The N-terminal domain of Cyclin A2 mediates its interaction with RhoA and restores cytoplasmic bundles in Cyclin A2–deficient cells. (A) Schematic representation of Cyclin A2 deletion mutants. The first one encompasses the N-terminal part of Cyclin A2 upstream of the Cyclin box (N-ter). The second contains the C-terminal part of the protein including the Cyclin box (C-ter). The latter construct contains amino acids 1–7, allowing proper expression of the protein. Flag tag was fused to the C-terminal part of each fragment. (B) Interaction of RhoA with Cyclin A2 fragments. Immunoprecipitations (IP) using anti-Flag–agarose affinity gel were performed on NIH3T3 cells cotransfected with plasmids encoding RhoA-EGFP and N-terminal, C-terminal, or full-length Cyclin A2. Binding of RhoA to the different fragments was detected by immunoblotting using anti-GFP antibodies. Molecular markers are given in kilodaltons. (C) Cyclin A2 fragments were expressed in Cyclin A2–deficient NIH3T3 cells and detected by immunofluorescence using anti-Flag antibody (green), whereas F-Actin was detected using phalloidin staining (red). Bars, 20 µm. Graph refers to the percentage of transfected cells harboring a reversion of the cortical phenotype. Data are represented as means ± SEM (**, P = 0.006; n = 3).