Smurf2 is a cell cycle–regulated protein localizing specifically in the centrosome, mitotic midzone, and midbody. (a) HeLa cells were synchronized by a double-thymidine protocol. At the indicated hours after release from the block, cells were analyzed by immunoblotting for the proteins shown. (b) Immunofluorescence microscopy for γ-tubulin (green) and Smurf2 (red) in HeLa cells at interphase and metaphase. Polyclonal anti-Smurf2 antibody was used for Smurf2 staining. DAPI was used to stain chromosomal DNA (blue). The close-up images are shown with threefold higher magnification. (c) Immunofluorescent staining for α-tubulin (green) and Smurf2 (red) in human mammary epithelial MCF-10A cells during mitosis. Bars, 10 μm.

Smurf2 silencing leads to chromosomal misalignment at metaphase and premature onset of anaphase with defective chromosome segregation. HeLa cells stably expressing GFP-H2B were transfected with Smurf2 siRNA #1, and chromosomal movement was monitored 24–25 h after transfection by time-lapse fluorescence microscopy. Quantified data are representative of at least two independent experiments. (a) Defects in chromosomal alignment in Smurf2-depleted cells at metaphase. Metaphase defects in cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (siNS, open bars) were categorized into the two indicated groups. (b) Unaligned chromosomes (*) and lack of metaphase plates (#) during mitosis with Smurf2 depletion. Arrowheads denote lagging chromosomes. (c) Chromosomal segregation defects in GFP-H2B HeLa cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (open bars) at anaphase. (a and c) Data are means ± SEM (error bars) from three independent experiments. (d) Lagging chromosomes (arrowheads, top row), major segregation defects (arrowheads, middle row), and lack of segregation in GFP-H2B HeLa cells with Smurf2 depletion. (e) Premature anaphase onset in Smurf2-depleted cells. The metaphase–anaphase transition of GFP-H2B HeLa cells was monitored by time-lapse microscopy, and the time from nuclear envelope breakdown (NEBD) until anaphase onset was determined. (f) Representative pictures of the metaphase–anaphase transition. AO, anaphase onset. Bars, 10 μm.

Smurf2 is required for stability and functional localization of Mad2. (a) Smurf2 silencing rapidly down-regulates Mad2 protein but not mRNA. HeLa cells in asynchronous culture were transfected with Smurf2 siRNA or nonspecific dsRNAs (siNS) and harvested at the indicated times after transfection for Western blotting and RT-PCR. (b) Smurf2 silencing results in increased polyubiquitination of Mad2 followed by proteasomal degradation. HeLa cells were transfected with a Mad2 expression plasmid and Smurf2 siRNA #1 or control dsRNA. Cells were then treated with 2 μM MG132 for 4 h and analyzed by immunoprecipitation (IP) with Mad2 antibody followed by Western blotting using the indicated antibodies. (c) Smurf2 physically interacts with Mad2. Untransfected HeLa cells were released from synchronization at S phase with double-thymidine treatment and were harvested at the indicated times for immunoprecipitation followed by Western blotting. Ig, Ig heavy chain; NS, nonspecific band. (d) Ectopic expression of Mad2 is unable to restore the spindle assembly checkpoint in Smurf2-depleted cells. Western blotting for mitotic regulators in HeLa cells transfected with the indicated plasmid and siRNA. Cells were synchronized with thymidine treatment, released into a nocodazole-containing medium, and harvested at the indicated times after release (see Fig. 4 b and Materials and methods). (e) Ectopically expressed Mad2 localizes at centromeres in nocodazole-treated control cells but not in Smurf2-depleted cells. Immunofluorescence microscopy with anti-Mad2 and ACAs 10 h after release from the thymidine block. The close-up images are shown with threefold higher magnifications. Bars, 10 μm.

Putative roles of Smurf2 in regulation of the spindle assembly checkpoint.

Smurf2 silencing inhibits mitotic progression and cytokinesis. (a) Smurf2 levels determined by immunoblotting in HeLa cells (top) and U2OS cells (bottom) 48 h after transfection with anti–Smurf2 siRNA (siSm2) or nonspecific dsRNA (siNS). UN, untransfected control. (b) Morphology of multinucleated HeLa cells 48 h after transfection with siRNA #1. Hoechst was used for DNA staining. (c) Percentages of multinucleated cells in HeLa cultures at the indicated hours after transfection with siNS (open bars) or Smurf2 siRNA #1 (shaded bars). At least 300 cells were counted per time point per cohort. Mean of data from two independent experiments are shown. See Fig. S2 a (available at http://www.jcb.org/cgi/content/full/jcb.200801049/DC1) for raw data from the individual experiments. (d) Percentages of cells that failed cytokinesis in siNS- or siSm2-transfected HeLa cultures quantified from the time-lapse microscopy. At least 65 mitotic cells per cohort (siNS vs. siSmurf2) were analyzed for the progression of cytokinesis. (e) Representative time-lapse pictures of siSm2-transfected HeLa cells that showed impaired mitotic progression and failed cytokinesis. Bars: (b) 50 μm; (e) 10 μm.

Smurf2 is a novel regulator of the spindle assembly checkpoint. (a) Smurf2 localizes at centromeres when the spindle checkpoint is active. HeLa cells were synchronized by a double-thymidine protocol (see Materials and methods). Cells were then released into a fresh medium (time 0), and 3 h later nocodazole was added. Immunofluorescence microscopy after staining with ACA, anti-Smurf2 antibody, and DAPI for DNA. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole. (b) Smurf2 depletion results in failed accumulation of APC/C-Cdc20 substrates during nocodazole-induced metaphase arrest. Immunoblotting was performed for mitotic regulators in HeLa cells transfected with Smurf2 siRNA #1 or nonspecific dsRNA (siNS). After release from the thymidine block, cells were incubated for the indicated hours in the presence of nocodazole. (c) Smurf2-depleted cells override nocodazole-induced spindle assembly checkpoint, resulting in tetraploidy. Control (siNS) and Smurf2-depleted cells were fixed at the indicated times and subjected to flow cytometry. (d) Cosilencing of Cdc20 restores mitotic accumulation of cyclin B1 and securin in Smurf2-depleted HeLa cells. Cells were synchronized and released into nocodazole-containing medium as described in b. (e and f) Smurf2 depletion results in loss of Mad2 from centromeres, whereas centromere localization of BubR1 is unaffected. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole, and immunofluorescence microscopy was performed. (a, e, and f) The close-up images are shown with threefold higher magnification. Bars, 10 μm.

Expression of a catalytically inactive Smurf2 impairs the spindle assembly checkpoint with Mad2 destabilization. (a) Immunoblotting for mitotic regulators in HeLa cells transfected with Flag-tagged Smurf2(C716A) or a control plasmid. Cells were synchronized at S phase with thymidine treatment, released into a nocodazole-containing medium, and harvested after incubation at the indicated times after release (see Materials and methods). OE, overexpression. (b) Chromosomal misalignment at metaphase and lagging chromosomes at anaphase observed in cells expressing Smurf2(C716A). Immunofluorescence microscopy of cells stained with anti-Flag antibody for exogenously expressed Smurf2, ACA, and DAPI. Cells were harvested 8.5 h after release from the thymidine block. The second row shows misaligned chromosomes at metaphase, and the bottom row shows lagging chromosomes at anaphase in cells expressing Smurf2(C716A). Bars, 10 μm. (c) Percentages of cells with chromosomal defects in control cultures (open bars) and Smurf2(C716A)-transfected cultures (shaded bars). Data are means ± SEM (error bars) from three independent experiments.