Tara, a Novel F-Actin Binding Protein, Associates with the Trio Guanine Nucleotide Exchange Factor and Regulates Actin Cytoskeletal Organization

RESEARCH ARTICLE 389 Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization

Katja Seipel1,2, Stephen P. O’Brien1, Elizabeth Iannotti1, Quintus G. Medley1,2 and Michel Streuli1,2,* 1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA 2Department of Pathology, Harvard Medical School, Boston, MA 02115, USA *Author for correspondence (e-mail: [email protected])

Accepted 13 November 2000 Journal of Cell Science 114, 389-399 © The Company of Biologists Ltd

SUMMARY

Reorganization of the actin cytoskeleton is essential to similar to the pattern of myosin II. Furthermore, a direct numerous cellular processes including cell locomotion and interaction between Tara and F-actin is indicated by in cytokinesis. This actin remodeling is regulated in part by vitro binding studies. Cells that transiently or stably Rho family GTPases. Previous studies implicated Trio, a overexpress Tara display an extensively flattened cell Dbl-homology guanine nucleotide exchange factor with two morphology with enhanced stress fibers and cortical F- exchange factor domains, in regulating actin cytoskeleton actin. Tara expression does not alter the ability of the cell reorganization, cell motility and cell growth via activation to attach or to initially spread, but rather increases cell of Rho GTPases. Trio is essential for mouse embryonic spreading following these initial events. Tara stabilizes F- development and Trio-deficiency is associated with actin structures as indicated by the relative resistance abnormal skeletal muscle and neural tissue development. of Tara-expressing cells to the F-actin destabilizer Furthermore, genetic analyses in Caenorhabditis elegans Latrunculin B. We propose that Tara regulates actin and Drosophila demonstrate a role for trio-like genes in cell cytoskeletal organization by directly binding and migration and axon guidance. Herein we characterize a stabilizing F-actin, and that the localized formation of novel Trio-binding protein, Tara, that is comprised of an Tara and Trio complexes functions to coordinate actin N-terminal pleckstrin homology domain and a C-terminal remodeling. coiled-coil region. Trio and Tara associate as assessed by the yeast interaction-trap assays and mammalian co- immunoprecipitation studies. Ectopically expressed Tara Key words: Rho, Rac, Dbl homology, Trio, Actin-binding protein, localizes to F-actin in a periodic pattern that is highly Pleckstrin homology, Coiled-coil

INTRODUCTION correlated with loss of cell contractility (Daniels and Bokoch, 1999; Sanders et al., 1999; van Leeuwen et al., 1999). Key The actin cytoskeleton is a dynamic structure that is upstream regulators of actomyosin-based contraction are extensively remodeled in migrating and proliferating cells. members of the Rho family of GTPases, including Rho and This actin remodeling, which involves the assembly and Rac (Hall, 1998; Van Aelst and D’Souza-Schorey, 1997). Rac disassembly of filamentous (F)-actin, is regulated by diverse activation leads to membrane ruffling and cell spreading actin binding proteins, including proteins that function in F- concomitant with loss of contractility and reduced cell actin formation, F-actin stabilization and destabilization, adhesion to the substratum (Nobes and Hall, 1995; van generation of supramolecular cytoskeletal structures, and Leeuwen et al., 1999), whereas Rho activation leads to regulation of cell contraction (Ayscough, 1998; Borisy and bundling of F-actin and myosin II into stress fibers, formation Svitkina, 2000; Cooper and Schafer, 2000). In migrating cells, of focal adhesions, and increased cell contractility (Amano actin polymerization drives the formation and extension of the et al., 1996; Chrzanowska-Wodnicka and Burridge, 1996; lammellipodial leading edge, while the activation of myosin Kimura et al., 1996; Nobes and Hall, 1995). These processes motors, particularly myosin II, is largely responsible for occur in a spatial and temporal sequence in migrating cells, creating the traction force needed for cell movement under the control of the coordinated activation of Rho GTPases (Condeelis, 1993; Lauffenburger and Horwitz, 1996; (Hall, 1998; Van Aelst and D’Souza-Schorey, 1997). Mitchison and Cramer, 1996). In non-muscle cells, myosin Several Rho GTPase effector targets have been identified contractile activity is regulated mainly by serine/threonine that regulate actomyosin-based contractility (Aspenstrom, phosphorylation. Phosphorylation of the regulatory myosin 1999; Schoenwaelder and Burridge, 1999). Rho kinase light chain increases myosin activity, as well as the association increases myosin II activity and cell contraction by inhibiting of myosin with F-actin, and leads to cell contraction, whereas phosphatase activity towards the myosin light chain (MLC) phosphorylation of the non-muscle myosin II heavy chain is (Kimura et al., 1996). Rho kinase also increases myosin 390 JOURNAL OF CELL SCIENCE 114 (2) activity by directly phosphorylating the MLC (Amano et al., as described (Gyuris et al., 1993; Serra-Pages et al., 1995). The human 1996). Rho also mediates formation of stress fibers by fibroblast cell WI-38 (ATCC CCL 75) cDNA library used for the activating Dia1, which may localize profilin-bound actin to interaction-trap assay was kindly provided by Dr Claude Sardet. The sites of F-actin assembly (Watanabe et al., 1999). The target of extent of the Trio peptide (amino acids 1207-1715) fused to the LexA Rac and Cdc42 GTPases, p21 activated kinase (Pak), peptide is schematically shown in Fig. 1A. The yeast strain EGY48 negatively regulates MLC kinase (MLCK) activity (Daniels et (MATa trp1 ura3 his3 LEU2::pLexAop6-LEU2), containing the plasmid pSH18-34, which includes the reporter lacΖ gene under the al., 1998; Manser et al., 1997; Sells et al., 1997). Furthermore, control of a modified Gal1 promoter, was used as host for all Rac activation leads to myosin heavy chain (MHC) interaction-trap assays. phosphorylation, perhaps mediated by Pak and Ca2+/ calmodulin kinases (van Leeuwen et al., 1999). Thus, Rac Plasmid constructions and DNA sequencing activation leads to reduced MLC phosphorylation, increased cDNA clones encoding Tara were isolated from a skeletal muscle MHC phosphorylation and loss of cell contractility, whereas λgt10 library (Clontech Laboratories, Palo Alto, CA) using standard Rho activation controls myosin activity by increasing MLC techniques and sequenced using the dideoxy method of sequencing. phosphorylation levels. The amino acids of Tara or Trio encoded by various expression vectors Rho GTPases are activated by Dbl-homology guanine or interaction-trap baits are given in Figs 1, 2. The following nucleotide exchange factors (DH-GEFs) (Cerione and Zheng, mammalian expression vectors were used: pMT.2, pMT.HA, pMT.GST, and pSP.SRα.2-HA. pMT.HA and pSP.SRα.2-HA encode 1996; Zohn et al., 1998). Among the DH-GEF family, Trio is initiator methionine codons followed by a HA epitope tag sequence unusual in that it contains two GEF domains, each specific for immediately upstream of the cloning site (Serra-Pages et al., 1995), different Rho family GTPases both in vitro and in vivo and pMT.GST encodes an initiator methionine codon followed by the (Bellanger et al., 1998; Blangy et al., 2000; Debant et al., GST coding sequence, followed by a cloning site. pMT.Tara encodes 1996; Seipel et al., 1999). Trio regulates actin cytoskeletal full-length Tara (amino acids 1-593), pMT.GST.H2 encodes a GST reorganization and cell growth and migration, possibly by fusion protein containing Tara amino acids 13-385, pMT.GST.Tara integrating diverse signals needed to coordinate actin encodes a GST fusion protein containing amino acids 1-593, cytoskeleton remodeling involved in cell migration and growth pGEX.Tara encodes a GST fusion protein containing amino acids 13- 385, pSP.SRa.Tara encodes full-length Tara (amino acids 1-593), and (Seipel et al., 1999). Absence of Trio causes mouse embryonic α lethality associated with abnormal development of skeletal pSP.SR .Tara.HA encodes full-length Tara (amino acids 1-593) with the HA epitope sequence located at the C-terminus. pMT2.HA.Trio muscle and neural tissues (O’Brien et al., 2000). In C. elegans, encodes HA-tagged full-length Trio (amino acids 1-3038; Seipel et the Trio-like unc-73 gene is necessary for both cell migration al., 1999). The human Tara cDNA sequence (GenBank Accession and axon guidance (Steven et al., 1998). Drosophila Trio was Number AF281030) is essentially identical to the previously shown to regulate axon guidance, and is functionally linked to deposited sequence, CAB42898. Rac, Pak, Abl protein tyrosine kinase, DLAR transmembrane protein tyrosine phosphatase, and the adaptor proteins Dock Cell labeling, protein analysis and protein purification and Ena (Awasaki et al., 2000; Bateman et al., 2000; Liebl et Cell proteins were metabolically labeled with [35S]methionine and al., 2000; Newsome et al., 2000). Mammalian Trio, but not C. [35S]cysteine (DuPont NEN, Boston, MA) in L-methionine- and L- elegans UNC-73 or Drosophila Trio, contains a kinase domain cysteine-free RPMI media (Life Technologies, Gaithersburg, MD) that is most closely related to Ca2+/calmodulin dependant containing 2% dialyzed fetal calf serum (FCS), 2 mM Hepes (pH 7.2) for 4 hours at 37oC. Following labeling, cells were washed in PBS, and kinases, but its substrates remain to be defined (Debant et al., lysed in NP-40 lysis buffer (1% Nonidet P-40 (NP-40), 150 mM NaCl, 1996). A mammalian neuronal-specific Trio-like protein, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA) containing 1 mM termed Kalirin, may function in the secretion of bioactive phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin and 10 peptides (Alam et al., 1997; Johnson et al., 2000; Mains et al., µg/ml aprotinin. Insoluble material was removed from the lysates by 1999). centrifugation in a microcentrifuge. Cell lysates were then precleared To learn more about Trio function, we screened for proteins with 25 µl Protein G-Sepharose slurry (Pharmacia) for 1 hour. For GST that interact with a portion of Trio that includes the N-terminal pull-downs, 0.5 ml of the lysates were incubated with 25 µl GEF-D1 domain. Herein, we describe and characterize the glutathione-Sepharose beads for 1 hour at 4oC. Following washing of the beads five times with PBS-0.1% NP-40, samples were separated Trio-binding protein Tara, which consists of an N-terminal µ pleckstrin homology (PH) domain and a C-terminal coiled-coil by SDS/PAGE. For immunoprecipitations, ~1 g anti-Tara.397 mAb, ~1 µg control isotype-matched mAb or 1 µl of ascites fluid of anti- region. Ectopically expressed Tara localizes in a periodic hemagglutinin mAb 12CA5 and 25 µl Protein G-sepharose slurry were fashion to filamentous (F)-actin and enhances cell spreading, added per 0.5 ml precleared lysate for 1 hour. Immunoprecipitates were suggesting that Tara regulates cell morphology. HeLa cell lines then washed with buffer containing 0.1% NP-40, 0.05% SDS, 150 mM stably expressing Tara are more resistant to Latrunculin B- NaCl and 50 mM Tris-HCl (pH 8.0). Immunoprecipitated proteins mediated F-actin destabilization. In vitro binding studies were analyzed using SDS-PAGE analysis with reducing conditions, indicate a direct interaction between F-actin and Tara. Based followed by autoradiography. Immunoblotting analysis was carried on these results, we propose that Tara regulates actin out using standard techniques. Proteins from cell lysates or cytoskeletal organization by stabilizing F-actin. immunoprecipitated proteins from non-labeled cells were resolved by SDS-PAGE, then transferred to nitrocellulose and immunoblots were probed with the anti-Tara.27 mAb, the anti-HA mAb HA 11, or the anti-GST mAb 112. Immunoblots were developed with ImmunoPure MATERIALS AND METHODS Protein A/G-horseradish peroxidase (Pierce, Rockford, IL) and the chemiluminescence reagent, luminol, essentially as described by the Interaction-trap assay supplier (DuPont NEN). For F-actin co-sedimentation assays, GST- Plasmid DNAs and yeast strains used for the interaction trap assay Tara protein was produced in COS7 cells using transient transfections. were provided by Dr Roger Brent and colleagues and used essentially Cells were lysed in 1% NP40 buffer, and then extracts were incubated Tara binds F-actin and enhances cell spreading 391 with glutathione-Sepharose beads for 1 hour at 4oC. Following the independent cell lines each) were treated for 30 minutes either with incubation, glutathione-Sepharose beads were recovered, washed six DMEM containing 10% FCS and 200 nM Latrunculin B (Calbiochem, times with PBS-0.1% NP-40. Tara protein was released by thrombin La Jolla, CA) or with 0.1% ethanol in DMEM as control. protease digestion for 1 hour at room temperature, and beads were removed by centrifugation. GST-Tara.13-385 fusion protein was Immunofluorescence produced in Escherichia coli, and the fusion protein was recovered by Cells were stained for Tara, Trio, F-actin, non-muscle myosin II heavy glutathione-Sepharose chromatography. Tara.13-385 protein was chain, α-actinin, vimentin or tubulin using the anti-Tara.397 mAb, released by thrombin digestion using conditions similar to those anti-Trio.18 mAb, phalloidin-TRITC (Sigma), anti-myosin II heavy described above. The amount of purified Tara.13-385 used for the F- chain mAb, anti-α-actinin mAb, the anti-HA mAbs 12CA5 and HA actin co-sedimentation assay (~0.5 µg) was approximated using bovine 11, anti-vimentin, anti-tubulin or isotype-matched control mAbs. serum albumin (BSA) standards. The full-length Tara protein was Cells were plated on glass coverslips ~18 hours following transfection highly susceptible to protease degradation in E. coli and could not be and grown for 1-2 days prior to staining. Cells were fixed in 2% produced in sufficient quantities. paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS for 10 minutes at room temperature (RT), incubated for 1 hour Northern blot analysis in blocking buffer (PBS containing 5% (v/v) normal goat serum) Northern blot analysis was carried out using a human multiple tissue supplemented with 0.1% sodium azide. Following incubation with northern blot (Clontech) and a multiple cell line blot (Clontech), both primary antibodies, cells were washed with PBS and then incubated of which contain 2 µg of poly(A)+ selected RNA from different human in blocking buffer containing FITC-labeled and Texas Red-labeled, tissues or cell lines per lane, and were hybridized with a random isotype-specific secondary antibodies (Southern Biotechnology primed [32P]-α-dCTP labeled Tara cDNA probe (nucleotides 222- Associates, Birmingham, AL), or phalloidin-TRITC. Following 1779) according to the manufacturer’s instructions. incubation with the secondary mAbs, slides were washed with PBS, then mounted in a polyvinyl alcohol medium and viewed on a Nikon Monoclonal antibodies Eclipse E800 microscope equipped for epifluorescence. The following mAbs were used for immunoprecipitations, immunoblotting and immunofluorescence: anti-Tara.397 and anti- F-actin co-sedimentation assays Tara.27 mAbs (see below), anti-Trio.18 mAb (see below), anti-HA F-actin co-sedimentation assays were done essentially as described by 12CA5 (Boeringer Mannheim, Indianapolis, IN), anti-HA HA 11 the manufacturer (Cytoskeleton, Denver, CO). Briefly, protein (BabCO, Richmond, CA), rabbit anti-non-myosin II heavy chain preparations were incubated with 40 µg freshly polymerized actin (F- BT561 (Biomedical Technologies, Stoughton, MA), anti-GST 112 actin) for 1 hour at RT. Following incubation, the protein plus F-actin (Immunogen Inc., Cambridge, MA), anti-actin (Sigma, St. Louis, MO), solution was subjected to centrifugation (160,000 g) to pellet F-actin anti-vimentin LN-6 (Sigma), anti-tubulin DM1A (Sigma) or anti-α- and protein bound to F-actin. After solubilzation of the pellet fraction actinin BM75.2 (Sigma). To generate the anti-Tara mAbs, mice were in a volume equal to the initial incubation volume, equal volumes of immunized with E. coli-derived GST-Tara.13-385 fusion protein. To the pellet and supernatant fractions were analyzed by SDS-PAGE. this end, E. coli NM522 cells were transfected with the pGEX.Tara.13- 385 expression plasmid, and GST-Tara.13-385 fusion protein was GenBank Accession Number purified from bacterial lysates using Glutathione Sepharose 4B The GenBank Accession Number for Tara is AF281030. (Pharmacia) chromatography. The resulting bead-protein complexes were used to immunize BALB/c mice. Splenocytes from immunized mice were fused to NS-1 myeloma cells and HAT-resistant hybridomas RESULTS were initially selected using enzyme-linked immunosorbant assay (ELISA). The anti-Trio.18 mAb was generated in a similar manner Trio interacts with Tara, a broadly expressed PH using as antigen GST-Trio.1848-2438 (Trio amino acids 1848-2438). domain-containing coiled-coil protein Cells and transfections Trio is a complex protein containing two DH-GEF domains, a COS7 cells were grown in RPMI medium, containing 10% FCS, 50 PSK domain, and multiple auxiliary domains (Fig. 1A) µg/ml gentamicin sulfate and 2 mM L-glutamine). COS7 cell transient (Debant et al., 1996). To learn more about the function of Trio, transfections were performed via the DEAE-dextran/DMSO method we performed an interaction-trap screen to identify Trio- using pMT.HA.Trio deletion mutants (see Fig. 1B), pMT.GST.H2 (Tara, binding proteins using a portion of Trio, termed TGD1, α amino acids 13-385), pSP.SR .Tara and MT.HA.Trio plasmid DNAs, containing amino acids 1207-1715 (Fig. 1A) as bait to screen and cells were harvested ~36 hours after transfection. The construct a human fibroblast cDNA library (Debant et al., 1996; Gyuris pMT2.HA.Trio contains the full-length Trio cDNA encoding Trio amino acids 1-3038 (Seipel et al., 1999). HeLa cells were grown in DMEM et al., 1993; Serra-Pages et al., 1995). This region of Trio was containing 10% FCS, 50 µg/ml gentamicin sulfate, and 2 mM L- chosen as bait because ectopic expression of TGD1, which glutamine. Cells were transfected using the Ca2+ phosphate method with includes the GEF-D1, PH-D1 and SH3-D1 domains, regulates pSP.SRα.Tara, pSP.SRα.Tara.HA and pSP.SRα.Trio plasmid DNA. actin cytoskeletal organization, cell growth and cell migration HeLa cell lines stably expressing Tara-HA (designated HeLa(+Tara)) (Seipel et al., 1999). A cDNA designated H2 was isolated, were established by electroporation with pSP.SRα.Tara.HA plasmid which encodes a peptide that efficiently bound the Trio.1207- DNA, together with pSP.SV.neo plasmid DNA, which contains the 1715 bait, but did not bind the Trio.1848-2438 bait (see Fig. neomycin-resistance gene. Control HeLa cells were established with 1A). To determine whether the H2 peptide also bound Trio in α pSP.SR .HA plasmid DNA together with pSP.SV.neo plasmid DNA. mammalian cells, co-precipitation studies were performed Resulting clones were selected in medium supplemented with 0.5 mg/ml using extracts prepared from [35S]methionine and Geneticin (Life Technologies). The expression levels of Tara-HA 35 and endogenous Tara were determined by anti-HA and anti-Tara [ S]cysteine metabolically labeled cells (Fig. 1B). To this end, immunoblotting and densitometric scanning of the autoradiographs. For a glutathione S-transferase (GST)-tagged H2 peptide (GST- serum starvation, HeLa-derived cell lines were washed with PBS, then H2) was transiently expressed in COS7 cells, together with incubated in DMEM media lacking FCS for 24 hours. Following serum various hemagglutinin (HA)-tagged Trio deletion constructs starvation, HeLa(+Tara) or control HeLa cell lines (at least three (see Fig. 1B). GST-H2 was then recovered using gluthathione 392 JOURNAL OF CELL SCIENCE 114 (2) sufficient for the H2 and Trio interaction. Taken together, these results indicate that the H2 peptide binds Trio in mammalian cells, as well as in the interaction-trap assay. The primary structure of the 593 amino acid H2 gene product was deduced following sequence determination of overlapping cDNA clones (Fig. 2A). This full-length gene product was designated Tara for Trio-associated repeat on actin (see below). The original interaction-trap H2 peptide contains Tara amino acids 13-385. A search of protein databases (GenBank, PIR, Swiss-Prot and EMBL) revealed that the Tara sequence was unique, but had a high degree of similarity with p116RIP (46% overall amino acid identity; Gebbink et al., 1997), and a lower level of sequence identity with a number of proteins that either contain pleckstrin homology (PH) domains or coiled-coil regions (Lupas, 1996; Musacchio et al., 1993; Shaw, 1996). The Tara C- terminal half was predicted to form a coiled-coil structure using an algorithm to detect coiled-coil regions (Fig. 2B; Lupas et al., 1991). Indeed, co-precipitation studies using tagged versions of Tara demonstrated that Tara amino acids 333-416, which are part of the coiled-coil region, are required for dimerization (data not shown). The Tara PH domain had the highest sequence similarity to the PH domains of p116 RIP (Gebbink et al., 1997), the Etk/Bmx protein tyrosine kinase (Qiu et al., 1998) and bovine myosin X (Yonezawa et al., 2000; Fig. 2C). p116RIP was shown to inhibit RhoA-stimulated contractility and to promote neurite outgrowth in N1E-115 cells, and was isolated by virtue of its binding to RhoA (Gebbink et al., 1997). The overall predicted structure of Tara, with its N-terminal PH domain and C-terminal coiled-coil region, is shown schematically in Fig. 2B. Fig. 1. Identification of a Trio interacting peptide, H2. (A) Isolation To characterize Tara protein, anti-Tara monoclonal of the Trio-binding peptide H2 using the interaction-trap assay. The antibodies, termed anti-Tara.27 and anti-Tara.397 were structure of the 3038 amino acid Trio protein is shown schematically developed using GST-Tara.13-385 fusion protein as antigen. at the top of the figure and below is shown the Trio.1207-1715 Immunoblotting experiments using the anti-Tara.27 mAb interaction bait, as well as the control Trio.1848-2438 bait. The interaction-trap assay for protein-protein interaction was as described and extracts prepared from HeLa cells demonstrated that in the Materials and Methods. SP, spectrin like domain, GEF-D, endogenous Tara has an apparent molecular mass of about guanine nucleotide exchange factor domain; PH, pleckstrin 80,000 even though the expected molecular mass of Tara is homology domain; SH3-D, src-homology 3 domain; Ig, only 68,000 (Fig. 2E, lane 2). Because endogenous Tara co- immunoglobulin-like domain; PSK, serine/threonine kinase. (B) Co- migrated with COS cell-derived recombinant full-length precipitation of Trio and the Trio interaction-trap assay positive Tara (Fig. 2E, lane 3), this discrepancy was due to the clone, H2, from mammalian cells. The GST-H2 fusion construct and electrophoretic properties of Tara, and not the absence of various HA-tagged Trio deletion constructs (the extent of the Trio additional coding sequence. Attempts to co-immunoprecipitate sequences contained in the constructs is given at the top of the figure) endogenous Trio and Tara were unsuccessful, possibly because were co-expressed in COS7 cells. Following cell labeling with of the low level of Tara and Trio expression in diverse cell types [35S]methionine and [35S]cysteine, cell extracts were prepared, GST- H2 was recovered, and associated labeled proteins were analyzed by (data not shown). However, co-precipitation analysis of HeLa SDS-PAGE and autoradiography (lanes 1-5). Control anti-HA cells ectopically expressing HA-tagged full-length Tara (HA- immunoprecipitated proteins from whole cell lysates are shown in Tara) and HA-Trio.1118-1919 demonstrated that full-length lanes 6-10. The expected position of the various Trio deletion Tara, like the H2 peptide, bound Trio (Fig. 2F). Tara neither constructs are indicated by asterisks. Molecular mass standards in modulated Trio GEF-D1 activity in in vitro GEF assays nor kDa are shown on the left of the figure, and the position of the GST- bound RhoA or Rac1 (data not shown). H2 fusion protein is indicated by an arrow. Northern blot analysis demonstrated that Tara mRNA is broadly expressed among human tissues and cell lines, with the highest relative expression levels seen in heart and placenta sepharose beads, and GST-H2-associated proteins were (Fig. 3). Of the cell lines tested, HeLa, SW-480 and G361 cells analyzed by SDS-PAGE. The ~70 kDa GST-H2 protein bound contained the largest amount of Tara mRNA. The most to HA-tagged Trio.1118-1919, Trio.1118-1681, and Trio.1118- abundant Tara mRNA species in all of the samples had a length 1551, but did not bind Trio.1227-1681 or Trio.1227-1551 (Fig. of about 2.8 kb, whereas minor mRNA species between 2.0 to 1B, lanes 1-5). Western blot analysis using the anti-HA mAb 4.4 kb were present in several of the mRNA samples (Fig. 3). confirmed that the various HA-tagged Trio deletion constructs were expressed (Fig. 1B, lanes 6-10). Based on the interaction- Ectopically expressed Tara enhances cell spreading trap assay and the co-precipitation studies, Trio amino acids and partially co-localizes with Trio 1207-1551, comprising the GEF-D1 and PH-D1 domains, are To gain insight into its possible functions, Tara was ectopically Tara binds F-actin and enhances cell spreading 393

Fig. 2. Characterization of the Trio- binding protein Tara. (A) Deduced 593 amino acid sequence of the human Tara protein based on the nucleotide sequence of Tara/H2 cDNA clones (GenBank Accession Number AF281030). The standard one letter amino acid code is used. (B) The C-terminal region of Tara is predicted to form a coiled-coil structure. Coiled-coil probabilities (x-axis) were calculated for each residue (y-axis) using a window of 28 amino acids (Lupas et al., 1991). (C) The N-terminal region of Tara contains a PH domain. Shown is an alignment of the Tara PH domain sequence with the PH domain sequences of p116RIP (amino acids 381-481; Gebbink et al., 1997), Etk/Bmx (amino acids 28-133, Qiu et al., 1998) and bovine myosin X (amino acids 1201-1343; GenBank Accession number T18519). At the bottom of the alignment are indicated consensus amino acid residues, with residues conserved in three out of four sequences shown in lower case, and absolutely conserved residues shown in upper case. (D) The overall structure of Tara is schematically shown with the relative length of the N- terminal PH domain and the C-terminal coiled coil region. (E) Tara has an apparent molecular mass of 80 kDa. Shown is an immunoblot analysis of endogenous Tara immunoprecipitated from HeLa cells (lane 2) or of recombinant Tara immunoprecipitated from COS7 cells (lane 3). A control Ig immunoprecipitate is shown in lane 1. Molecular mass standards in kDa are shown on the left of the figure, and the position of the Tara and Ig proteins are indicated on the right of the panel. (F) Tara binds Trio.1118-1919 in mammalian cells. Shown is a SDS-PAGE analysis demonstrating co-immunoprecipitation of HA-Tara and HA-Trio.1118-1919. COS7 cells were transfected with vectors encoding either HA-Tara and HA-Trio.1118-1919 (lanes 2 and 4) or HA and HA-Trio.1118-1919 (lanes 1 and 3). Following cell labeling with [35S]methionine and [35S]cysteine, cell extracts were prepared, and anti-Trio mAb (lanes 1 and 2) or control anti- HA mAb (lanes 3 and 4) precipitations were performed. The 97 kDa molecular mass standard is shown on the left of the figure, and the positions of the HA-Trio.1118-1919 and HA-Tara proteins are indicated by arrows. expressed in HeLa cells and Tara localization and cell HeLa cells that were transfected with a plasmid encoding green morphology were assessed by immunofluorescence analysis. fluorescent protein (gfp; green, part B), suggesting that Tara As seen in Fig. 4, Tara expression (red, part A) enhanced regulates cytoskeletal organization and cell contraction. The spreading with cells appearing larger compared to control ability of Tara to enhance cell spreading was also seen in other 394 JOURNAL OF CELL SCIENCE 114 (2) concentrated at membrane ruffles (Fig. 4C). In cells expressing both Trio (red, Fig. 4D) and Tara (green, Fig. 4E), there was partial co-localization of Trio and Tara, particularly at membrane ruffles (yellow/orange, Fig. 4F). Cells expressing both Trio and Tara had the Trio-mediated pancake-like Fig. 3. Tara mRNA is widely appearance, with Tara localization seen in the filamentous expressed in human tissues. pattern as well as in vesicular structures. Possibly, Tara and Northern blot analysis of 2 µg Trio interact at specific sites such as membrane ruffles to of poly(A)+ RNA isolated from regulate actin cytoskeletal reorganization. However, given the the human tissues or human limited co-localization of ectopically expressed Tara and Trio, cell lines indicated at the top of a more detailed study is required to elucidate the physiological the panels (promyelocytic leukemia, HL60; epitheliod relevance of this interaction. carcinoma HeLa S3; chronic myelogenous leukemia, K- Tara is associated with F-actin 562; lymphoblastic leukemia, The filamentous expression pattern of Tara suggested that Tara MOLT-4, Burkitt’s lymphoma may localize to F-actin. To test this possibility, HeLa cells Raji; colorectal ectopically expressing Tara, or control HeLa cells, were stained adenocarcinoma, SW80; lung for F-actin using rhodamin-conjugated phalloidin, as well as carcinoma, A549; and for Tara using the anti-Tara.397 mAb. As seen in Fig. 5, the F- melanoma, G361) using a actin staining pattern (red, part A) and the Tara staining pattern radiolabeled Tara cDNA probe. (green, part B) were largely overlapping (orange-yellow, part Size markers in kb are shown on the right of the panels. C). Indeed, higher magnification images of the F-actin and Tara staining (Fig. 5D-F) highlighted the co-localization of F-actin and Tara. As observed in Fig. 4, ectopic Tara expression caused cell types, including MCF7 and N1E-115 cells (data not the HeLa cells to display enhanced spread morphology shown). The Tara protein itself was observed in a filamentous compared with the mock transfected HeLa cells. A similar pattern, as well as in vesicular structures (Fig. 4A). A similar filamentous staining pattern was observed in other cell types staining pattern was observed in other cell types (data not (data not shown), as well as in HeLa cells expressing HA- shown). Because the size and number of Tara-positive tagged Tara and stained with anti-HA mAb instead of the anti- vesicular structures correlated with Tara expression levels, the Tara mAb (Fig. 5G-I). There was no significant co-localization vesicular localization of Tara might have been an of Tara with either vimentin or tubulin, indicating that Tara overexpression artifact. Attempts to visualize endogenous Tara does not localize to intermediate filaments or microtubules in HeLa cells and a number of other cell types was (data not shown). Taken together, these results strongly argue unsuccessful, possibly due to limited expression levels of that Tara is associated with F-actin. endogenous Tara. The periodicity of Tara localization on F-actin, which The association between Tara and Trio was also tested by occurred at about 350 nm intervals, was reminiscent of the immunofluorescence co-localization studies using HeLa cells staining pattern of myosin II (Verkhovsky et al., 1995). Indeed, ectopically expressing full-length Trio (amino acids 1-3038) co-staining of ectopically expressed Tara (red, Fig. 6A) and and Tara (Fig. 4). Trio expression (green) alone caused the cells endogenous non-muscle myosin II heavy chain (green, Fig. to spread with a circular, pancake-like morphology (Fig. 4C) 6B) demonstrated that these two proteins had a nearly identical (Seipel et al., 1999). Trio was present throughout the cell and localization in HeLa cells (Fig. 6C; inset shows a fivefold

Fig. 4. Ectopic expression of Tara in HeLa cells causes cell spreading and partial co-localization with Trio. Shown are immunofluorescence images of HeLa cells transiently transfected with (A) Tara, (B) green fluorescence protein (gfp) to visualize HeLa cell morphology, (C) HA-Trio (Trio amino acids 1-3038) or (D,E) Tara and HA-Trio, and stained for (A,E) Tara or (C,D) Trio expression. Co-localization of Tara and HA-Trio expression is apparent in the (F) merged image of parts D,E, where coincidence of red and green appears yellow/orange. Tara and Trio co-localize predominately at the cell edges (see arrows). Ectopic expression of Tara, compared with gfp expression, enhances HeLa cell spreading. HeLa cell transfections and immunofluorescence were as described in Materials and Methods. All images are of identical magnification. Scale bar: 25 µm. Tara binds F-actin and enhances cell spreading 395

Fig. 5. Ectopically expressed Tara localizes to F-actin in HeLa cells. Shown are immunofluorescence images of HeLa cells transiently transfected with Tara (A-F) or with Tara-HA (G-I) and stained for (B,E) Tara (green), using anti-Tara mAb, (H) Tara (green), using anti-HA mAb, or (A,D,G) F-actin, using rhodamin-labeled phalloidin (red). Merged images of A,B, D,E or G,H are shown in panels C,F,I, respectively, with coincidence of red and green appearing yellow/orange. D-F are 2.5-fold magnified images of A-C. Ectopic expression of both Tara and Tara- HA caused enhanced cell spreading, and both Tara and Tara-HA predominantly localize to F-actin in a periodic fashion. HeLa cell transfections and immunofluorescence were as described in Materials and Methods. The intensely red staining cells in A are non-Tara expressing (i.e., non- transfected) HeLa cells. In order to obtain a reasonable F-actin signal in the well-spread, Tara-expressing cells, images were overexposed which resulted in intense phalloidin (red) staining of less spread and thicker, non- transfected cells. Scale bar: 50 µm for A-C,G-I; 20 µm for D-F. enlargement). Furthermore, co-staining of ectopically localized to the F-actin pellet fraction (lane 12), as determined expressed Tara (red, Fig. 6D) and endogenous α-actinin (green, by Coomassie Blue staining of gels. Purified COS cell-derived Fig. 6E) revealed that α-actinin and Tara alternate along actin Tara (see Materials and Methods) also bound F-actin as fibers particularly at the leading edge of the cell (Fig. 6F, inset determined using the F-actin sedimentation assay (Fig. 7B). The shows a fivefold enlargement). α-actinin, but not Tara, was presence of misfolded protein could account for the finding that observed at focal contacts located at the edges of cells (see Fig. only about half of the Tara preparations bound F-actin. Co- 6E,F). The co-localization of Tara with the actin-binding immunoprecipitation experiments did not demonstrate any proteins myosin and α-actinin further supported the idea that interaction between Tara and G-actin, thus indicating that Tara Tara is a F-actin associated protein. To test whether Tara binds specifically to F-actin and not G-actin (data not shown). directly bound myosin II or α-actinin, co-immunoprecipitation Furthermore, purified, E. coli-derived Tara.13-385 protein (Tara experiments were performed, but failed to demonstrate a direct amino acids 13-385; see Materials and Methods) efficiently interaction between myosin and Tara or between α-actinin and Tara (data not shown). Tara binds F-actin in vitro To test whether Tara bound directly to F-actin, co-sedimentation assays were performed using F-actin and COS cell-derived Tara (Fig. 7A). In the absence of F-actin, Tara was located exclusively in the soluble fraction (Fig. 7A, lane 1). However, in the presence of F-actin, about half of the Tara protein was located in the pellet fraction, as determined by immunoblot analysis. α-Actinin was used as positive control for the F-actin sedimentation assay and BSA as negative control. As seen in Fig. 7A, the known actin- binding protein α-actinin almost exclusively localized to the F-actin pellet fraction (lane 10), whereas <5% of the BSA

Fig. 6. Tara co-localizes with myosin II on actin fibers. Shown are immunofluorescence images of HeLa cells transiently transfected with Tara, and stained for (A,D) Tara (red), (B) myosin II heavy chain (green), or (E) α-actinin (green). Merged images of A,B, or D,E are shown in panels C,F, respectively, with coincidence of red and green appearing yellow/orange. Ectopically expressed Tara displays a very high degree of co-localization with myosin II in bead-like structures along F-actin. The expression pattern of Tara and the actin-binding protein α-actinin along F-actin are largely alternating, with some co-localization occurring. Tara does not co- localize with α-actinin at focal adhesions. HeLa cell transfections and immunofluorescence were as described in Materials and Methods. All images are of identical magnification, except the insets in C,F, which are magnified fivefold. Scale bar: 20 µm. 396 JOURNAL OF CELL SCIENCE 114 (2)

Fig. 7. Tara binds F-actin in vitro. (A) COS cell-derived Tara binds F-actin using a co-sedimentation assay. Shown is an anti- Tara immunoblot (lanes 1-4) or Coomassie Blue-stained gels (lanes 5-12) of the F-actin co-sedimentation assay soluble (S) and pellet (P) fractions. F-actin co-sedimentation assays were as described in Materials and Methods using COS7 cell-derived recombinant Tara in the form of a whole-cell lysate (lanes 1-8), purified α-actinin (lanes 9-10) or purified BSA (lanes 11-12). For the assay, recombinant Tara, the known actin-binding protein α-actinin, or control BSA were incubated in the presence (lanes 3, 4, 7-12) or absence (lanes 1, 2, 5, 6) of F-actin, and following incubation, F-actin was pelleted by centrifugation. Pelleted proteins were solubilized in a volume equal to the assay volume and then equal volumes of the pellet and soluble fraction were analyzed by anti-Tara immunoblotting or by Coomassie Blue staining. The positions of Tara and G-actin are indicated at the left and the positions of α-actinin, BSA, G-actin are shown on the right. (B) Partially purified recombinant Tara binds F-actin. Shown is an anti-Tara immunoblot (lanes 1-4) of the F-actin co-sedimentation assay soluble (S) and pellet (P) fractions using partially purified Tara protein. COS7 cell-derived GST-Tara fusion protein was isolated by Glutathione-Sepharose chromatography, and Tara was isolated following incubation with thrombin as described in the Material and Methods. The position of Tara is shown on the left. (C) E. coli-derived Tara.13-385 binds F-actin. Shown is an anti-Tara immunoblot (lanes 1-4) of the F-actin co- sedimentation assay soluble (S) and pellet (P) fractions using ~0.5 µg E. coli-derived, purified Tara.13-385 (Tara amino acids 13-385) protein. E. coli-derived GST-Tara.13-385 fusion protein was isolated by glutathione-sepharose chromatography, and Tara.13-385 was isolated following incubation with thrombin as described in Material and Methods. The position of Tara.13-385 is shown on the right. (D) A Coomassie Blue stained gel of E. coli-derived, Tara.13-385 protein used for F-actin co-sedimentation assays. In addition to Tara.13-385, there was a ~80 kDa contaminating protein present in the preparations. The identity of the Tara.13-385 protein was confirmed by immunoblotting with an anti-Tara mAb (Fig. 7C, and data not shown). Molecular mass standards in kDa are shown on the left of the panel and the position of Tara.13-385 is shown on the right. bound F-actin (Fig. 7C,D). However, it was not possible to staining. LatB treatment of control HeLa cells resulted in determine the stoichiometry of the Tara.13-385 and F-actin nearly complete disappearance of F-actin with only punctate interaction, because the low yields of Tara.13-385 protein structures remaining (Fig. 8E). In contrast, LatB-treated precluded performing the F-actin co-sedimentation assays with HeLa(+Tara) cells retained cortical F-actin staining, although saturating amounts of Tara.13-385. Taken together, the the overall amount of F-actin was reduced. These results immunofluorescence localization of Tara to F-actin, the co- suggest that Tara functions to stabilize F-actin. Furthermore, localization of Tara with myosin II and the in vitro co- the HeLa(+Tara) cells displayed a flattened morphology sedimentation of Tara with F-actin strongly argue that the N- compared with the control HeLa cells. Thus, both transient terminal region of Tara (i.e., amino acids 13-385) directly binds and stable expression of Tara alters the actin cytoskeletal F-actin. organization and confers an enhanced spread morphology. To establish whether Tara expression altered cell attachment HeLa cells stably overexpressing Tara exhibit or the rate of cell spreading, HeLa(+Tara) and control cells altered cell morphology and redistributed F-actin were stained for F-actin at various times following cell plating To gain additional insight into the possible function of Tara, (Fig. 9). HeLa(+Tara) and control HeLa cells attached at a HeLa cell lines stably expressing C-terminally HA-tagged Tara similar rate (Fig. 9A,B) and there was little difference in the (Tara-HA) were established and characterized. These cell lines, extent of cell spreading during the first 8 hours after plating referred to as HeLa(+Tara) cells, express ~fivefold more Tara- (Fig. 9A-F). However, by 24 hours about half of the HA than endogenous Tara as determined by immunoblotting HeLa(+Tara) cells were more spread than control HeLa cells and densitometric scanning (data not shown). Compared to (Fig. 9G,H). Enhanced F-actin in stress fibers was already control HeLa cells, the HeLa(+Tara) displayed enhanced F- evident in the HeLa(+Tara) cells by 4 hours, and by 24 hours actin staining (Fig. 8). Altered F-actin staining was evident in HeLa(+Tara) cells contained prominent stress fibers. Enhanced HeLa(+Tara) cells compared with control HeLa cells in serum- cortical F-actin was also noticeable 4 and 8 hours after plating free media (Fig. 8A,B), in cells induced for 30 minutes by in the HeLa(+Tara) cells, but by 24 hours the level of cortical factors present in FCS (Fig. 8C,D) or in cells treated with the F-actin was relatively normal (Fig. 9C-H). The finding that the G-actin sequestering drug Latrunculin B (Lat B) (Fig. 8E,F). HeLa(+Tara) cells initially attached and spread in a manner Under all three culture conditions the HeLa(+Tara) cells had similar to the control HeLa cells suggests that ectopical Tara more prominent F-actin staining, particularly cortical F-actin expression does not significantly affect the rate of cell Tara binds F-actin and enhances cell spreading 397 attachment or initial spreading, but rather that Tara Burridge, 1996; Rameh and Cantley, 1999; Steimle et al., 1999; overexpression alters the actin cytoskeleton to promote Yao et al., 1999), it is also possible that the PH domain enhanced cell spreading. regulates the Tara F-actin binding activity. Alternatively, the Tara PH domain could mediate protein-protein interactions, including the direct binding to actin, as was reported for some DISCUSSION PH domains. The coiled-coil region of Tara was shown to dimerize and thus enables Tara dimerization or formation of We report the identification of a novel F-actin and Trio GEF- higher order structures. Coiled-coils can form rod-like binding protein termed Tara, which consists of a PH domain structures that may be important for specific function and/or and a coiled-coil region. Ectopic expression of Tara alters facilitate binding to other proteins (Lupas, 1996). Indeed, a actin cytoskeletal organization, promotes cell spreading, and number of actin binding proteins are coiled-coil proteins, increases F-actin stability, suggesting that Tara regulates actin including myosin II, caldesmon and α-actinin. cytoskeletal organization. F-actin-binding proteins perform Tara was isolated by virtue of its binding to a subregion of many functions, including actin bundling, crosslinking, Trio that includes GEF-D1. This region of Trio is of particular stiffening, contracting, capping and fragmenting. Our data interest as previous studies demonstrated that Trio GEF-D1 suggest that Tara likely mediates F-actin bundling, crosslinking activates Rac1 and RhoG in vitro and in vivo (Bellanger et al., and/or stiffening, as ectopically expressed Tara enhanced 1998; Blangy et al., 2000; Debant et al., 1996), and that and stabilized F-actin structures. The relative resistance of ectopically expressed Trio GEF-D1 promotes cell spreading, Tara expressing cells to Latrunculin B-mediated F-actin membrane ruffling and cell migration (Seipel et al., 1999). destabilization argues that Tara stabilizes F-actin structures. Furthermore, activation of either Rac or Rho by the first or Alternatively, or in addition, Tara may serve as a linker second Trio GEF domain may coordinate actin cytoskeletal protein to recruit proteins required for F-actin formation and turnover. Tara belongs to a subgroup of actin-binding proteins that lack conventional actin-binding domains, and in some of these proteins actin- binding activity was mapped to coiled-coil regions (Sobue and Sellers, 1991) or PH domains (Yao et al., 1999). Consistent with previously identified F-actin bundling and crosslinking proteins, which contain either multiple actin-binding domains or are able to dimerize (Ayscough, 1998), Tara contains at least two actin-binding sites per coiled-coil dimer. Tara and myosin II co-localize along F- actin in a periodic fashion. A similar pattern was previously observed for myosin II, but the basis for this particular spacing is unknown (Kolega, 1998; Verkhovsky et al., 1995). The co-localization of Tara and myosin II suggests that Tara and myosin II directly interact, but we did not detect a physical interaction between these two proteins. Tara is most similar in overall structure and amino acid identity to p116RIP, a RhoA- binding protein that regulates actin remodeling in neurites by inhibiting RhoA-induced contractility (Gebbink et al., 1997). However, we did not observe Rho family GTPase binding to Tara (data not shown), suggesting that although Tara and p116RIP are structurally similar they may have different modes of action. Based on the characterization of several PH domains (Shaw, 1996), the Tara PH Fig. 8. Altered F-actin in HeLa cells stably expressing Tara. Immunofluorescence domain may constitute a phospholipid binding images of (A,C,E) control HeLa cells or (B,D,E) HeLa(+Tara) cells, which stably domain that mediates binding of Tara to the express recombinant Tara-HA at levels ~ fivefold above endogenous Tara levels, stained for F-actin using rhodamin-labeled phalloidin. Cells were cultured in serum-free plasma membrane. Tara may thus link the medium for 1 day before induction and then incubated for 30 minutes with (A,B) serum- plasma membrane with the actin cytoskeleton. free media as control, (C,D) media containing10% FCS or (E,F) serum-free media As the activity of a number of proteins, containing 200 nM Latrunculin B. Results are representative of at least three including actin binding proteins, is modulated independent HeLa(+Tara) and control HeLa cell lines. All images are of identical by phospholipid binding (Gilmore and exposure times and magnification. Scale bar: 50 µm. 398 JOURNAL OF CELL SCIENCE 114 (2) prominent in the Tara-expressing cells. Tara and Trio may thus coordinate the amount of F-actin present in stress fibers, within the cortex, and at the leading edge of the cell. This function would be especially important during cell locomotion, a cellular process where communication between various regions of the cell is absolutely essential for directed cell movement (Lauffenburger and Horwitz, 1996; Mitchison and Cramer, 1996).

We thank Drs Nancy Kedersha and Haruo Saito for critical review of the manuscript; and Dr Roger Brent and colleagues for plasmid DNAs and yeast strains used for the interaction-trap assay. This work was supported by grants CA55547 and CA75091 from the National Institutes of Health. M. S. is a Leukemia and Lymphoma Society Scholar.

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