Spatial positive feedback at the onset of mitosis.

Cell Culture, Synchronization, and Transfection HeLa cells (ATCC) and Tet-On Advanced HeLa cells (Invitrogen) were cultured at 37°C, 5% CO2 in DMEM (Invitrogen) supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 μg/ml) and G418 (100 μg/ml, for Tet-On Advanced HeLa cells). Cells were synchronized at the G1/S phase of the cell cycle by double thymidine block. Transfection of cDNAs was typically performed using either Fugene 6 (Roche) (for DNA transfection) or Gene Silencer (Genlantis) (for small interfering RNAs, siRNA, transfection or double DNA/siRNA transfection) according to the manufacturers’ instructions. Transfection of 50 μM dextran-70-kDa-Texas Red was done by suspended drop electroporation (SDE) as described in ( Guignet and Meyer, 2008 ).

Microscopy and Data Analysis

Time-lapse live cell imaging was performed on either an ImageXpress Micro inverted epifluorescence microscope (Molecular Devices) or on a Nikon Eclipse TI with perfect focus, both equipped with temperature, humidity and CO 2 control. ImageXpress images were acquired with 20x or 40x plan fluorescence objectives. Excitation (Ex) and emission (Em) filters sets (Chroma Technology Corporation) were as follows: CFP, 427-10nm (Ex), 483-32 nm (Em); YFP, 504-12 nm (Ex), 542-27 nm (Em); mCherry, 589-15 nm (Ex), 632-22 nm (Em). Nikon Eclipse TI images were acquired using either 40x NA 1.3 Plan Flour or 60x NA 1.3 Plan Apo objectives. Excitation (Ex) and emission (Em) filters sets were as follows: CFP, 430-24 nm (Ex), 470-24 nm (Em); YFP, 500-20 nm (Ex), 535-30 nm (Em); mCherry, 572-35 nm (Ex), 632-60 nm (Em). Micromanager 1.3 was used for acquisition of time-lapse images. A typical experiment would monitor cells from 15–20 h with images taken every 3–5 min. Confocal microscopy was performed on a Leica TCS SP2 ABOS equipped with a 63x 1.3 NA oil immersion objective. DAPI was excited using a 405 nm diode laser; Alexa 488 or YFP were excited with a 488 nm or 514 nm Ar laser and Alexa 647 was excited with a 633 nm HeNe laser line. All data analysis was done with scripts written in Matlab (Mathworks) or using Cell Profiler (Broad Institute) and ImageJ (National Institutes of Health). Cdk1-Cyclin B1 Autophosphorylation For cyclin B1 auto-phosphorylation experiments where we varied the concentration of Cdk1-cyclin B1, purified human Cdk1-cyclin B1 protein complex was incubated at the indicated concentrations at 25°C for 1, 2 or 5 min with kinase buffer (20 mM HEPES/NaOH pH 7.4, 10 mM MgCl 2, 1 mM DTT), 15 μM ATP and phosphatase inhibitor cocktail (Sigma) in a total volume of 20 μl. For Cyclin B1 auto-phosphorylation experiments where we varied kinase reaction volumes, purified human Cdk1-cyclin B1 protein complex was incubated at 25°C for 1, 2 or 5min with kinase buffer (20 mM HEPES/NaOH pH 7.4, 10 mM MgCl 2, 1 mM DTT), 15 μM ATP and phosphatase inhibitor cocktail (Sigma) in a total volume of 10, 20, 40, 80 or 160 μl. Reactions were stopped by addition of Laemmli sample buffer and analyzed by gel electrophoresis and blotting followed by phospho-imaging. Nuclear/Cytoplasmic Cell Fractionation HeLa cell nuclear and cytoplasmic fractions were isolated with NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) according to the manufacturer’ instructions. Total protein amount in the nuclear and the cytosolic lysates was quantified by BCA assay for protein quantitation. Typically, 15–20 μg or 100–200 μg of total protein for each fraction was used for western blot analysis or immunoprecipitation, respectively. For Cdk1-cyclin B1 nuclear/cytosolic kinase assays, 100 μg of total protein was used to immunoprecipitate cyclin B1 using anti-cyclin B1 antibody (Santa Cruz). Kinase assays were performed using purified histone H1 (Upstate) as a general Cdk1-cyclin B1 substrate and the resulting 32 P-labeled histone was detected and quantified by SDS gel electrophoresis and blotting followed by phosphorimaging.

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