Figure 4. Aurora B-dependent phosphorylation of CHMP4C S210 activates the NoCut abscission checkpoint.

A. β-galactosidase assay from yeast co-transformed with the indicated VP16 and GAL4-fused constructs (n=3±S.D.). B. Cell lysates and glutathione-bound fractions from 293T cells transfected with the indicated fusion proteins were examined by western blotting with α-HA. C. HeLa mCh-Tub cells stably expressing HA-CHMP4C^R were fixed and stained with α-HA and α-Aurora B. D. Asynchronous and mitotic lysates of HeLa mCh-Tub cells stably expressing HA-CHMP4C^R were immunoprecipitated with α–HA and treated as indicated and examined by blotting with α–HA, α-CHMP4C, α-CHMP4B, α–CEP55 and α–HSP90. E. F. Asynchronous or mitotically arrested HeLa mCh-Tub cells stably expressing HA-CHMP4C^R were either released into media containing DMSO or the phosphatase inhbitor Okadaic Acid (OA) for the indicated times (E) or were treated overnight during the nocodazole arrest with inhibitors of MEK (U0126), PI 3-kinase (LY294002) or Aurora B (ZM447439) (F). Cell lysates were examined by blotting with α–HA and α–HSP90. G. H. Proteins were immunoprecipitated from 293T cells with α–HA and subjected to an in-vitro kinase assay with recombinant Aurora B. Incorporated ³²P was visualized by phosphorimaging, blotting with α–HA allowed detection of immunoprecipitates. I-K. Asynchronous cultures of HeLa mCh-Tub cells stably expressing HA, HA-CHMP4C^R, HA-CHMP4C^R δINS, or HA-CHMP4C^R S210A were transfected with the indicated siRNA. Resolved cell lysates were examined by blotting with α-CHMP4C, α-HA and α-HSP90 (I). Alternatively, cells were imaged live (J, K) and abscission time (Luciferase, 104±35 minutes, n = 244, CHMP4c siRNA, 71±37 minutes, n = 260; CHMP4c siRNA and HA-CHMP4C^R, 118±52 minutes, n = 269; CHMP4C siRNA and HA-CHMP4C^R δINS, 81±40 minutes, n = 264; CHMP4C siRNA and HA-CHMP4C^R S210A, 91±38 minutes, n = 268) quantified across 7 independent experiments. L, HeLa cells stably expressing YFP-LAP2β and either HA or HA-CHMP4C^R, HA-CHMP4C^R δINS, or HA-CHMP4C^R S210A were treated with the indicated siRNA, imaged live and the timing of YFP-LAP2β bridge resolution (Luciferase, 628 ± 382 minutes, n = 41; CHMP4c siRNA, 413 ± 292 minutes, n = 41; CHMP4c siRNA and HA-CHMP4C^R, 698 ± 332 minutes, n = 41; CHMP4C siRNA and HA-CHMP4C^R δINS, 402 ± 259 minutes, n = 36; CHMP4C siRNA and HA-CHMP4C^R S210A, 421 ± 295 minutes, n = 40) quantified from 2 independent experiments.