RESEARCH ARTICLE 549 The flightless I colocalizes with - and microtubule-based structures in motile Swiss 3T3 fibroblasts: evidence for the involvement of PI 3-kinase and Ras-related small GTPases

Deborah A. Davy1,*, Hugh D. Campbell2, Shelley Fountain2, Danielle de Jong2 and Michael F. Crouch1,* 1Molecular Signalling Group, Division of Neuroscience, John Curtin School of Medical Research and 2Molecular Genetics and Evolution Group and Centre for Molecular Structure and Function, Research School of Biological Sciences, Australian National University, Canberra, Australia 2600 *Authors for correspondence (email: [email protected]; [email protected])

Accepted 17 November 2000 Journal of Cell Science 114, 549-562 © The Company of Biologists Ltd

SUMMARY

The flightless I protein contains an actin-binding domain protein colocalizes with β-tubulin- and actin-based with homology to the family and is likely to be structures. Members of the small GTPase family, also involved in actin cytoskeletal rearrangements. It has been implicated in cytoskeletal control, were found to colocalize suggested that this protein is involved in linking the with flightless I in migrating Swiss 3T3 fibroblasts. cytoskeletal network with signal transduction pathways. LY294002, a specific inhibitor of PI 3-kinase, inhibits the We have developed antibodies directed toward the leucine translocation of flightless I to actin-based structures. Our rich repeat and gelsolin-like domains of the human and results suggest that PI 3-kinase and the small GTPases, mouse homologues of flightless I that specifically recognize Ras, RhoA and Cdc42 may be part of a common functional expressed and endogenous forms of the protein. We have pathway involved in Fliih-mediated cytoskeletal regulation. also constructed a flightless I-enhanced green fluorescent Functionally, we suggest that flightless I may act to prepare fusion vector and used this to examine the localization of actin filaments or provide factors required for cytoskeletal the expressed protein in Swiss 3T3 fibroblasts. The rearrangements necessary for cell migration and/or flightless I protein localizes predominantly to the nucleus adhesion. and translocates to the cytoplasm following serum stimulation. In cells stimulated to migrate, the flightless I Key words: Flightless I, Fliih, , GTPase, Actin, Tubulin

INTRODUCTION moderate and severe fliI mutants indicate a possible role for the fliI protein in regulating actin cytoskeletal rearrangements. The flightless I (fliI) encodes a A 380 leucine-rich repeat region (LRR) (Kobe 1,256 amino acid protein with a predicted molecular mass of and Deisenhofer, 1995a), characterizes the N-terminal domain 143,672 Da (Campbell et al., 1993). It has highly conserved of the fliI protein. This motif is thought to play a role in specific homologues in (49% amino acid protein-protein interactions, that in some cases are responsible sequence identity to Drosophila), mouse (95% identity to for a role in signal transduction (Kobe and Deisenhofer, human) (Campbell et al., 2000) and human (58% identity to 1995b). This amphipathic / motif (Kobe Drosophila) (Campbell et al., 1993; Campbell et al., 1997). and Deisenhofer, 1994; Kobe and Deisenhofer, 1995b) was first Northern analysis of human FLII mRNA expression patterns observed in the α2-glycoprotein (Takahashi et al., 1985) and showed the highest level in skeletal muscle, but the gene was has since been identified in hormone receptors (McFarland et expressed at varying levels in all tissues examined (Campbell al., 1989), certain (Tan et al., 1990), tyrosine kinase et al., 1997). Viable mutations of the D. melanogaster fliI receptors (Martin-Zanca et al., 1989) and molecules cause ultrastructural defects in the indirect flight muscles responsible for cell adhesion (Hashimoto et al., 1988). A (Deak et al., 1982; Miklos and de Couet, 1990), with frayed variety of LRR functions have been characterized (Kobe and and disorganized myofibrils and Z bands that appear abnormal Deisenhofer, 1994), including the LRR region in yeast or absent. Null alleles of fliI are homozygous lethal (Perrimon adenylate cyclase which is known to interact with Ras (Suzuki et al., 1989). In contrast to normal embryonic development et al., 1990). (Miller and Kiehart, 1995), mutant embryos have an irregular C-terminal to the LRR region lie two large duplicated cytoskeleton and arrangement of nuclei at the cell cortex, domains each characterized by three tandemly repeated followed by poorly coordinated membrane invaginations sequences of between 125 and 150 amino acids (Campbell et (Kajava et al., 1995; Straub et al., 1996). These developmental al., 1993; Way and Weeds, 1988). This region shows significant events require an intact cytoskeleton. The phenotypes of homology to members of the gelsolin-family of actin-binding 550 JOURNAL OF CELL SCIENCE 114 (3) (ABPs) (Campbell et al., 1997; Kwiatkowski et al., 50 µg of peptide was incubated with 50 µl of the antibody for one 1986; Hartwig and Kwiatkowski, 1991). The gelsolin family hour at room temperature. This preparation was then used in the same members are ABPs that can bind monomeric actin subunits manner as antibodies that were not blocked. promoting polymerization (Janmey et al., 1985; Yin, 1986; Construction of a containing the complete human Kwiatkowski et al., 1989). Members of this family can cap FLII coding region (Lind et al., 1987) and/or sever actin filaments (Kwiatkowski The previously reported human FLII cDNA (phfli2) (Campbell et al., et al., 1989; Arora and McCulloch, 1996; Chaponnier et al., 1993) was almost full length, but was missing a part of the ATG 1986). ABPs are important for actin cytoskeletal rearrangements initiation codon. A human EST clone, Image Consortium clone and are regulated by micromolar concentrations of calcium 135082 (GenBank accession no. R33910), was previously identified (Janmey et al., 1985; Pope et al., 1995; Yin et al., 1988; Young as containing the ATG initiation codon and 37 bp of 5′ untranslated et al., 1994) and phosphoinositide binding (Janmey and Stossel, sequence (Campbell et al., 1997). However, this EST clone contained 1989; Lassing and Lindberg, 1985; Lu et al., 1996). an insert of only 800 bp. Clone 135082 plasmid DNA was digested The cytoskeletal network plays important roles in the with XhoI and end repaired with T4 DNA polymerase. The DNA was maintenance of cell shape, the transport and anchoring of digested with BspEI and the 300 bp XhoI-BspEI fragment was gel cellular components involved in cellular adhesion, and purified. Plasmid phfli2 was digested with EcoRV and BspEI, migration. While significant progress has been made in dephosphorylated with calf intestinal alkaline phosphatase, and the 6.8 kb fragment was gel purified. These two fragments were ligated identifying the biochemical signals that are involved in together to yield phfli2FL. The insert in phfli2FL was sequenced from regulating the cytoskeleton, the relationship between them and both ends by dye primer sequencing (ABI Prism) using the –21 M13 the impact on cellular functions are not well defined. Recent forward and reverse primers, confirming the expected structure. The evidence has shown that members of the Ras-related GTPase 5′ sequence of the phfli2FL cDNA up to and across the BspEI site was family are responsible for the regulation of a variety of actin- identical to the previously determined cDNA and genomic sequences based structures (Allen et al., 1997; Nobes and Hall, 1995; (Campbell et al., 1993; Campbell et al., 1997). Tapon and Hall, 1997; Rodriguez-Viciana et al., 1997). In addition to this, signaling pathways involved in the regulation In vitro transcription/translation of phfli2FL of proteins that influence cytoskeletal dynamics are The transcription/translation of phfli2FL plasmid DNA was achieved increasingly seen to involve phosphoinositide 3-kinase (PI 3- using the TNT T7 Quick Coupled Translation/Transcription System (Promega). [35S]methionine was used to produce a radioactive form kinase) (Derman et al., 1997; Rodriguez-Viciana et al., 1997; of protein that can, when appropriately prepared, be visualized using Johanson et al., 1999; Hill et al., 2000). autoradiography. A 5µl aliquot from each reaction was added to SDS- The work presented here describes the development of PAGE sample buffer (300 mM Tris, 50 mM DTT, 15% glycerol, 2% flightless I-specific antibodies and the identification and SDS, bromophenol blue) plus 15 mg/ml dithiothreitol (DTT), or 1 ml examination of the subcellular distribution of the mouse of lysis buffer containing inhibitors (see Cell Harvest methods). flightless I protein (Fliih) protein in quiescent and serum- Samples containing lysis buffer and 5 µl of transcription/translation stimulated Swiss 3T3 fibroblasts. We also examine potential reaction were heated for 30 minutes at 70°C and precleared by interacting proteins and regulatory molecules for Fliih. The incubation with Protein A-Sepharose beads only for one hour at 4°C. FliL or FliG antibodies (1:50) were added and samples were incubated results of our experiments imply a role for flightless I in ° the regulation of cytoskeletal rearrangements involved in overnight at 4 C on a rotating mixer. Protein A-Sepharose beads were hydrated with Tris buffered saline (TBS: 50 mM Tris-HCl, pH 7.4, cytokinesis and cell migration. 0.2 M NaCl) and 5 mg (w/v) was added to each sample. The samples were mixed for 1 hour at 4°C. The beads were pelleted by centrifugation for 45 seconds at 17,400 g, and the supernatant MATERIALS AND METHODS discarded. The Protein A-Sepharose-antibody-antigen pellets were rinsed 3 times with 1 ml of TBS. SDS-PAGE sample buffer was added Immunization with flightless I-specific-peptide L to each pellet and samples were analyzed by SDS-PAGE. The SDS- The cDNA sequences of flightless I and homologues have been PAGE gel was dried prior to autoradiography. determined (Campbell et al., 1993) (D. melanogaster GenBank accession no. U01182, C. elegans GenBank U01183 and Homo Bacterial expression of flightless I sapiens GenBank U01184; U80184). Peptide L corresponding to a Two primers were prepared that enabled amplification of the human sequence (CKLEHLSVSHN, amino acids 57-66) within the LRR (L) FLII coding region for insertion into an E. coli expression vector. domain of human FLII was synthesized (Biomolecular Resource Primer HDC128 (5′-AGA GCG GCC ATA TGG AGG CCA CCG Facility, JCSMR), for subsequent production of anti-peptide GGG TGC TGC CG-3′) was designed to reconstruct the translation antibodies. An antibody against peptide G within the gelsolin-like initiation codon and add an overlapping NdeI site. Primer HDC129 domain (CSHFKRKFIIH, amino acids 1032-1041) has been (5′-GCC AGC ATC GAT TAG GCC AGG GCC TTG CAG AAG previously described (Davy et al., 2000). Peptide L was conjugated to GCG-3′) was designed to match the 3′ end of the coding region and keyhole limpet hemocyanin (Sigma), using the protocol described by add a flanking ClaI site. Each primer (10 pmol) was used in a 50 µl Goldsmith and coworkers (Goldsmith et al., 1987). The N-terminal reaction with 1 ng phfli2 cDNA (Campbell et al., 1993) as template cysteine was added to the peptide for coupling purposes. A New in a Perkin-Elmer 9600 PCR machine using the Expand Long Zealand White rabbit was injected subcutaneously and serum was Template PCR system (Boehringer Mannheim) mixture of Taq and collected following clot retraction and stored at −70°C. The rabbit was Pwo DNA polymerases in Expand buffer 1 (50 mM Tris-HCl, pH 9.2 boosted at regular intervals and further samples were collected. (25°C), 14 mM (NH4)2SO4 1.75 mM MgCl2). TaqStart antibody (Clontech) was used to obtain a hot start. After 1 minute at 94°C, 25 Preparation of the flightless I anti-peptide antibody cycles of 94°C (30 seconds), and 68°C (3 minutes), were carried out. The FliL antibodies were affinity-purified using 1 ml, NHS-activated, The reaction product was digested with NdeI and ClaI, purified by gel HiTrap Affinity columns (Pharmacia, Sweden) coupled with peptide electrophoresis and the 3.8 kb fragment was ligated between the NdeI L, according to the manufacturer’s instructions. For peptide blocking, and ClaI sites of pETMCSI. This vector is a derivative of the pET T7 The flightless I protein 551 promoter vectors (Studier et al., 1990) and was a generous gift from were subsequently fixed with 2% paraformaldehyde, mounted onto Dr Nick Dixon, Research School of Chemistry, ANU. For the slides and EGFP-fluorescence visualized with the FITC wavelength expression, 6 independent isolates of this construct were transformed (Ex: 494 nm; Em: 520 nm) using confocal microscopy. into BL21(DE3)/pLysS. Transformants were then grown at 37°C to an OD595 of 0.5 and induced by addition of 1 mM isopropyl-beta-D- Immunohistochemistry thiogalactopyranoside (IPTG). Cultures were incubated at 37°C for 2 Cells were seeded into 12-well plates, as above. Fibroblasts were seeded hours after induction and cells harvested by centrifugation. Control at 7.5×103 cells/ml, maintained in 10% FCS/DMEM and allowed to cultures were treated identically except that IPTG was not added. The attach overnight. The medium was then replaced with 0% FCS/DMEM number of cells was normalized by OD measurements taken when and the incubation continued for a further 24 hours. Cells were cells were harvested. Samples were analyzed by SDS-PAGE and subsequently activated with 10% FCS with or without preincubation western transfer. with test agents, LY294002 (25 µM, Biomol, USA) or rapamycin (100 nM, ICN, USA), as indicated. Cells were fixed with 2% Mammalian cell culture and harvest paraformaldehyde for 15 minutes at room temperature, and then washed Swiss 3T3 fibroblasts (Commonwealth Serum Laboratories, 5 times with cold PBS. Cells were permeabilized with PBS/1.0% Australia) were cultured and harvested as described previously BSA/0.1% SDS for 15 minutes followed by blocking for 1 hour with (Crouch and Simson, 1997). PBS/1.0% BSA. The appropriate antibody (1:100) was diluted in PBS/1.0% BSA and added to each well and incubation continued at Fractionation 4°C overnight. Wells were rinsed 5 times with cold PBS then incubated Lysates were fractionated as described previously by Franze- with FITC-labeled secondary antibodies (1:100) (Jackson Fernandez and Pogo (Franze-Fernandez and Pogo, 1971) into the Immunoresearch, USA) in PBS/1.0% BSA for 1 hour at room nuclear (N), cytoskeletal (C) or membrane/cytosol (M) fractions. The temperature. Fibroblasts were also double-labeled in some instances resulting fractions were used for immunoprecipitation procedures. with two antibodies. In these cases, Texas Red-labeled secondary antibody (Jackson Immunoresearch, USA) was used in conjunction Immunoprecipitation with the FITC-labeled secondary antibody. There was no crossover of Appropriate antibodies were added to cell lysates and incubated fluorescence between the FITC (Ex: 494 nm; Em: 520 nm) and Texas overnight on a rotating mixer. Protein A-Sepharose beads were added Red channels (Ex: 596 nm; Em: 618 nm). Cellular fluorescence was and samples mixed for 1 hour. Beads were pelleted by centrifugation visualized and recorded with a Leica (Germany) confocal microscope. for 45 seconds at 17,400 g, and the supernatant discarded. Protein A- Commercial antibodies used were against gelsolin and Ras Sepharose-antibody-antigen pellets were rinsed 3 times with 1 ml Tris (Transduction Laboratories), RhoA, Cdc42, and Rac1 (Santa Cruz). buffered saline (50 mM Tris-HCl, pH 7.4, 0.2 M NaCl). Staining of filamentous actin was achieved with the use of Texas Red Phalloidin (Molecular Probes). Phalloidin was dried under nitrogen and SDS-PAGE redissolved in PBS/1.0% BSA. Phalloidin (25 U/ml) was added to each Proteins were electrophoresed on 7% SDS-polyacrylamide gels and well and the same protocol as for secondary antibodies was followed. western transferred to nitrocellulose membrane (Schleicher and Schuell, BA-85, Germany). All samples for electrophoresis required the addition of SDS-PAGE sample buffer plus DTT, and were heated RESULTS for 5 minutes at 100°C prior to loading. The blot was incubated with the appropriate antibody overnight at 4°C. Detection of antigens after The FliL antibody specifically recognizes a 145 kDa the primary antibody incubation was performed using horseradish protein following the in vitro transcription and peroxidase-labeled secondary antibodies followed by enhanced translation of human FLII cDNA chemiluminescent detection (ECL; Amersham, UK). A plasmid containing the complete coding region of human Construction of human FLII-EGFP fusion vector FLII was used for in vitro-coupled transcription/translation The human FLII coding region was fused in frame to the C terminus reactions containing the T7 RNA polymerase for the of the coding region for the enhanced green fluorescent protein transcription step. The construct, named phfli2FL, was verified (EGFP) as follows. The plasmid phfli2FL containing the reconstructed by restriction analysis and end-sequencing. In phfli2FL, the T7 full-length human FLII cDNA was digested with NcoI and end- RNA polymerase promoter in the pBluescript SK-vector is repaired. Following BamHI digestion, the 4.1 kb fragment containing located upstream from the 5′ end of the FLII cDNA. One the FLII coding region was purified by gel electrophoresis. The vector labeled protein present following completion of the reaction pEGFP-C1 (Clontech) was digested with SalI, end-repaired, digested had a molecular mass of 145 kDa (Fig. 1A, lane1), and was with BamHI, purified and ligated with the FLII fragment to yield pEGFP-C1-FLII. The structure of the construct was verified by specifically recognized by the anti-peptide antibodies, FliL (Fig. restriction analysis and end-sequencing using the EGFP-C sequencing 1A, lane 2) and FliG (not shown). The immunoprecipitation of primer (Clontech). this protein was blocked when antibodies were incubated with peptide L, prior to use (Fig. 1A, lane 3). The 145 kDa protein Transient transfection of Swiss 3T3 fibroblasts with was absent in samples that contained the product of a pEGFP-C1-FLII luciferase-encoding control cDNA (Fig. 1A, lane 4) when Swiss 3T3 fibroblasts were seeded in a 12-well plate, each well probed with the FliL antibody (Fig. 1A, lane 5), or samples containing a coverslip, at 2×103 cells/ml, maintained in 10% that did not contain cDNA (not shown). Thus, the FliL and FliG FCS/DMEM containing antibiotics (/streptomycin), and antibodies were specific for the 145 kDa protein encoded by allowed to attach overnight. The medium was then changed to 10% FLII cDNA. FCS/DMEM without antibiotics and allowed to equilibrate for 4 hours. The pEGFP-C1-FLII construct was incubated in the presence Immunoprecipitation of a 145 kDa protein from E. of FuGene (Boehringer Mannheim), according to the manufacturer’s instructions, and this mix was then added to the cultured cells. The coli cells expressing human FLII empty pEGFP-C1 vector was used as the control. Cells were allowed E. coli cells transformed with a T7 promoter expression vector to incubate without a change in medium for a further 48 hours. Cells containing FLII cDNA were grown in liquid culture and 552 JOURNAL OF CELL SCIENCE 114 (3)

Fig. 1. Detection of the expressed product from human FLII cDNA and the endogenous Fliih protein in Swiss 3T3 fibroblasts. (A) [35S]Methionine-labeled FLII protein produced by the transcription/translation of FLII cDNA immuno-reacts with the FliL antibody. A 145 kDa protein was present (as indicated), amongst many others, in samples containing the product of the transcription/translation of FLII cDNA (lane 1). Immunoprecipitation (IP) of whole sample with FliL antibody revealed a single 145 kDa protein (lane 2, arrow). When the FliL antibody was preincubated with peptide L for 1 hour prior to use, the 145 kDa protein was absent (lane 3). The transcription/translation product of a luciferase control plasmid (lane 4) or the immunoprecipitation of this sample with FliL (lane 5) did not produce a 145 kDa protein. (B) Expression in E. coli of a 145 kDa protein that is recognized by the FliL antibody. E. coli clones transfected with the FLII cDNA expression construct were either left untreated (lanes 1, 3 and 5), or treated with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) (lanes 2, 4 and 6) for two hours. The FliL antibody was used for immunoblotting after western transfer. A 145 kDa protein is indicated (arrow). Data are representative of 6 individual clones. (C) A 145 kDa protein (arrow) present in Swiss 3T3 fibroblasts immunoreacts with FliG antibody. Cells were left unstimulated (lanes 1 and 3) or stimulated for 16 hours (lanes 2 and 4) with 10% FCS. The FliG antibody was used for immunoblotting. This protein was absent when blots were probed with peptide G-blocked FliG antibody (lanes 3 and 4). Data are representative of 3 experiments. In all cases, the alternative antibody gave similar results (not shown).

when antibodies were blocked with the appropriate peptide (not shown). The lower molecular mass bands may be degradation products or are possibly truncated forms of FLII that can occur when translation is disrupted during the reaction. The identity of the nonspecific 110 kDa protein is unknown, but this was present in both induced and non-induced cells and was recognized non-specifically by the secondary antibody. Identification and immunoprecipitation of a 145 kDa protein from Swiss 3T3 fibroblasts The FliG antibody identified a 145 kDa protein in whole cell lysate from Swiss 3T3 fibroblasts (Fliih) (Fig. 1C, lanes 1 and 2) that was specifically blocked with peptide G (Fig. 1C, lanes 3 and 4). Similar results were seen when the FliL antibody was used (not shown). The increase in the amount of protein in stimulated cells (Fig. 1C, lane 2) reflects protein synthesis following the long stimulation time. FliL or FliG antibodies were also used to immunoprecipitate (IP) protein from Swiss 3T3 mouse fibroblasts. The FliL antibody immunoprecipitated a protein with a molecular mass of approximately 145 kDa that was recognized following western transfer by both the FliL (Fig. 2) and FliG antibodies (not shown). A protein of identical molecular mass, immunoprecipitated by FliG antibodies, was recognized by both antibodies following western transfer (not shown). In quiescent cells, the FliL/FliG-immunoreactive protein was predominantly found in the nuclear (Fig. 2, lane 1) and induced with IPTG for 2 hours, or left uninduced, prior to lysis. membrane/cytosol (Fig. 2, lane 3) fractions, with a lesser amount Cell lysates were prepared from six independent E. coli clones associated with the cytoskeleton (Fig. 2, lane 5). Stimulation and analyzed by 7% SDS-PAGE. Western blots were with 10% FCS induced a marked decrease in the amount of immunoblotted (IB) with the FliL antibody (Fig. 1B) or FliG protein accumulated in the nucleus (Fig. 2, lane 2), and a (not shown) antibodies. A 145 kDa protein was identified in concurrent increase in Fliih associated with the cytoskeleton samples that had been induced with IPTG (Fig. 1B, lanes 2, 4 (Fig. 2, lane 6). There was little change in the amount of Fliih and 6), but not in lysates from cells that were not induced (Fig. in the membrane/cytosol fraction (Fig. 2, lane 4) following 1B, lanes 1, 3 and 5). The 145 kDa protein was also absent stimulation. The evidence leads us to conclude that the rabbit The flightless I protein 553 the plasma membrane (Fig. 3B). We believe these structures to be actin arcs (see Fig. 4G). Fliih also localized to the actin-rich plasma membrane underlying the leading edge (Fig. 3B). We also found Fliih localized to membrane ruffles, actin-rich structures that form on the dorsal surface of the lamellipodia in motile cells (Fig. 3C). Similar results were seen when the FliG antibody (not shown) was used. Fliih colocalizes with cytoskeletal structures associated with migration In view of the likely actin-binding properties of the Fliih protein (Liu and Yin, 1998), fibroblasts were simultaneously labeled with Texas Red-phalloidin (Fig. 4A,D,G) and FliL (Fig. 4B,E,H) to examine colocalization sites between the polymerized actin-based network and Fliih. Phalloidin labeling of unstimulated cells revealed short actin filaments (Fig. 4A) in a disorganized fashion throughout the cytoplasm of the cell. Fliih predominantly localized to the nuclear/perinuclear region (N) with evidence of a filamentous network extending into Fig. 2. A 145 kDa protein immunoreactive to FliL antibody is the cytoplasm (Fig. 4B). The Fliih-based network did not identified in nuclear, cytoskeletal and membrane/cytosol fractions in extensively colocalize with phalloidin-defined filaments in Swiss 3T3 fibroblasts. Unstimulated (lanes 1,3 and 5) or cells unstimulated cells, a result evident when images were merged activated with 10% FCS for 16 hours (lanes 2,4 and 6) were using Adobe Photoshop (Fig. 4C), although there was some fractionated into nuclear (N), cytoskeletal (C) and membrane/cytosol evidence for colocalization on particular filaments (compare (M) fractions. Lysate was immunoprecipitated with the FliL Fig. 4A-C). antibody, proteins separated by SDS-PAGE and western transferred, Following stimulation with serum, phalloidin-defined stress and blots probed with FliL antibody. In all cases, blots were incubated with either FliL or FliG antibody (1:500). Positions of fibers can be seen extending across the cell (Fig. 4D). The Fliih molecular mass standards (Novex) are as indicated (kDa). Antibody protein translocated from the nuclear/perinuclear region toward heavy chain is indicated. In all cases the alternate antibody gave the periphery of the cell (Fig. 4E). The Fliih-defined filaments similar results (not shown). were perpendicular to the actin-based stress fiber network. Despite some apparent colocalization between Fliih with actin- based networks, represented by the population of yellow anti-peptide FliL and FliG antibodies specifically recognize the filaments seen when the images were merged (Fig. 4F), we translated human phfli2FL product, the human FLII protein believe that the Fliih-based network is not coincident with expressed in bacterial cells and the endogenous murine Fliih stress fibers. We do note, however, discrete points of apparent protein in cultured Swiss 3T3 fibroblasts. In the latter, Fliih colocalization between Fliih-defined filaments and the terminal appeared to undergo a stimulus-induced subcellular ends of stress fibers (Fig. 4F). The actin-rich leading edge translocation from the nucleus to the cytoskeleton. (Fig. 4G), the actin arc and membrane ruffles are structures normally associated with migration. Phalloidin-loaded cells Fliih localization in quiescent and stimulated Swiss simultaneously labeled with the FliL antibody (Fig. 4H) 3T3 fibroblasts revealed discrete regions of colocalization (Fig. 4I) at actin Swiss 3T3 fibroblasts were fixed with 2% paraformaldehyde arcs, membrane ruffles and the leading edge of migrating cells. and subsequently incubated with affinity-purified FliL (Fig. 3) FliG antibodies gave identical staining patterns to FliL or FliG (not shown) antibodies followed by FITC-labeled antibodies for all conditions, and the specificities of both the secondary anti-rabbit antibodies. Quiescent Swiss 3T3 antibodies was further established by showing that fibroblasts were flat and circular in shape with no evidence of preincubation of the antibodies with their cognate peptides a polarized morphology (Fig. 3A) or structures normally blocked the signal (not shown). associated with motile fibroblasts. Unstimulated cells accumulated Fliih predominantly in the nuclear (N) and GFP-C1-FLII colocalizes with actin-based structures perinuclear regions. Fliih could be seen extending in a We have constructed the vector pEGFP-C1-FLII by fusing the filamentous arrangement from the nuclear/perinuclear region human FLII cDNA in frame to the C terminus of the coding into the cytoplasm of the cell. We consistently noted pockets region for the enhanced green fluorescent protein (EGFP). The of increased fluorescence within the nucleus in regions construct prepared for this work contains the entire coding reminiscent of nucleoli. The significance of this nuclear region for human FLII including the ATG initiation codon. localization is unknown. This is fused in frame to the 3′ end of the coding region for To examine the localization of Fliih following stimulation, EGFP via a 15 amino acid region encoded by the polylinker of quiescent cells were activated with 10% FCS, prior to pEGFP-C1. The human cytomegalovirus immediate early immunohistochemistry. Fliih remained localized to the promoter drives expression. The plasmid also contains the neo nuclear/perinuclear region in cell populations that were gene under the control of the SV40 early promoter. We used confluent, and therefore non-migratory (not shown). In this vector to transiently transfect Swiss 3T3 fibroblasts and migratory cells, Fliih localized to discrete bands posterior to subsequently examined the localization of EGFP-tagged FLII 554 JOURNAL OF CELL SCIENCE 114 (3)

Fig. 3. Localization of the Fliih protein in Swiss 3T3 fibroblasts. Cells either remained unstimulated (A) or were activated for 16-18 hours with 10% FCS (B and C), and prepared for immunohistochemistry. Fluorescence was visualized using confocal microscopy. ‘N’ identifies the nucleus. Bars: 50 µm (A,B,D); 30 µm (C). Images are representative of 3 experiments. White arrows indicate (A) Fliih-defined filaments and (C) membrane ruffles on the dorsal surface. (B) Large black arrows indicate actin arcs, thin black arrows indicate nuclear accumulations and the white arrow indicates the peripheral membrane. protein. Fibroblasts were transiently transfected with the Fliih colocalizes with β-tubulin in Swiss 3T3 EGFP-C1 vector (not shown) or with the EGFP-C1-FLII fibroblasts construct (Fig. 5). Cells were labeled with Texas Red The failure of Fliih-defined filaments to colocalize with actin phalloidin (Fig. 5A and D) and the localization of the GFP-C1- filaments led us to examine the possibility that translocation FLII protein (Fig. 5B and E) was examined using the FITC from the nuclear/perinuclear region is mediated by the β- channel. We show a phalloidin-labeled cell that is not tubulin-based network. We simultaneously labeled activated transfected and subsequently fails to emit an FITC-sensitive fibroblasts with β-tubulin (Fig. 6A) and FliL (Fig. 6B) signal (Fig. 5A and B, arrow). In transfected cells, we see antibodies and examined the resulting fluorescent images for EGFP-C1-FLII localizing to the peripheral membrane (Fig. regions of colocalization (Fig. 6C). It appeared that Fliih 5B) and membrane ruffles (Fig. 5E) of migrating cells. When colocalized with a population of microtubules (yellow images were merged, yellow regions reveal sites of filaments) extending into a polarized region of the cell colocalization between the GFP-C1-FLII protein and actin-rich following serum stimulation. regions of motile cells (Fig. 5C and F). GFP alone did not We also examined other β-tubulin-based structures including localize with F-actin, but rather showed a mainly nuclear the mitotic spindle, a structure involved in localization (not shown). separation and cell division. In Fig. 6D, the β-tubulin-based

Fig. 4. Colocalization between actin-based structures and Fliih. Swiss 3T3 fibroblasts were left unstimulated (A-C) or were stimulated with 10% FCS for 16-18 hours (D-I) and prepared for immunohistochemistry. Cells were labeled with Texas-Red phalloidin (A, D and G) and Fliih antibodies (B, E and H). Fliih antibodies were detected using FITC-conjugated anti- rabbit secondary antibodies. The images were recorded by confocal fluorescent microscopy. The images showing the localization of actin (red) and Fliih (green) were merged (C, F and I) revealing regions of colocalization (yellow regions). ‘N’ indicates the nucleus. Images are representative of 3 experiments. White arrows indicate (A) phalloidin-defined filaments, (B) Fliih-defined structures or (F) points of colocalization between phalloidin- and Fliih- defined filaments. Bars: 75 µm (A-F); 150 µm (G-I). The flightless I protein 555

Fig. 5. The expressed EGFP-FLII fusion protein colocalizes with actin-based structures in Swiss 3T3 fibroblasts. Cells were prepared for fluorescence histochemistry to examine the localization of Texas Red-phalloidin-defined filaments (A and D) using confocal microscopy. The localization of the EGFP-C1-FLII protein was visualized by confocal microscopy using the FITC channel (B and E). Regions of colocalization (C and F) are represented in yellow. A non-transfected cell (white arrow) can be seen nearby (A and C). This cell is labeled with Texas Red phalloidin but fails to emit a GFP signal. Images are representative of 2 experiments. Bars, 50 µm. mitotic spindle is evident in a dividing cell. Two intensely antibodies. Cdc42 localized predominantly to the perinuclear fluorescent regions at opposing poles of the mitotic spindle region in serum-starved cells and translocated in a filamentous could be seen when the localization of Fliih was visualized arrangement when cells were stimulated for 1-4 hours with (Fig. 6E). When images were merged (Fig. 6F), these appeared 10% FCS (not shown). In cells activated with 10% FCS for 16- to be centrosomes at the spindle poles. Fliih was also found 18 hours, Cdc42 (Fig. 8A,D) and Fliih (Fig. 8B,E) antibodies concentrated at the midbody of telophase cells. Fliih was colocalized (Fig. 8C,F) at the actin arc and the leading edge of absent from the actin-rich contractile ring (Fig. 6G) but was motile cells. enriched on the microtubular midbody alone (Fig. 6H). Higher Fibroblasts were also double-labeled with Ras (Fig. 8G) and magnification of this midbody region more clearly shows Fliih (Fig. 8H) antibodies, and subsequent confocal images the localization of Fliih (Fig. 6I) to these β-tubulin-based merged (Fig. 8I). Ras was seen in a diffuse punctate structures (not shown) in Swiss 3T3 fibroblasts. At this stage, distribution throughout the cytoplasm of the cell, with regions most of the Fliih was again localized to the nuclear region (Fig. of increased fluorescence localized at actin arcs and the leading 6H). edge of migrating cells. When the images were merged it was clear that Fliih and Ras colocalized at an actin arc and the Localization of gelsolin, Ras and small Ras-related leading edge. Sites of Fliih enrichment within the nucleus were GTPases in Swiss 3T3 fibroblasts in relation to Fliih clearly evident in Fig. 8B and Fig. 8K. Quiescent and stimulated cells were fixed then incubated Swiss 3T3 fibroblasts were simultaneously labeled with overnight with an antibody directed to gelsolin and RhoA (Fig. 8J) and Fliih (Fig. 8K) antibodies. RhoA localized subsequently incubated with secondary Texas-Red labeled to the perinuclear region in quiescent cells and did not anti-mouse antibodies. Gelsolin localized to short, fragmented colocalize with filamentous Fliih in the early stages of actin filaments in quiescent fibroblasts (Fig. 7A). The activation (not shown). Following long-term serum association of gelsolin with fragmented F-actin decreased after stimulation, RhoA predominantly localized in a punctate activation with 10% FCS, yielding a more punctate distribution distribution throughout the cytoplasm of the cell. However, throughout the cytoplasm (Fig. 7B). regions of enrichment were evident on actin arcs and the Similar results were seen when fibroblasts were stimulated leading edge of migrating cells. When images were merged with thrombin (not shown). Swiss 3T3 fibroblasts were (Fig. 8L), RhoA and Fliih colocalized on the actin arc and the simultaneously labeled with Fliih and Cdc42 antibodies and actin-rich leading edge of motile fibroblasts. RhoA was subsequently incubated with Texas-Red and FITC-secondary also seen localized to membrane ruffles associated with 556 JOURNAL OF CELL SCIENCE 114 (3)

Fig. 6. Colocalization of β- tubulin and Fliih in Swiss 3T3 fibroblasts. Swiss 3T3 fibroblasts were simultaneously labeled with β-tubulin (A and D) and FliL (B and E) antibodies following stimulation with 10% FCS for 16-18 hours. Regions of colocalization (C and F) are yellow when images are merged. (G-I) Cells were labeled with FliL antibodies and the region of actomyosin ring formation (G) and the microtubular midbody (H and I) in telophase cells examined. Bars: 75 µm (A-C), 50 µm (D-H), 10 µm (I). Images are representative of 3 experiments. lamellipodia (not shown). In contrast, RhoB and Rac1 showed labeled with the FliL antibody and subsequently exposed to no evidence of colocalization with Fliih-defined filaments, on secondary antibodies and fluorescence visualized using actin arcs or at the leading edge of migrating cells (not shown) confocal microscopy. The translocation of Fliih to the leading following similar serum activation times. edge was not inhibited by rapamycin (Fig. 9A), but LY294002 markedly inhibited the translocation of Fliih to the leading The effect of rapamycin and LY294002 and Fliih edge of migrating cells (Fig. 9B). We noted an almost complete translocation absence of actin arcs in cells treated with rapamycin or Fibroblasts were pretreated with rapamycin or LY294002 for LY294002, and the development of lengthy tails in both 30 minutes prior to activation with 10% FCS. Cells were rapamycin- and LY294002-treated cells.

Fig. 7. Localization of gelsolin in quiescent and stimulated Swiss 3T3 fibroblasts. Quiescent cells were either left unstimulated (A) or were activated with 10% FCS for 8 hours (B) and prepared for immunohistochemistry. Cells were probed with gelsolin antibody. N indicates the nucleus. Bar, 50 µm. Images are representative of 2 experiments. The flightless I protein 557

Fig. 8. Colocalization between Fliih and members of the small GTPase family. Swiss 3T3 fibroblasts were stimulated with 10% FCS for 16-18 hours and prepared for immunohistochemistry. Cells were simultaneously labeled with Cdc42 (A and D/d), Ras (G) or RhoA (J) and FliL (B, E/e, H and K) antibodies. When images are merged (C, F/f, I and L) yellow represents regions of colocalization, the black arrows identify actin arcs and white arrows indicate the leading edge. Bars: 75 µm (A-L); 10 µm (d-f). Images are representative of 5 experiments.

Fig. 9. The effect of rapamycin or LY294002 on the translocation of the Fliih protein in stimulated Swiss 3T3 fibroblasts. Cells were treated with rapamycin (A) or LY294002 (B) for 30 minutes prior to stimulation (for 8 hours) with 10% FCS. Fibroblasts were subsequently prepared for immunohistochemistry and labeled with the FliL antibody. N indicates the nucleus. Large white arrow (A) indicates the leading edge. The small white arrows (A and B) indicate elongated tails. Bars, 25 µm. Images are representative of 3 experiments. 558 JOURNAL OF CELL SCIENCE 114 (3) DISCUSSION rich cortical layer at the leading edge of migrating cells. This localization was confirmed when cells were transfected with The characterization of the Drosophila melanogaster fliI pEGFP-C1-FLII. protein, and conserved homologues, suggests that this protein The leading edge of polarized, motile cells is a functionally may play a role in linking the cytoskeleton to intracellular distinct region, the most conspicuous feature being proteins involved in signal transduction (Liu and Yin, 1998; lamellipodia (Small, 1994). Polymerization of growing actin Goshima et al., 1999; Campbell et al., 1993; Claudianos and filaments (Schafer et al., 1998; Cooper and Schafer, 2000; Campbell, 1995). To investigate the role of flightless I, we have Mikhailov and Gundersen, 1998; Mitchison and Cramer, 1996) produced two anti-peptide antibodies that recognize defined creates a propulsive force that pushes forward lamellae of regions of the human FLII and mouse Fliih sequences. FliL and motile cells (Mallavarapu, 1999). Subsequent rearrangements FliG (Davy et al., 2000) antibodies recognize a 145 kDa protein of the plasma membrane enable the formation of new cell- as measured by western blotting and immunoprecipitation substrate contacts (Nabi, 1999) at the leading edge necessary methods. Fliih antibodies specifically react with a 145 kDa for traction (Cheresh et al., 1999; Drubin and Nelson, 1996). FLII protein synthesized in vitro from cDNA or prepared by Like gelsolin, flightless I is able to sever filaments, at least in bacterial expression in E. coli. A protein of identical size is the case of the C. elegans protein (Goshima et al., 1999), and also specifically detected in mouse cell extracts. All of the this may account functionally for the localization of Fliih to evidence strongly indicates that this is the Fliih protein. In cytoskeletal structures. Whereas gelsolin links to fragmented support of this, the 145 kDa immunoreactive protein is reduced actin filaments in unstimulated cells and is displaced by growth by 50% in liver extracts from heterozygous Fliih mutant mice factor stimulation, Fliih is induced to migrate to highly specific generated by gene targeting (H. Campbell et al., unpublished cytoskeletal structures within lamellae under the same results). The development of flightless I-specific antibodies conditions. This suggests that despite , the provides the opportunity to characterize the intracellular actin-binding and -severing activities of flightless I and other response to different cellular conditions and to investigate the gelsolin-family members target different actin-based structures localization of Fliih in Swiss 3T3 fibroblasts. in the cell. In summary, Fliih localizes to cytoskeletal-based In quiescent Swiss 3T3 fibroblasts, Fliih predominantly structures associated with migrating cells, is critically localizes to the nuclear/perinuclear region, as is the case for positioned to play a role in cytoskeletal regulation and may do some other actin-binding proteins (Pope et al., 1998; so by binding to and severing actin filaments. Wulfkuhle et al., 1999; Matsuzaki et al., 1988). The nuclear Microtubules are believed to lend structural support and staining is punctuated by dense foci of Fliih protein, which may have a role in the directed movement of many cell types represent the accumulation of Fliih within nucleoli (Shaw and (Conrad et al., 1989). Another less appreciated role is their Jordan, 1995; Wulfkuhle et al., 1999; Matsuzaki et al., 1988). function as a physical anchor and long-range transport Proteins larger than 45 kDa require a suitable nuclear substrate for key mediators of protein expression (Knowles et localization signal (Garcia-Bustos et al., 1991; Newmeyer and al., 1996; Bassell and Singer, 1997; Bassell et al., 1994). After Forbes, 1988; Nigg et al., 1991) to target them to the nucleus translocation from the nucleus, Fliih localizes to actin-based and in some cases a nuclear export signal (Dingwall and structures but does not appear to translocate via the actin-based Laskey, 1992; Goldfarb, 1991) is required to exit the nucleus. network. It is possible that the movement of Fliih may relate Examination of the human and mouse flightless I sequences to the translocation of factors via microtubules to the actin reveal putative nuclear localization (e.g. 1035KRKFIIHRG- cytoskeleton. There is precedent for this hypothesis, the best KRK1046) and export (150LTDLLYLDL158) signals that are described examples being the localization of β-actin mRNA to highly conserved between species for all known sequences. the cell periphery of motile cells (Kislauskis et al., 1993; Fliih can be induced to translocate out of the nucleus when Latham et al., 1994) and the localization of proteins required cells are stimulated, although it remains unclear whether the for myofibrillar repair (Russell and Dix, 1992) and genesis putative flightless I nuclear localization and export signals are (Morris and Fulton, 1994). Studies have also suggested that the functional. presence of microtubules in developing indirect flight muscles The flightless I protein features both an actin-binding and in Drosophila melanogaster may provide factors for the leucine-rich-repeat domain (Claudianos and Campbell, 1995; formation of myofibrils (Fernandes et al., 1991; Reedy and Campbell et al., 1993). This has led to the concept that Beall, 1993). Since myofibrillar structure is severely disrupted flightless I may be an intermediate protein directly linking the in flightless I mutants a role for flightless I in the development cytoskeleton to signaling proteins such as Ras (Claudianos and of these structures is possible. Additionally, the recent Campbell, 1995). In support of this hypothesis, it has been identification of flightless I-binding partners that have shown that flightless I is able to bind actin in vitro (Liu and homology to transcription factors (Liu and Yin, 1998; Fong and Yin, 1998), and others have found that the C. elegans form of de Couet, 1999) or may be involved in binding dsRNA (Wilson expressed protein interacts with Ras both in vitro and in vivo et al., 1998), supports the suggestion that Fliih may be part of in the yeast system (Goshima et al., 1999). We have a ribonucleoprotein complex responsible for the delivery of demonstrated the colocalization of the flightless I protein factors via microtubules, and the subsequent attachment to with actin in Drosophila and mouse embryos using an actin-based structures. immunohistochemical approach (Davy et al., 2000). The We also find that flightless I localizes to β-tubulin-based present study has identified Fliih localizing to actin-based structures known to be involved in cell division and structures associated with motility, such as actin arcs (Heath cytokinesis. Fliih localizes to centrosomes, structures that and Holifield, 1993; Soranno and Bell, 1982) and membrane radiate a microtubular network forming the mitotic spindle ruffles (Cheresh et al., 1999). Fliih also localizes to the actin- which is responsible for the regulation of chromosome The flightless I protein 559 separation during cell division (Field et al., 1999) and discrete functional role, microinjection of dominant negative Rho or accumulations within the midbody, the final remnant that constitutively active Cdc42 into Drosophila melanogaster connects daughter cells (Glotzer, 1997; Mandato et al., 2000). embryos results in similar effects to those seen in flightless I The Fliih protein is distributed throughout the cytoplasm null mutants (Crawford et al., 1998). This includes disruption during early stages of cell separation, but is clearly localized of actomyosin furrow canal formation during Drosophila to the nucleus when cytokinesis is nearing completion. The cellularization and embryonic lethality prior to gastrulation. presence of Fliih at these dynamic sites supports the view that This evidence, together with the colocalization between Fliih it may be required for cytokinesis. Such a role could explain and GTPase proteins described here, suggests that flightless I, the embryonic lethality observed with homozygous Fliih Ras, RhoA and/or Cdc42 may be functionally linked in the knockout embryos (H. Campbell et al., unpublished results). control of cytoskeletal rearrangements. The phenotype of Drosophila melanogaster fliI null mutants Phosphoinositides, particularly phosphatidylinositol 4,5- (Straub et al., 1996) supports the suggestion that Fliih plays a bisphosphate and phosphatidylinositol 3,4,5-triphosphate role in cytokinesis. Cellularization is a specialized form of (Singh et al., 1996; Yin et al., 1988), can regulate gelsolin and cytokinesis (Miller and Kiehart, 1995; Loncar and Singer, other ABPs (Yin et al., 1981; Young et al., 1994; Janmey and 1995; Miller, 1995) involving microtubule- (Warn and Warn, Stossel, 1987). Cell locomotion and changes in cell shape are 1986; Foe and Alberts, 1983) and actin- (Warn and McGrath, generally accompanied by synthesis and hydrolysis of inositol 1983; Miller and Kiehart, 1995; Warn and Robert-Nicoud, and a rapid, but transient increase in the 1990) based structures for successful completion. We have intracellular calcium levels (Janmey, 1994). Fliih localization shown that flightless I localizes to cellularization structures in to the leading edge was absent when PI 3-kinase was inhibited D. melanogaster syncytial embryos (Davy et al., 2000), which by pretreatment with LY294002. Our results also show that lends support to the view that Fliih may also be involved in exposure to LY294002 inhibits the translocation of Fliih into cytokinetic processes in Swiss 3T3 fibroblasts. the cytoplasm following stimulation. The involvement of PI 3- Cytoskeletal rearrangements that result in the formation of kinase-regulated pathways mediating nuclear localization and specialized migratory structures are known to be regulated, in export has previously been described (Klingenberg et al., 2000; part, by members of the small GTPase family, including Ras Belkowski et al., 1999). GTPase proteins can lie upstream or (Bar Sagi and Feramisco, 1986), RhoA (Ridley and Hall, 1992) downstream of phosphoinositide activity (Rodriguez-Viciana and Cdc42 (Nobes and Hall, 1995). The involvement of et al., 1994; Reif et al., 1996), and the inhibition of Fliih different GTPases in cytoskeletal regulation has also been translocation may be due to a direct inhibition of PI 3-kinase described in other cell types (Ziman et al., 1993; Eaton et al., or GTPase activity, or may be an indirect effect due to the 1995; Chen et al., 1996; Faix et al., 1998; Johnson, 1999). A disruption of actin filaments (Johanson et al., 1999). These hierarchical relationship is believed to exist between the small results suggest that Fliih may be a direct or indirect GTPases in the signaling control of cytoskeletal behavior in biochemical target of phosphorylated phosphoinositides or Swiss 3T3 fibroblasts (Ridley and Hall, 1992), although recent GTPase proteins, and we plan to investigate any direct physical evidence indicates a more complex situation (Lim et al., 1996; link or regulation of Fliih function by PI 3-kinase- Aspenström, 1999). We found that Rac1 and RhoB were phosphorylated products. diffusely distributed predominantly throughout the cytoplasm Work that describes the involvement of p70 S6 kinase in the of Swiss 3T3 fibroblasts and did not colocalize with Fliih. regulation of actin cytoskeletal rearrangements (Crouch, 1997; When cells were stimulated with 10% FCS, Ras, Cdc42 and Berven et al., 1999) led to speculation about the effect RhoA were induced to colocalize with Fliih on actin arcs and rapamycin may have on the flightless I protein in Swiss 3T3 the leading edge of lamellae. There is evidence of GTPase fibroblasts. Treatment with this inhibitor did not appear to activity modulating the behavior of proteins homologous to affect the translocation of Fliih to the leading edge and flightless I via a direct interaction (Azuma et al., 1998), therefore Fliih does not appear to play a role in rapamycin- however, the data emerging concerning the flightless I protein sensitive cytoskeletal rearrangements. It should be noted that is less clear. Although Goshima et al. (Goshima et al., 1999) cells treated with rapamycin and LY294002 failed to form actin show that an expressed form of C. elegans flightless I is able arcs and appeared to form elongated tails, a phenomenon to bind Ras, as predicted (Claudianos and Campbell, 1995), previously described (Berven et al., 1999). Liu and Yin have failed to show binding between flightless I In summary, we have identified a 145 kDa protein in Swiss and Ras or Ras-related GTPases using the two-hybrid system 3T3 fibroblasts and shown that Fliih localizes to a variety of (Liu and Yin, 1998). Classical GTPase cascades include Rac actin- and tubulin-based structures. Fliih colocalizes with Ras, (Ma et al., 1998a), a protein that does not appear to colocalize RhoA and Cdc42 implicating them in a common functional with Fliih. The failure to observe colocalization between pathway, and this anterior localization is inhibited by flightless I and Rac may be due to an interaction occurring at LY294002. Future work will involve investigating the time points that were not examined in this study. Alternatively, functional significance of this inhibition, the colocalization any PI 3-kinase-mediated regulation of Fliih may be via a Rac- between Fliih and GTPase proteins and any role this may play independent pathway (Lim et al., 1996; Ma et al., 1998b). It is in the transduction of a Fliih-mediated signal. We will also unclear as to whether the colocalization between Fliih and investigate the potential role of PI 3-kinase in Fliih-mediated GTPase proteins is representative of a direct interaction, regulation of cytoskeletal rearrangements. The actin severing although this remains a significant possibility. The effect of activity and ability to colocalize and/or interact with important these signaling molecules on Fliih need not be a direct one, and signal transduction molecules highlights the possibility of indeed these proteins may lie upstream or downstream and link an important role for flightless I in cell migration and to Fliih via intermediary proteins. In support of a common proliferation. 560 JOURNAL OF CELL SCIENCE 114 (3)

We thank Professors Ian Young and Ian Hendry for helpful Cellularization in Drosophila melanogaster is disrupted by the inhibition of discussions throughout the course of this work, and Dr Klaus Matthaei Rho activity and the activation of Cdc42 function. Dev. Biol. 204, 151-164. for advice concerning the construction of the EGFP-C1-FLII Crouch, M. F. and Simson, L. (1997). The G-protein Gi regulates mitosis but construct. We thank Katharina Heydon for advice on the use of the not DNA synthesis in growth factor-activated fibroblasts: a role for the confocal microscope, Dr Nick Dixon for the generous gift of the pET nuclear translocation of Gi. FASEB J. 11, 189-198. Crouch, M. F. (1997). Regulation of thrombin-induced stress fibre formation vector and valuable advice, and Dr Peter Milburn and Cameron in Swiss 3T3 fibroblasts by the 70-kDa S6 kinase. Biochem. Biophys. Res. McCrae of the ANU Biomolecular Resource Facility for running the Commun. 233, 193-199. sequencers. 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