The flightless I protein colocalizes with actin- and microtubule-based structures in motile Swiss 3T3 fibroblasts: evidence for the involvement of PI3-kinase and Ras-related small GTPases

The flightless I protein contains an actin-binding domain with homology to the gelsolin family and is likely to be involved in actin cytoskeletal rear rangements. It has been suggested that this protein is involved in linking the cytoskeletal network with signal transduction pathways. We have develop ed antibodies directed toward the leucine rich repeat and gelsolin-like dom ains of the human and mouse homologues of flightless I that specifically re cognize expressed and endogenous forms of the protein. We have also constru cted a flightless I-enhanced green fluorescent fusion vector acid used this to examine the localization of the expressed protein in Swiss 3T3 fibrobla sts. The flightless I protein localizes predominantly to the nucleus and tr anslocates to the cytoplasm following serum stimulation, In cells stimulate d to migrate, the flightless I protein colocalizes with beta -tubulin- and actin-based structures. Members of the small GTPase family, also implicated in cytoskeletal control, were found to colocalize with flightless I in mig rating Swiss 3T3 fibroblasts. LY294002, a specific inhibitor of PI 3-kinase , inhibits the translocation of flightless I to actin-based structures. Our results suggest that PI 3-kinase and the small GTPases, Pas, RhoA and Cdc4 2 may be part of a common functional pathway involved in Fliih-mediated cyt oskeletal regulation. Functionally, we suggest that flightless I may act to prepare actin filaments or provide factors required for cytoskeletal rearr angements necessary for cell migration and/or adhesion.