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First published online 26 February 2008
doi: 10.1242/jcs.021584

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Chemical genetic analysis of the regulatory role of Cdc2p in the S. pombe septation initiation network

¹ Cell cycle control laboratory, ISREC, 1066 Epalinges, Switzerland
² Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Switzerland
³ Department of Chemistry, University of Wisconsin-Oshkosh, 800 Algoma Boulevard, Oshkosh, WI 54901, USA

View larger version (56K): [in this window] [in a new window]   Fig. 1. The cdc2-as mutant is sensitive to analogue. (A) Wild-type and cdc2-as cells were grown in YE medium to early exponential phase and analogue was added to a concentration of 1 µM; the controls received an equal volume of DMSO. Cell number was determined at intervals. (B) Micrographs of the cells described in panel A, stained with DAPI and Calcofluor. Cells were incubated for 3.5 hours at 25°C after addition of analogue. (C) FACS analysis of the cells described in (A). HU 2.5h are cells treated for 2.5 hours with 12 mM hydroxyurea, to generate G1 and G2 peaks as 1C and 2C references. Note that in exponential growth, an S. pombe FACS profile shows only a single peak, corresponding to 2C DNA content. This is because G1 is very short and cells have completed S-phase before the daughters separate. (D) Samples from the experiment shown in panel A were fixed and stained with Rhodamine-conjugated phalloidin to reveal F-actin. Scale bars: 10 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 2. The effects of analogue on cdc2-as division and kinase activity. (A,B) The indicated cells were grown in YE medium to early exponential phase and analogue was added to the indicated final concentrations; the controls received an equal volume of DMSO. Samples were removed at the indicated times and analysed by FACS. (C) A protein extract was prepared from cdc2-as 10 minutes after release from the analogue arrest. As a control, protein extracts were prepared from cdc2+ cdc25-22 arrest-release-synchronised cells (20 minute time point, when cells are in early mitosis and Cdc2p activity is high) and assayed. Analogue, or an equivalent volume of solvent, was added to the extracts at the indicated concentrations, and an H1 kinase assay was performed. The upper panel shows a scanned autoradiograph of the phosphorylated H1. The middle panels show the Coomassie-Blue-stained histone H1 and the bottom panels show a western blot of the extracts used for the assay, probed with TAT-1 antibody as loading control. Note that the kinase activity is reduced by addition of analogue to the cdc2-as extract, but not the cdc2+ extract.

View larger version (60K): [in this window] [in a new window]   Fig. 3. The effects of cdc2-as on meiosis, and rescue by increased expression of cdc13. (A) A cross of h+ cdc2-as with h- cdc2-as lys1-131 was performed at 25°C. After 2 days, the mating mixture was suspended in PBS, fixed, and stained with DAPI and Calcofluor. The outcomes of the mating are indicated on the histogram. The figures indicate the percentage of each outcome class; more than 300 spore-containing cells were scored. The combined DAPI and transmission image of a dyad is shown. (B) Small cells were selected from an exponentially growing cdc2-as culture by centrifugal elutriation, and inoculated into fresh medium in the presence of 10 µM analogue or DMSO and incubated for 3 hours at 36°C. Cells were fixed and stained with DAPI and Calcofluor. (C) cdc2-as cells were transformed with a plasmid containing the genomic copy of cdc13 or empty vector. Colonies were allowed to form on EMM2 medium at 25°C, replicated to 36°C and incubated at 36°C overnight. Colonies were photographed. The inset shows a magnification of the edge of the colony. (D) The cells described in panel C were treated with the indicated concentrations of analogue at 25°C and samples were analysed by FACS. Scale bars: 10 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 4. The analogue-induced cell cycle arrest is reversible. (A) Exponentially growing cdc2-as cells were treated with 1 µM analogue for 3.5 hours at 25°C. Cells were washed and resuspended in fresh medium. Samples were removed and stained with DAPI and Calcofluor (DNA) or Rhodamine-conjugated Phalloidin (F-Actin). (B) The progression of the cells described in panel A through mitosis was plotted. Cells with two nuclei and no division septum were scored as binucleate. Scale bars: 10 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Inactivation of Cdc2p-as promotes recruitment of GFP-Sid1p to the SPB. Small cells of the strain cdc2-as GFP-sid1 rlc1-GFP pREP3X-mad2 were selected by centrifugal elutriation. A sample was taken when cells were in mitosis, and analogue or DMSO was added to aliquots of the remaining cells. Ten minutes later, cells were harvested, fixed and stained to examine -tubulin and GFP by indirect immunofluorescence. (A,B,C) DAPI, -tubulin and GFP signals, and a merge. The arrows point to the GFP-Sid1p signal on the SPB. The ring signal is Rlc1-GFP, which serves as a marker for entry into mitosis. Scale bars: 10 µm. (D) Magnifications of the nuclear region of the micrographs shown in panels A-C. (E) Graph of the percentage of cells with GFP-Sid1p associated with the SPB, for a given spindle length, in each of the samples. The number of cells with spindles scored in each sample was more than 200.

View larger version (47K): [in this window] [in a new window]   Fig. 6. Analysis of the effects of Cdc2p-as inactivation upon the regulation of cytokinesis. (A) Small cells of the strain cdc2-as mob1-GFP pREP3X-mad2 were selected by centrifugal elutriation. A sample was taken when cells were in mitosis, and analogue or DMSO was added aliquots of the remaining cells. Ten minutes later, cells were harvested, fixed and stained to examine -tubulin and GFP by indirect immunofluorescence. The images show representative DAPI staining, -tubulin and GFP indirect immunofluorescence in the three samples. For the T10 analogue panels, the left-hand image shows a cell with a single Mob1p-GFP ring, whereas the right hand images show a cell with a double ring. Note that Mob1p-GFP is present on the SPBs throughout mitosis. Quantification is presented in Table 2. (B) cdc2-as cdc13-GFP pREP3X-mad2 cells were treated as described in the legend for panel A. (C) Cells of the strain cdc2-as cdc7p-GFP pREP3X-mad2 were treated as described in the legend for panel A. A sample was examined 20 minutes after addition of analogue. Cells were fixed and stained with DAPI and Calcofluor. Scale bars: 10 µm.

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