RESEARCH ARTICLE 549 The flightless I protein colocalizes with actin- 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 gelsolin 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, Cytoskeleton, GTPase, Actin, Tubulin INTRODUCTION moderate and severe fliI mutants indicate a possible role for the fliI protein in regulating actin cytoskeletal rearrangements. The Drosophila melanogaster flightless I (fliI) gene encodes a A 380 amino acid 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 Caenorhabditis elegans (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 beta sheet/alpha helix 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 enzymes (Tan et al., 1990), tyrosine kinase et al., 1997). Viable mutations of the D. melanogaster fliI locus 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) proteins (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 plasmid 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).