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First published online 17 June 2008
doi: 10.1242/jcs.031591

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UbcH10 has a rate-limiting role in G1 phase but might not act in the spindle checkpoint or as part of an autonomous oscillator

Adam Walker^*,, Claire Acquaviva^*,, Takahiro Matsusaka^*, Lars Koop and Jonathon Pines^¶

Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

View larger version (43K): [in this window] [in a new window]   Fig. 1. UbcH10 is degraded in mitosis. (A) hTert-RPE cells cells were arrested in mitosis with 15 µM colcemid, collected by mitotic shake-off and released into fresh medium. Samples were taken at the indicated times and analysed by propidium iodide staining and flow cytometry (right panel) and for protein levels by immunoblotting with the relevant antibodies (left panel, molecular mass markers in kDa on the right). (B) HeLa cells were arrested in mitosis with 20 ng/ml nocodazole, collected by mitotic shake-off and released into fresh medium. Samples were taken at the indicated times and analysed using propidium iodide staining and flow cytometry (right panel), and using immunoblotting with the relevant antibodies to evaluate protein levels (left panel, molecular mass markers in kDa on the right). (C) Main panel (left). Asynchronous hTert-RPE cells were fixed and stained for immunofluorescence with anti-UbcH10 and anti-cyclin B1 antibodies and with Hoechst 33342 to visualise the DNA. Merged images on the top. Scale bar, 30 µm. The dot plot shows the levels of UbcH10 fluorescence in various mitotic cells measured using ImageJ software (arbitrary units). The small bars in the dot plot show the minimum and maximum values, and the box shows the first and third quartiles. The horizontal bar in the box is the median value. Suspected outliers (open circles) as determined by statistical analysis are shown. The two samples were compared applying Student's t-test and are significantly different from each other (P<0.001). P/PM/M, prophase + prometaphase + metaphase cells; T/G1, telophase + G1 cells that had not yet completed separation. More than 200 cells were analysed in three separate experiments. (D) Venus-tagged UbcH10 begins to be degraded in anaphase. (Top) Graph showing G2-phase HeLa cells that had been injected with an expression construct encoding venus-tagged UbcH10 (5 ng/µl). Degradation profiles of Venus-UbcH10 in four different cells (, , x, ) representative of 38 cells analysed. Venus-UbcH10 fluorescence was measured at 3-minute intervals as cells progressed through mitosis. Time 0 was set to nuclear envelope breakdown (NEBD). (Bottom) DIC and fluorescence images (taken every 3 minutes) of the cell represented by in the graph above. The cell is shown at different points of the cell cycle: PM, prometaphase; M, metaphase; A, anaphase.

View larger version (33K): [in this window] [in a new window]   Fig. 2. UbcH10 levels increase at a similar time to cyclin A in late G1 phase. (A) HeLa cells were released from a dimethylenastron-induced prometaphase block and samples taken at the indicated times (hrs). Samples were analysed by propidium iodide staining and flow cytometry (top) and by immunoblotting with the indicated antibodies (bottom). Results are representative of two independent experiments. (B) Serum-starved hTert-RPE cells were released into the cell cycle by adding 20% serum and harvested at the indicated time points (hrs). Samples were analysed by propidium iodide staining and flow cytometry (bottom) and by immunoblotting with the indicated antibodies (top). Results are representative of five independent experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Overexpressing UbcH10 does not overcome the spindle-assembly checkpoint. (A) HeLa cells that overexpress UbcH10 arrest in mitosis in response to nocodazole at a frequency similar to that of control cells. Cells were treated with 50 ng/ml nocodazole and entry into and exit from mitosis was monitored by time-lapse DIC microscopy with images taken every 15 minutes (not shown). The graph shows the mean values and ±s.e.m. of mitotic cells counted at hourly intervals from three independent experiments are shown. (B) A minority of cells that overexpress UbcH10 undergo mitotic slippage. HeLa cells that had been transfected to express UbcH10 and GFP under an IRES were incubated in 50 ng/ml nocodazole and monitored by fluorescence and DIC microscopy. Images were taken at 5-minute intervals. The cell shown is representative of 24 cells out of 224, and exits mitosis after more than 3 hours. Bar, 10 µm. (C) Cells with reduced levels of Mad2 cannot arrest in mitosis in response to nocodazole. HeLa cells were transfected with 200 nM siRNA targeting Mad2 and 48 hours later monitored by time-lapse DIC microscopy in the presence of 50 ng/ml nocodazole. The cell shown is representative of 56 cells out of 564 cells analysed. Bar, 10 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Cells depleted of UbcH10 degrade cyclin A correctly in mitosis but not in the following interphase. (A) Cells depleted of UbcH10 degrade cyclin A with normal kinetics. HeLa cells treated with 40 nM of GAPDH (control) or UbcH10 siRNA were injected in late G2 phase with a plasmid encoding cyclin A tagged with Venus (5 ng/µl) and followed by time-lapse fluorescence and DIC microscopy. Images were taken every 3 minutes (exposure 200 mseconds), and the total fluorescence minus background was measured for each cell in successive images of a time series and plotted against time. The degradation curves shown are representative of nine control cells and 16 UbcH10 siRNA-treated cells in three independent experiments. Cells expressing low and high levels of cyclin-A–GFP were analysed to exclude any effects caused by overexpressing cyclin A. Four control cells expressing similar levels of cyclin A to four UbcH10-depleted cells are shown. Time 0 represents time of nuclear envelope breakdown. (B) Depleting UbcH10 causes cells to prematurely begin DNA synthesis, which requires cyclin A. hTERT-RPE cells were transfected with 40 nM control siRNA (GAPDH), and siRNA targeting UbcH10 or UbcH10+CycA, and synchronised in G0 by serum starvation for 24 hours. Cells were stimulated to re-enter the cell cycle by adding serum and harvested at the indicated time points. (i) Progression through the cell cycle was assayed by propidium iodide staining and flow cytometry analysis. (ii) Incorporation of BrdU that had been added 30 minutes before each time point. (iii) Protein levels were assayed by immunoblotting with the indicated antibodies. C, asynchronous cells. Results are representative of three independent experiments.

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