Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis.

Cell Culture

HeLa cells were grown in DME supplemented with 5% newborn calf serum, 5% FCS, and antibiotics. HeLa cells were synchronized using a thymidine-aphidicolin protocol ( Pines and Hunter 1989 ). Cells were cultured on the microscope stage in a CO 2 -independent medium without phenol red (GIBCO BRL). The medium was overlaid with mineral oil (Sigma Chemical Co.) to prevent evaporation. Hydroxyurea was used at a final concentration of 2.5 mM and was added immediately after release from the aphidicolin block.

Plasmid Constructs and Protein Purification

The cyclin B1-MmGFP, cdc25C(S216G), and Wee1 constructs have been previously described ( Hagting et al. 1998 ). Cyclin B1 R42A -MmGFP was the gift of Paul Clute (Wellcome/CRC Institute). Cdc25B3 and Cdc25C were tagged at the amino terminus with MmGFP ( Zernicka-Goetz et al. 1996 , Zernicka-Goetz et al. 1997 ) by PCR using Taq polymerase and cloned into the pCMX vector ( Umesono et al. 1991 ). The stop codon of GFP was mutated to a Hind III site to link it with the first amino acid of Cdc25B or Cdc25C creating a 3–amino acid linker (Gly-Ile-Pro). Myc-tagged cdc25B2 ( Lammer et al. 1998 ) was cloned into the pCDNA3 vector for expression in HeLa cells. All constructs were sequenced using an Applied Biosystems DNA sequencer. Cyclin B1–cdc2 K33R complexes were expressed in and purified from baculovirus-infected cells as described ( Hagting et al. 1998 ). Cyclin B1 F146A was expressed in Escherichia coli and purified as described ( Hagting et al. 1999 ).

Microinjection and PCC Detection

Constructs expressing cDNAs under the control of the cytomegalovirus promoter were microinjected into cell nuclei using an Eppendorf semi-automatic microinjection apparatus. To assay for condensed chromatin, Hoechst 33342 was added to cells at a concentration of 1 μg/ml at the end of the experiment. Injected cells were identified by green fluorescent protein (GFP) fluorescence and those that had rounded up with abnormally condensed chromatin were scored. At least 50 cells were scored for each injected construct and experiment. Apoptotic cells were assayed using the apoptosis detection kit (R&D Systems, Inc.) and HeLa cells treated with cycloheximide plus tumor necrosis factor α were used as positive controls.

Time-lapse Differential Interference Contrast

(DIC) and Fluorescence Imaging To visualize GFP-chimeras in living cells, cells were cultured on an inverted Leica DMIRB/E microscope using the ΔTC3 system (Bioptechs) to maintain cells at 37°C. Images were captured with a PentaMax CCD camera (Princeton Instruments) fitted to the lateral photo port. GFP- and yellow fluorescent protein (YFP)–chimeras were detected with custom filter sets JP1 and JP2 (Chroma Technology Corp.) and two Lambda 10-2 filter wheels (Sutter Instrument) controlled by a PowerWave computer (PowerComputing). One filter wheel was used to control the wavelength of the excitation light. The other filter wheel controlled the wavelength of the emission light and also the polarizer for DIC images. To distinguish between GFP and YFP we used the JP3 filter set as described ( Hagting et al. 1999 ). Images were collected and processed using IP Lab Spectrum software (Scanalytics Inc.) and exported to Adobe Photoshop for printing.

Immunofluorescence and Confocal Imaging

For β-tubulin and MPM2 staining, cells were fixed with 3% PFA/Triton X-100 and stained as described ( Pines 1997 ) 3–4 h after microinjection. Tubulin was detected using an anti–β-tubulin mAb (Nycomed Amersham) and mitotic epitopes were detected using the MPM2 mAb (Upstate Biotechnology, Inc.). To detect myc-cdc25B2, pCDNA3/myc-cdc25B2 was microinjected (0.1 μg/μl) and cells were fixed with methanol/acetone (1:1) 3 h after injection and stained with the mAb 9E10 (gift of Erich Nigg, University of Geneva, Geneva). A Cy5 conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc.) was used as the secondary antibody. Cells were analyzed by confocal laser scanning microscopy using a Bio-Rad 1024 confocal microscope set on 10% laser power and Kalman averaging. Stacks of images were projected using Lasersharp software (Bio-Rad Laboratories) and exported to Adobe Photoshop for processing and printing. Glutathione-S-transferase (GST) Pulldowns Human cdc25B2, cdc25B3, cdc25C, and cyclin B1 were in vitro translated from pBSK/cdc25B cDNA using the TNT-coupled reticulocyte system (Promega Corp.). GST-cyclin B1, GST-Cdc25B2, GST-Cdc25B3, and GST were expressed in BL21(DE3) cells using the pGEX-4T expression vector and purified on GSH-Sepharose.