be a zeppelin-shaped dimer with one F-actin-binding site per monomer (Millard et al., 2005). In isolation the IMD can IRTKS is widely distributed and is an insulin receptor bundle actin filaments and induce filopodia-like protrusions, substrate however, mutation of the actin binding sites abrogates We used peptides based on the sequence of human and mouse protrusion formation by both the isolated IMD and by full- IRTKS (see Materials and Methods) to raise a polyclonal length IRSp53 (Millard et al., 2005). The structure of the IMD antibody, which recognised a band of 60 kDa in extracts from is closely related to that of the BAR domain, a function of the mouse myoblast cell line C2C12 (see Fig. S1A in which is to tubulate membranes (Gallop and McMahon, 2005). supplementary material). Immunoprecipitations from C2C12 Intriguingly, recent data has indicated that the IRSp53 IMD extract were performed using this antibody and the precipitated also has the capacity to tubulate membranes and it has been material subjected to SDS-PAGE. A band of 60 kDa was suggested that this activity contributes to protrusion formation excised from the gel and analysed by mass spectrometry after (Suetsugu et al., 2006). Like IRSp53, MIM-B can bundle trypsin digestion. Three of the five peptide sequences derived filaments via its IMD, however, outside the IMD, MIM-B has were found to match the murine form of the novel protein (Fig.

Journal of Cell Science little sequence conservation with IRSp53, suggesting a S1B in supplementary material). The tissue distribution of functional divergence (Yamagishi et al., 2004). Unlike IRSp53, IRTKS was studied by immunoblotting murine tissue extracts. MIM-B does not possess an SH3 domain, instead it contains Western blots were inconclusive as the IRTKS antibody an actin monomer binding WASP homology 2 (WH2) domain produced a high background signal in some tissues (not and a region that binds receptor protein tyrosine phosphatase shown), equally, attempts at northern blotting failed. We could, ␦ (Gonzalez-Quevedo et al., 2005; Mattila et al., 2003; however, detect IRTKS by immunoprecipitating IRTKS from Woodings et al., 2003). Overexpression of MIM-B results in the tissue extracts using the IRTKS antibody covalently linked the formation of structures resembling actin microspikes and to protein G beads, and then blotting the immunoprecipitated lamellipodia, but not long, filopodia-like protrusions as material. Clear expression of IRTKS was observed in bladder, observed on overexpression of IRSp53 (Mattila et al., 2003; liver, testes, heart and lung, and trace amounts were found in Woodings et al., 2003). Yamagishi et al. reported that five spleen, brain and skeletal muscle (Fig. S1C in supplementary mammalian proteins contain IMDs, however, of these, only material). None was detected in intestine or kidney. This shows IRSp53 and MIM have been studied, the remaining three being that IRTKS has widespread tissue distribution in mouse. predicted proteins, based on cDNA sequences (Yamagishi et IRSp53 was originally identified as a substrate for the insulin al., 2004). Of these three proteins, two are closely related to receptor (Yeh et al., 1996), so we tested whether IRTKS shared IRSp53 and one to MIM-B. In this study we have characterised this characteristic. COS7 cells were co-transfected with Myc- insulin receptor tyrosine kinase substrate (IRTKS), one of the IRTKS and the insulin receptor ␤-subunit. The transfected two IRSp53-related IMD proteins. We find that IRTKS has cells were stimulated with insulin and Myc-IRTKS properties clearly distinct from those of IRSp53. immunoprecipitated from the extract, and probed for the presence of phosphotyrosine by immunoblotting. We observed Results tyrosine phosphorylation of IRTKS, which was dependent on Identification of IRTKS the presence of the insulin receptor and stimulation with In a search for IRSp53-related proteins we identified a human insulin, indicating that IRTKS is an insulin receptor substrate cDNA sequence (accession number: NM_018842) containing (Fig. S1D in supplementary material). an open reading frame, translation of which would result in a 511 amino acid protein with 39% identity and 59% similarity IRTKS binds Rac, but not Cdc42 to human IRSp53 (also known as BAI1-associated protein IRSp53 has been shown to bind to Rac via a site within the N- IRTKS: a novel IMD-containing actin regulator 1665

A

IRSp53 MSLSRSEEMHRLTENVYKTIMEQFNPSLRNFIAMGKNYEKALAGVTYAAKGYFDALVKMGELASESQGSK70 IRTKS MSRGP-EEVNRLTESTYRNVMEQFNPGLRNLINLGKNYEKAVNAMILAGKAYYDGVAKIGEIATGSPVST69

IRSp53 ELGDVLFQMAEVHRQIQNQLEEMLKSFHNELLTQLEQKVELDSRYLSAALKKYQTEQRSKGDALDKCQAE 140 IRTKS ELGHVLIEISSTHKKLNESLDENFKKFHKEIIHELEKKIELDVKYMNATLKRYQTEHKNKLESLEKSQAE 139

IRSp53 LKKLRKKSQGSKNPQKYSDKELQYIDAISNKQGELENYVSDGYKTALTEERRRFCFLVEKQCAVAKNSAA 210 IRTKS LKKIRRKSQGSRNALKYEHKEIEYVETVTSRQSEIQKFIADGCKEALLEEKRRFCFLVDKHCGFANHIHY 209

IRSp53 YHSKGKELLAQKLPLWQQACADPSKIPERAVQLMQQVASNGATLPSALSASKSNLVISDPIPGAKPLPVP 280 IRTKS YHLQSAELLNSKLPRWQETCVDAIKVPEKIMNMIEEIKTPASTPVSGTPQASPMIERSNVVRKDYDTLSK 279

IRSp53 PELAPFVGRMSAQESTPIMNGVTGPDGEDYSPWADRKAAQPKSLSPPQSQSKLSDSYSNTLPVRKSVTPK 350 IRTKS CSPKMPPAPSGRAYTSPLIDMFNNPATAAPNSQRVN------315

IRSp53 NSYATTENKTLPRSSSMAAGLERNGRMRVKAIFSHAAGDNSTLLSFKEGDLITLLVPEARDGWHYGESEK 420 IRTKS NSTGTSEDPSLQRSVSVATGLNMMKKQKVKTIFPHTAGSNKTLLSFAQGDVITLLIPEEKDGWLYGEHDV 385

IRSp53 TKMRGWFPFSYTRVLDSDGSDRLHMSLQQG---KSSSTGNLLDKDDLAIPPPDYGAASRAFPAQTASGFK 487 IRTKS SKARGWFPSSYTKLLEENETEAVTVPTPSPTPVRSISTVNLSENSSVVIPPPDYLECLSMGAAADRRADS 455

IRSp53 QRPYSVAVPAFSQGLDDYGARSMSSGSGTLVSTV 521 IRTKS ARTTSTFKAPASKPETAAPNDANGTAKPPFLSGENPFATVKLRPTVTNDRSAPIIR 511

B

IRTKS TAKPPFLSGENPFATVKLRPTVTNDRSAPIIR 511 IRSp53-M DDYGARSMSRNPFAHVQLKPTVTNDRSAPLLS 534 IRSp53-L DDYGARSMSRNPFAHVQLKPTVTNDRCDLSAQGPEGREHGDGSARTLAGR 552 Fig. 2. GTPase binding of IRSp53 and IRTKS. IRSp53-T DDYGARSMSSADVEVARF 520 GST-fused constitutively active (L61) or IRSp53-S DDYGARSMSSGSGTLVSTV 521 dominant negative (N17) Cdc42 and Rac bound to glutathione beads were incubated with extract Fig. 1. Alignment of the protein sequences of IRSp53 and IRTKS. (A) Human IRSp53 from COS7 cells expressing Myc-tagged

Journal of Cell Science (splice variant S, accession no. BAC57948) aligned with human IRTKS (NP_061330). constructs (as indicated) or with untransfected Identical residues are highlighted in black. The region boxed with a solid line is the C2C12 extract (endogenous). Bead-bound IMD, that with a dash-dot line is the partial CRIB motif of IRSp53, that with a dashed material was analysed by immunoblotting line is the SH3 domain and that with a dotted line is the putative WW domain interacting alongside an equivalent fraction of the original motif. (B) Alignment of C-terminal region of IRTKS with the variable C-terminal extracts. Blots were probed with anti-Myc regions of four splice variants of IRSp53; IRSp53-L (BAC57945), IRSp53-M (9E10). FL, full-length protein; aa, amino acid (BAC57946), IRSp53-T (BAC57947) and IRSp53-S (BAC57948). Splice variant names residues present in truncated constructs. IRTKS are as defined by Miyahara et al. (Miyahara et al., 2003). Dashed box indicates region exhibits IMD-mediated binding to both Rac present in all splice variants of IRSp53. mutants.

terminal 230 amino acids and also to Cdc42 via a partial CRIB (Fig. 2). As observed for IRSp53, binding of IRTKS to Rac motif located between residues 268 and 280 (Govind et al., was mediated by the N-terminal 240 amino acids. The GTPase 2001; Krugmann et al., 2001; Miki et al., 2000). The Rac binding characteristics of endogenous IRTKS were also binding site is within the IMD, which is well conserved studied using extract from C2C12 cells. Binding to both Rac between IRSp53 and IRTKS, whereas the Cdc42 binding site mutants, but not to either Cdc42 mutant was detected, is found within a region of poor conservation between the two consistent with the results obtained with the exogenously proteins. Binding to the two GTPases was studied by GST expressed Myc-IRTKS. pulldown assay using Myc-tagged IRSp53 and IRTKS expressed in COS7 cells and GST-GTPases expressed in E. Expression of IRTKS in COS7 cells affects the actin coli. Full-length IRSp53 bound to the constitutively active L61 cytoskeleton mutant of Cdc42 but not the dominant negative N17 mutant, We compared the effects on the actin cytoskeleton of whereas binding to both Rac mutants could be detected (Fig. expression of IRTKS and IRSp53 in COS7 cells. Expression 2). The Rac-binding site on IRSp53 was located within the N- of IRSp53 results in the formation of numerous long, often terminal 241 amino acids, and the Cdc42-binding site was wavy filopodia-like extensions, to which IRSp53 is localised within amino acids 242-521, consistent with published data. (Fig. 3A,B) (Govind et al., 2001; Krugmann et al., 2001; Full-length IRTKS bound similarly to both L61 and N17 Rac Millard et al., 2005; Yamagishi et al., 2004). Overexpression mutants, but no binding to either Cdc42 mutant was detected of IRTKS resulted in a clearly distinct actin phenotype. In cells 1666 Journal of Cell Science 120 (9)

expressing relatively low levels of Myc-IRTKS numerous small actin microspikes were observed at the cell periphery (Fig. 3C,E). There was also a notable clearing of F-actin from the cytoplasm, which could be the result of reorganisation of the filaments or depolymerisation, but it was not possible to determine the cause. At higher expression levels, the actin microspikes induced by IRTKS appeared to coalesce into clusters of brightly stained protrusions (Fig. 3D,F). In cells expressing the highest IRTKS levels, the majority of cellular F-actin was observed within these actin clusters. In confluent monolayers of IRTKS-expressing cells, the actin clusters were notably abundant along cell junctions (Fig. 3G,H). In all cases the Myc-IRTKS was localised to these actin structures, but not necessarily concentrated there. Dense clusters of F-actin such as those observed in Myc-IRTKS-expressing cells were never observed on overexpression of IRSp53. Expression of a construct lacking the IMD of IRTKS failed to induce formation of the actin clusters (data not shown), indicating that the IMD is required for formation of the clusters. Although it would also be desirable to study the effects of knockdown of IRTKS expression on the actin cytoskeleton, we have, thus far, been unable to achieve more than about 50% knockdown, which does not result in a detectable phenotype (data not shown).

The C-terminus of IRTKS modulates its effects on the actin cytoskeleton Despite a high level of sequence similarity, overexpression of IRSp53 and IRTKS resulted in very different effects on the actin cytoskeleton. We reasoned that this difference in behaviour may be caused by a region of sequence divergence between the proteins. A notable difference between the form of IRSp53 studied here and IRTKS is the presence of an extension at the C terminus of IRTKS (Fig. 1). As previously noted, this C-terminal region has sequence similarity with the

Journal of Cell Science WH2 domain, a common actin monomer-binding motif. This C-terminal region is similar to, but distinct from, that of the L and M forms of IRSp53 (Fig. 1), which we address later in this manuscript. An IRTKS construct lacking the C-terminal 24 amino acids was expressed in COS7 cells. Expression of this construct did not result in the formation of actin clusters, as Fig. 3. Expression of IRSp53 and IRTKS in COS7 cells. COS7 cells was observed for full-length IRTKS. Instead, this construct were transfected with Myc-tagged IRSp53 and IRTKS constructs induced the formation of long filopodia, reminiscent of those and 24 hours later fixed and stained with 9E10 and Alexa Fluor induced by IRSp53 (Fig. 3I,J). This suggested that this C- 488-anti mouse and TRITC-phalloidin. Left hand panels show terminal (Ct) extension of IRTKS is important for the staining of the Myc-tagged proteins and the right hand panels show formation of the actin clusters. We prepared full-length IRSp53 the same field of view with F-actin stained. Cells expressing Myc- fused to the Ct extension of IRTKS (amino acids 483-511). IRSp53 exhibit long filopodia-like extensions (A,B). Cells expressing low levels of Myc-IRTKS exhibit short actin microspikes Expression of this construct (IRSp53+Ct) resulted in the (C,E), which at higher expression levels coalesce into clusters of formation of actin clusters similar to those observed on short spikes (D,F). Confluent cells expressing Myc-IRTKS have a expression of full-length IRTKS (Fig. 3K,L). These data high concentration of clustered actin bundles at cell junctions (G,H). suggest that the unique Ct extension of IRTKS is important in Cells expressing Myc-IRTKS with the Ct extension removed controlling the effect of IRTKS on the actin cytoskeleton and produce long wavy filopodia-like extensions and not actin clusters appears to convert a filopodia-inducing activity into an actin (I,J), whereas cells expressing Myc-IRSp53 with IRTKS Ct cluster-forming activity. extension fused produce actin clusters (K,L). Boxed insets are The L isoform of IRSp53 has a C-terminal region similar to magnifications of the indicated region of the image and highlight ␮ that of IRTKS, so we tested the effect of expression of this actin structures typically induced by the construct. Bars, 20 m. The isoform. We found that expression IRSp53-L resulted in a images shown are representative of more than three independent transfections per construct, where more than 100 cells have been phenotype indistinguishable from that of IRSp53-S (Fig. 4). In viewed per transfection. For IRSp53, over 80% of cells have the addition to (or perhaps because of) the different changes that described phenotype of long filopodial protrusions, and for IRSp60, expression of IRSp53 and IRTKS effect on the actin 30-40% of cells show no change in shape, whereas 60-70% have cytoskeleton, the cell shape also is affected differently by these short actin clusters and spikes. Bars, 20 ␮m. two proteins. Cells expressing IRSp53 S or L isoforms (Fig. 4) IRTKS: a novel IMD-containing actin regulator 1667

typically have long thin filopodial extensions and a diminished extent of the other proteins, indicating that perhaps the clusters cell surface area, as measured by drawing around the periphery contained some focal-complex-like structures, but were not of the cells (Fig. 4). The area of IRSp53 (S or L forms)- major sites of focal adhesion formation (Fig. 5I-L). We also expressing cells was roughly half of the control (GFP alone), observed similar co-localisation for IRSp53+Ct expression for IRTKS or IRSp53+Ct-expressing cells. This could be because VASP (Fig. S2A-D in supplementary material) and vinculin the long filopodial extensions make up half of the area in (Fig. S2E-H in supplementary material), cortactin (not shown) IRSp53-expressing cells, but we were not able to quantify and Arp2/3 complex (not shown), indicating that the structures these long protrusions. An alternative explanation is that formed by IRSp53+Ct and IRTKS expression were of similar IRSp53 expression reduces the surface area by membrane composition and appearance. tubulation or cell retraction. Since it was previously shown that the protrusion-generating To further characterise the actin clusters observed with ability of IRSp53 depends on four basic residues near the distal IRTKS expression, we also examined the location of VASP, poles of the IMD (Millard et al., 2005), we mutated the four vinculin, Arp2/3 complex and cortactin in IRTKS-expressing corresponding lysine residues in IRTKS IMD: K141E, K142E, cells. VASP is typically found in filopodia, focal adhesions and R145E, K146E and found that this protein could no longer lamellipodia, whereas cortactin localises in lamellipodia, and induce actin clusters (Fig. S3 in supplementary material). vinculin is found in focal complexes and focal adhesions, so these proteins served as markers of various cytoskeletal IRTKS possesses a functional IMD actin bundling structures. Fig. 5A-D shows that VASP localises abundantly in domain IRTKS-induced actin clusters. Cortactin (Fig. 5E-H) and The IMD of IRSp53 and MIM-B have previously been Arp2/3 complex (not shown), markers for dynamic actin demonstrated to bundle actin filaments in vitro (Millard et al., networks, were also highly enriched in these structures. 2005; Yamagishi et al., 2004). To test whether the Vinculin was present in patches coinciding with IRTKS- corresponding region of IRTKS bundles actin, amino acids 1- induced actin clusters, but did not generally co-localise to the 249 (IMD) were expressed as a GST-fusion in E. coli, and purified to homogeneity after cleavage from the GST. Bundling was then assessed using a low speed co- sedimentation assay in which F-actin was incubated with varying concentration of IRTKS IMD and then centrifugation at 10,000 g. The presence of IRTKS IMD resulted in a concentration-dependent increase in actin pelleting, indicating filament bundling by IRTKS IMD (Fig. S4A in supplementary material). This demonstrates the amino acids 1-249 of IRTKS constitutes a functional IMD. In addition, we wished to study the effect of the IRTKS Ct extension on the in vitro bundling characteristics of IRTKS, but we were unable to generate

Journal of Cell Science purified full length IRTKS recombinantly. We thus tested the ability of a shortened version of IRTKS, termed IMD-Ct, composed of the IMD of IRTKS (amino acids 1-253) fused to the Ct extension (amino acids 483-511). We did not find any difference in the bundling ability of IMD-Ct compared with IMD alone (Fig. S4B in supplementary material).

The IRTKS Ct extension modulates bundling by the IMD in vitro We wished to study the effect of the IRTKS Ct extension on the in vitro bundling characteristics of IRTKS. The IMD-Ct recombinant protein was incubated with actin filaments fluorescently labelled with Cy3 and the resulting mixture was then visualised using a fluorescence microscope. We and others have previously used this assay to observe bundles formed by the IMD of IRSp53 (Millard et al., 2005; Yamagishi et al., 2004). The IMD of IRTKS resulted in the formation of long bundles, frequently greater than 100 ␮m in length, which were Fig. 4. Cell area changes in cells expressing IRSp53 or IRTKS Myc- similar to those we have previously observed using the IMD tagged IRTKS, IRSp53-S, IRSp53-L, IRSp53+Ct or GFP (control) of IRSp53 (Fig. 6A) (Millard et al., 2005). By contrast, the were expressed in COS7 cells as shown. IRSp53-S and IRSp53-L bundles induced by the IMD-Ct were noticeably shorter constructs decreased the area of the cell body in contrast to IRTKS ␮ and IRSp53+Ct. IRSp53-S and IRSp53-L have significantly reduced (usually <20 m) and were frequently found in small clusters cell body areas compared with COS7 cells overexpressing only GFP. (Fig. 6B). The same was observed if the Ct extension of IRTKS The graph shows the mean of three independent experiments in was fused to the IMD of IRSp53 (data not shown). Without which 30 cells were measured for each construct per experiment ± added bundling protein, the individual filaments were too small s.e.m. *P<0.05 compared with GFP control cells. The cell body area and disordered to resolve (Fig. 6C). This demonstrates that the is drawn in red on each image. Bar, 10 ␮m. Ct extension of IRTKS modifies the actin bundling 1668 Journal of Cell Science 120 (9)

characteristics of the IMD. Notably, the effect of the Ct binding and are conserved in all WH2 domains (Paunola et al., extension on bundling in vitro mirrors that observed in vivo, 2002; Van Troys et al., 1996). The Ct extension of IRTKS lacks in that it switches from induction of long straight bundles (such one of these residues, an invariant isoleucine, suggesting it may as those found in long filopodia) to short, clustered bundles. not function as a conventional WH2 domain (Fig. 7A). We We were unable to produce recombinant IMD fused to the C- prepared a construct consisting of the Ct extension of IRTKS terminal region of IRSp53, so we cannot rule out the possibility (aa 483-511) fused to the C terminus of GST (GST-Ct) to test that addition of this short extension might also alter bundling for actin monomer binding. As a control, a construct consisting characteristics of the IMD. We did not observe any qualitative of the WH2 domain of Scar/WAVE1 (residues 496-528) fused differences between IRTKS IMD and IRTKS + Ct IMD in to the C terminus of GST (GST-ScarWH2) was used. In vitro, standard low-speed pelleting assays to measure actin bundling WH2 domains sequester monomers and this results in an (Fig. 6D,E), nor was this data noticeably different from that of increase in the apparent critical concentration, which is the IRSp53 IMD (Millard et al., 2005) or MIM IMD (Bompard et lowest actin concentration at which polymerisation can occur. al., 2005). It would be an interesting next step to characterise The critical concentration can be measured by recording the the IRTKS bundles by electron microscopy as has been done fluorescence of a range of concentrations of pyrene actin at for IRSp53 IMD (Yamagishi et al., 2004). equilibrium (Carlier et al., 1993). The addition of GST- ScarWH2 to actin resulted in a clear increase in the apparent Actin binding by the Ct extension of IRTKS critical concentration from 0.17 ␮M ± 0.01 to 0.65 ␮M ± 0.1 As previously noted by Yamagishi et al. alignment of the Ct (± s.d.; based on data from three experiments; Fig. 7B). By extension of IRTKS with other proteins shows that there is contrast, the GST-Ct had no significant effect on the apparent clear similarity between the Ct extension of IRTKS and the critical concentration (0.18 ␮M ± 0.06), suggesting no WH2 domain located at the C terminus of MIM, as well as the sequestration of monomers. This indicates that the Ct extension WH2 domain of WASP interacting protein (WIP) (Fig. 7A). of IRTKS is not a monomer sequestering WH2 motif. To WH2 domains are actin monomer-binding motifs found within address whether there might be direct actin monomer binding many actin regulators (Paavilainen et al., 2004; Paunola et al., between this WH2 motif and G-actin, we attempted to 2002). Mutational analysis of the WH2 domain of thymosin- synthesise peptides corresponding to the WH2 to allow ‘in- ␤4 identified four residues that are critical for actin monomer solution’ actin binding studies to fluorescent peptides. However, the peptides proved difficult to synthesise, precluding this analysis. Instead, we used a native gel shift assay with GST-Ct and GST-ScarWH2 to determine their ability to shift G-actin from its normal migration on the gel (Costa et al., 2004). Whereas 10 ␮M GST-ScarWH2 shifted 2.5 ␮M G-actin ␮ Journal of Cell Science quantitatively on this assay, 10 M GST- Ct caused only a slight smearing of the actin band and shifted very little to the higher mobility position (data not shown). We performed this experiment three times with similar results and we can conclude that GST-Ct likely has a very weak affinity for actin monomers, which may explain why it does not effectively sequester them. Further experiments to measure the affinities of the WH2 motifs in the context of the full- length proteins will need to be performed to completely resolve this issue. Since WH2 proteins have also been shown to be actin-filament-binding

Fig. 5. IRTKS co-localises with F-actin, cortactin, VASP and vinculin. COS7 cells were transfected with Myc-tagged IRTKS and labelled for Myc and F-actin, along with VASP, cortactin or vinculin. Myc-IRTKS co- localises with VASP (A-D, inset in D), cortactin (E-H, inset in H) and vinculin (I-L, inset in L) at sites of cell-cell contact where aggregates of F-actin (C,G,K) are observed. Bar, 20 ␮m. IRTKS: a novel IMD-containing actin regulator 1669

proteins in some cases (Irobi et al., 2004), we next tested the ability of the GST-Ct and GST-ScarWH2 to bind actin filaments using a co-sedimentation assay. Owing to the monomer-sequestering activity of GST-ScarWH2, the presence of this construct resulted in a reduction in the proportion of actin that was polymerised. To achieve consistent levels of F- actin we therefore stabilised filaments with phalloidin. This did not appreciably affect binding of either construct to filaments. We found that GST- Ct co-sedimented with actin filaments in a concentration-dependent and saturable manner, whereas little GST-ScarWH2 co-sedimented with filaments (Fig. 7C). A Kd of approximately 1 ␮M was obtained for the binding of GST-Ct to filaments. This suggests that the Ct extension of IRTKS may act as an F-actin binding motif.

Discussion The identification and characterisation of a new conserved actin bundling and membrane interacting domain, the IMD, has considerably progressed our Fig. 6. In vitro filament bundling. (A-C) Fluorescently labelled actin filaments understanding of how the IRSp53 and MIM proteins were incubated with IRTKS IMD (A), with IRTKS IMD-Ct (B) or with no function (Millard et al., 2005; Yamagishi et al., additions (C) then imaged using a fluorescence microscope. IMD induces long 2004). In this study, we describe the characterisation bundles, whereas IMD-Ct induces short, clustered bundles. Bars, 20 ␮m. of IRTKS, an IRSp53-related protein that possesses (D,E) 5 ␮M F-actin was incubated with the indicated concentrations of IRTKS an IMD and a WASP homology WH2 motif. We IMD or IMD + Ct and then centrifuged at 10 000 g. Pellets and supernatants propose that IRTKS has a wide tissue distribution were analysed by SDS-PAGE after staining with Coomassie Blue. There is an IMD concentration-dependent increase in pelleting of actin, indicating filament and different biochemical properties to IRSp53. bundling. Because IRSp53 has several splice variants and isoforms, it is important to carefully define probes used for detection of related proteins. To avoid confusion, we induces long filopodia-like protrusions, IRTKS expression have specifically determined that our antibody does not react results in clusters of short actin clusters around the cell with IRSp53 (not shown) and that it does immunoprecipitate a periphery. These clusters co-localise with endogenous

Journal of Cell Science protein matching the sequence of IRTKS, using Fourier cortactin, Arp2/3 complex and VASP, and to a lesser extent transform ion cyclotron resonance (FTICR) mass contain some vinculin. We find that a short extension at the C spectrometry. Therefore, we present evidence that although terminus of IRTKS is important in producing this effect. IRSp53 has been shown to be highly enriched in brain tissues Removal of the C-terminal extension, causes IRTKS to induce (Govind et al., 2001; Okamura-Oho et al., 2001), IRTKS is low long filopodia-like protrusions, reminiscent of those induced or nonexistent in brain but is present in bladder, liver, testes, by IRSp53, rather than actin clusters. Conversely, fusing the C heart and lung tissue. Like IRSp53, IRTKS can be tyrosine terminus of IRTKS on to IRSp53 causes actin clusters rather phosphorylated in an insulin-signalling-dependent manner. than filopodia to be induced by IRSp53. Notably, expression The IMD regions of IRTKS and IRSp53 appear to have of IRSp53-L, a splice variant of IRSp53 with a C-terminus largely the same properties biochemically and when homologous to that of IRTKS, does not result in the formation expressed in cells. We found that the N-terminal 249 amino of actin clusters, and it would be interesting to identify acids of IRTKS bundle actin filaments similarly to the precisely which residues are responsible for the divergent IRSp53 IMD and similarly to MIM IMD (Bompard et al., behaviours of these two proteins. We find that, when fused to 2005). The IMD of IRTKS also binds to Rac, in both the GTP the IMD, this extension also affects IMD-induced bundling in and GDP forms. Sequence conservation among IRSp53, vitro, causing the formation of shorter, often clustered bundles. IRTKS and MIM reveals a cluster of basic residues that are We attempted to test whether the shorter clusters might be important for the actin bundling (Millard et al., 2005), which formed as a result of severing or depolymerisation activity also appears to be important for the interaction with Rac of IRTKS, using pyrene-labelled actin fluorescence (Bompard, 2005) and probably with phosphoinositides depolymerisation assays, but we could not obtain reliable data, (Suetsugu et al., 2006). perhaps due to solubility problems with the IMD proteins at Expression of the IMD of MIM, IRSp53 (Yamagishi et al., low salt concentrations (Millard et al., 2005). When IMD-Ct 2004) or IRTKS (this study) produces a similar effect of is expressed in cells, similar structures to those observed with filopodia-like structures protruding from the periphery of IRTKS expression are also formed. This suggests that the C- expressing cells. However, expression of full-length IRSp53, terminal extension is important for the actin organising IRTKS and MIM cause clearly distinct changes in actin function of IRTKS. The Ct extension of IRTKS has sequence cytoskeletal organisation, suggesting that the divergent regions similarity to the WH2 motif, an actin monomer binding of these proteins modulate their behaviour. Whereas IRSp53 sequence (Paunola et al., 2002). However, we find that the Ct 1670 Journal of Cell Science 120 (9)

extension binds to F-actin but does not sequester monomers been shown to bind F-actin, but only relatively poorly to G- and only very weakly shifts the actin mobility in native gel actin (Van Troys et al., 2004). Furthermore, espins are actin- assays (data not shown). This is not without precedent, since bundling proteins that contain a WH2 motif, which although it one of four WH2 domains found within tetrathymosin␤ has binds to G-actin also aids in bundle formation (Loomis et al., 2006). It is not yet clear how the filament-binding activity of the Ct extension contributes to bundling or organisation of actin. One possibility is that interaction with a second filament binding site outside the IMD alters the orientation of bundled filaments to produce non-parallel, poorly ordered bundles. This could explain why IRTKS frequently induces clustered structures rather than distinct, parallel bundles. Another possibility is that filament binding by the Ct extension causes severing or destabilisation of filaments. Binding of ADF and/or cofilins to F-actin causes a small, destabilising twist in the filament, which leads to depolymerisation (Paavilainen et al., 2004). Filament severing would give rise to shortened bundles (Maciver et al., 1991). The regulation of IRSp53 and IRTKS by GTPases remains largely unexplored. Interestingly, IRSp53 binds to Cdc42, a known activator of filopodia formation, whereas IRTKS does not. However, the interaction with Cdc42 is not required for filopodia induction, as the IMD alone will produce filopodia- like protrusions when expressed in cells. All IMD proteins have numerous protein interaction motifs and as such act as scaffolds as well as effectors. Interactions with upstream regulators could recruit IMD proteins to particular cellular locations, for instance to filopodia by Cdc42 or to lamellipodia by Rac. The lack of a Cdc42-binding site in IRTKS may suggest that it plays a role in regulating lamellipodia, rather than filopodia. The relative importance of the bundling activity of the IMD with the activities of recruited binding partners or other regions of the proteins has not yet been determined, and will no doubt be the subject of future studies. Journal of Cell Science Materials and Methods Chemicals and reagents Monoclonal antibodies 9E10 (anti-Myc) and PY72 (anti-phosphotyrosine) and protein G beads were obtained from Cancer Research U.K. Anti-p34-Arc of the Arp2/3 complex and anti-cortactin were obtained from Upstate Biotechnology, VASP273 was a kind gift from Juergen Wehland (Braunschweig, Germany). Alexa Fluor 488-conjugated anti-mouse secondary antibody was obtained from Molecular Probes. Unless otherwise stated all chemicals and reagents were purchased from Sigma-Aldrich UK.

Fig. 7. Actin binding by the IRTKS Ct extension. (A) Alignment of Cell culture, transfection and immunocytochemistry IRTKS C-terminal extension with the WH2 domains of five human COS7 and C2C12 cells were grown in DMEM containing 10% FBS (Gibco-BRL), proteins. Residues conserved in at least three of the displayed WH2 supplemented with penicillin and streptomycin at 37°C, 5% CO2. Cells were domains are highlighted in black. Residues in IRTKS Ct conserved transfected using GeneJuice (Novagen) according to the manufacturer’s instructions in the WH2 domains are shown in grey. Residues critical for actin and fixed or lysed 24 hours later. Cells were fixed using 4% formaldehyde, blocked ␤ with 50 mM NH4Cl and permeabilised with 0.1% Triton X-100 as previously monomer binding by thymosin 4 are indicated with asterisks. described (Millard et al., 2005). After incubation in appropriate antibodies, (B) Measurement of actin critical concentration by monitoring coverslips were mounted in Mowiol and antifade (p-phenylenediamine) as pyrene-actin fluorescence at a range of actin concentrations, alone or previously described (Millard et al., 2005). in the presence of 2.5 ␮M GST-Ct or GST-ScarWH2. Polymerisation of actin results in an increase in the gradient of fluorescence change Plasmids with actin concentration and the critical concentration is derived cDNA encoding IRTKS (accession no. BC013888) was obtained from MRC gene service (Cambridge, UK). The coding sequence was amplified by PCR and cloned from the intercept of the pre- and post-polymerisation gradients. into pRK5Myc (Lamarche et al., 1996). IRSp53pRK5Myc and GTPase constructs Data shown are from three replicates of a single representative in pGEX2T were gifts from Dr Alan Hall (Krugmann et al., 2001). Truncated forms experiment. (C) Co-sedimentation assay at a range of F-actin of IRSp53 and IRTKS were generated by PCR amplification and products cloned concentrations showing binding of GST-Ct and GST-ScarWH2 to into pRK5Myc for mammalian expression or pGEX4T2 (Amersham Pharmacia) for ␤ phalloidin stabilised F-actin. The quantification is described in the bacterial expression. Insulin receptor subunit expression construct was a gift from Jeremy Tavare (University of Bristol, UK). Enzymes were purchased from New Materials and Methods. The binding curve fitted for GST-Ct gives a England Biolabs. Kd of approximately 1 ␮M. GST-ScarWH2 data could not be fitted to a binding curve. Data shown are from three replicates of a single Production of polyclonal antibodies representative experiment. Peptides CLIEISSTHKKLNESLD and CIEYVETVTSRQSEIQK (peptide 2 is IRTKS: a novel IMD-containing actin regulator 1671

identical in mouse and human, whereas peptide 1 has one S to T change at position was loaded on a 7.5% native gel as previously described (Costa et al., 2004; 15 in mouse) corresponding to residues 75-90 and 161-176, respectively, of human Palmgren et al., 2001). The gel was stained with Coomassie Blue dye and a shift IRTKS, were synthesised by Alta Biosciences (Birmingham UK). Peptides were upwards in the mobility of the G-actin was considered to indicate binding. coupled to maleimide-activated keyhole limpet haemocyanin (Pierce) according to the manufacturer’s instructions and conjugates were used to immunise rabbits F-actin bundling assays (Eurogentec). The same peptides were coupled to Affigel 10 (Bio-Rad) and used to IRTKS IMD and IRTKS IMD-Ct were produced and in E. coli and purified as affinity purify antisera. described previously for IRSp53 IMD (Millard et al., 2005). Low speed co- sedimentation assays were performed as previously described, except that Immunoprecipitation and mass spectrometry centrifugation was performed at 10 000 g instead of 16 000 g (Millard et al., 2005). Four 10 cm dishes of confluent undifferentiated C2C12 cells were lysed in 150 mM Bundling of fluorescent filaments was performed as previously described (Millard NaCl, 20 mM, Tris-HCl pH 7.5, 1% N-octyl-␤-D-glucopyranoside supplemented et al., 2005). with the protease inhibitors chymostatin, leupeptin, antipain, pepstatin A (all 1 ␮g/ml) and 1 mM phenylmethylsulfonyl fluoride. Lysates were incubated with 20 We thank Guillaume Bompard for careful reading of the manuscript ␮g of the affinity purified IRTKS antibody bound to protein G beads. Following and helpful suggestions throughout this study. We thank Tim Dafforn several washes with lysis buffer samples were boiled in sample buffer and analysed for help with the linear regression analysis. We thank Anthony Jones by SDS-PAGE. Following staining with Coomassie Blue, a 60 kDa band was and Helen Cooper for the FTICR analysis of IRTKS. This work was excised. Trypsin digestion of the sample and mass spectrometry using a MicroMass Q-TOF Global was carried out by University of Birmingham functional genomics supported by an MRC Senior fellowship to L.M.M. (G117/399). J.D. and proteomics laboratories. is supported by an AICR grant to L.M.M. (04-009).

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Elastic behavior of cross-linked and bundled actin networks. IRSp53-p60 constructs were transfected into COS7 cells, which, 24 hours later, Science 304, 1301-1305. were lysed in 20 mM Tris-HCl pH 7.5, 0.5% NP-40, 50 mM NaCl, 5% glycerol, Gonzalez-Quevedo, R., Shoffer, M., Horng, L. and Oro, A. E. (2005). Receptor Journal of Cell Science protease inhibitors (as above). An equal volume of cell lysate was added to each tyrosine phosphatase-dependent cytoskeletal remodeling by the hedgehog-responsive set of GST-GTPase beads. 20 ␮g of GST-GTPase was used per pulldown. Beads gene MIM/BEG4. J. Cell Biol. 168, 453-463. were rotated for 1 hour at 4°C and they were then washed with lysis buffer, boiled Govind, S., Kozma, R., Monfries, C., Lim, L. and Ahmed, S. (2001). Cdc42Hs in Laemmli buffer and subjected to SDS-PAGE and western blotting. facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin. J. 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