Reintroduction of both WT and Mut2–Cyclin A2 reverses F-Actin corticalization induced by Cyclin A2 depletion. (A) Schematic representation of mouse Cyclin A2 protein and modifications induced in this study. Mut1 and Mut2 show substitution of amino acid residues N171 or M200, L204, and W207, respectively, into alanines by site-directed mutagenesis. All constructs were C-terminally fused to three Flag tags. When required, nuclear localization (NLS) or nuclear export (NES) signals were introduced between the Cyclin A2 coding sequence and the Flag tags. The position of the shRNA sequence leading to undetectable levels of Cyclin A2 is indicated. (B) Analysis of binding and kinase-activating properties of Cyclin A2 mutants. Immunoprecipitation (IP) using anti-Flag–agarose affinity gel was performed on NIH3T3 cells extracts after transfection by WT–, Mut1–, or Mut2–Cyclin A2. Immune complexes were either submitted to Western blotting to identify binding partners or analyzed for their histone H1 kinase activity. Molecular markers are given in kilodaltons. (C) Cell cycle distribution of control and Cyclin A2–deficient cells infected with virus expressing wild-type (WT) and mutant Cyclin A2 (Mut2). Synchronized cells were analyzed 24 h after release from the proliferation block. Data are represented as means ± SEM (n = 3). (D) Immunofluorescence analysis of F-Actin organization of shCycA2 NIH3T3 cells transfected with plasmids encoding WT, Mut2, or Mut2 with NLS (Mut2-NLS) Cyclin A2 fused to a Flag tag. F-Actin was stained as in Fig. 1. Transfected Cyclin A2 proteins were detected with the anti-Flag antibody. Bars, 20 µm. The graph refers to the percentage of transfected cells harboring a reversion of the cortical phenotype (150–200 cells were scored for each case). Data are represented as means ± SEM (**, P = 0.0056; n = 4).