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First published online 3 January 2006
doi: 10.1242/jcs.02729

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Activity of Cdc2 and its interaction with the cyclin Cdc13 depend on the molecular chaperone Cdc37 in Schizosaccharomyces pombe

The Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JR, UK

View larger version (53K): [in a new window]   Fig. 1. (A) A schematic diagram indicating the location of mutations within the cdc37ts mutant alleles cdc37-681 (Leu285 to Pro), cdc37-184 (Ala287 to Asp), cdc37-13 (Glu237 to Lys and Tyr261 to His) and cdc37-J (Leu286 to Met, His305 to Leu and Arg314 to Gly). Alignment of the human and S. pombe Cdc37 protein sequences enabled the Hsp90-binding domains (Roe et al., 2004) (horizontal stripes) and the homodimerisation domain (Roe et al., 2004) (diagonal stripes) to be mapped from the human to the S. pombe protein. (B) The cdc37ts mutants and the cdc37+ strain ED1022 were streaked on YE plates and incubated at 28, 32 and 36°C for 4 days to examine the ability of each strain to form single colonies at different temperatures

View larger version (42K): [in a new window]   Fig. 2. (A) Analysis of cell number of cdc37-184, cdc37-681 and cdc37+ ED1022 strains. Strains were cultured at 28 and 36°C over an 8 hour time course, and samples taken at 2 hour intervals for determination of cell number using a Coulter electronic particle counter. (B) Comparison of Cdc37 protein levels in cdc37ts mutants and the cdc37+ strain ED1022 after 8 hours at 28 and 36°C. Western blot analysis was carried out on whole-cell protein extracts using the anti-S. pombe Cdc37 antibody. ß-tubulin was detected by TAT1 antibody and used as a loading control. (C) Cell morphology of cdc37ts mutants cdc37-184 and cdc37-681 and the cdc37+ strain ED1022 on YE plates incubated at 28 and 36°C for 24 hours. Bar, 10 µm. (D) Mean cell length of cdc37-184, cdc37-J, cdc2-33 and cdc2-L7 and cdc37+ cells (with s.d. bars). Strains were cultured in liquid YE at 28 and 36°C over an 8 hour time course and the lengths of 200 cells measured for each sample.

View larger version (45K): [in a new window]   Fig. 3. (A) Flow cytometry of cdc37-184, cdc37-J, cdc37+ ED1022 and cdc10-129 to determine the DNA content of cells. Strains were cultured in YE at 28 and 36°C over an 8 hour time course. Samples of cells were taken every 2 hours and processed for flow cytometry. (B) Frequency of phenotypes observed by DAPI staining of cdc37-184, cdc37-681 and cdc37+ cells. Samples of cells were taken every 2 hours, fixed in formaldehyde and stained with DAPI. Phenotypes 1,2 and 3 are shown in C. Phenotype 4 is a cell with a single nucleus and a septum, phenotype 5 is a cell with a septum cutting through a single nucleus and phenotype 6 is a cell with multiple septa. (C) Cellular phenotypes 1, 2 and 3 observed with DAPI staining of cdc37+ and mutants at both 28 and 36°C. (D,E,F) Immunofluorescence of microtubules, using the TAT1 antibody, of cdc37+ interphase microtubules (D), and arrested cdc37-184 (E) and cdc37-J (F) cells. Strains were cultured at 28 and 36°C for 8 hours. (G) Immunofluorescence of cdc37-184 and cdc37+ ED1022 cells with the anti-S. pombe Cdc37 antibody. Samples of cells were processed for immunofluorescence with anti-S. pombe Cdc37 antibody or depleted antibody (see Materials and Methods). Bars, 10 µm.

View larger version (25K): [in a new window]   Fig. 4. Effect of increased expression of Cdc2 in cdc37ts mutant strains cdc37-13 and cdc37-184. The multicopy plasmid pIRT2-cdc2 which carries a genomic cdc2 fragment was introduced into each strain and the resulting transformants tested for growth at 28 and 36°C by serial dilution.

View larger version (40K): [in a new window]   Fig. 5. (A) Cdc2 kinase activity and protein levels were assayed in cdc37-681, cdc37-184 and cdc37+ cells after growth at 28 and 36°C. Strains were cultured at 28 and 36°C over a 3 hour time course. Samples of cells were taken hourly and Cdc2 was affinity-precipitated on p13Suc1 beads. The kinase activity of Cdc2 was determined by its ability to phosphorylate histone H1. Cdc2 protein levels were determined by western blot with the anti-PSTAIR antibody and ß-tubulin detected by TAT1 antibodies as a loading control. The asterisk indicates the position of p31 which is also recognised by the anti-PSTAIR antibody (see text). (B) The level of Cdc2 protein in soluble and insoluble fractions of extracts of cdc37-184, cdc37-681 and cdc37+ cells grown at 28°C or incubated at 36°C for 3 hours was analysed. Native protein extracts were prepared and the soluble and insoluble fractions separated by centrifugation at 20,000 g for 5 minutes at 4°C. Western blot analysis with anti-PSTAIR antibody against Cdc2 and TAT1 antibody as a loading control. (C) Level of phosphorylation on Tyr15 of Cdc2 in cdc37-184, cdc37-681 and cdc37+ strains incubated at 28 and 36°C over a 3 hour time course. Samples of cells were taken hourly and denatured S. pombe proteins extracted and western blotted with antibody specific for Cdc2 phosphotyrosine 15.

View larger version (44K): [in a new window]   Fig. 6. (A) The interaction between Cdc2 and Cdc13 in cdc37-184, cdc37-681 and cdc37+ strains was analysed. Strains were cultured at 28 and 36°C over a 4 hour time course and samples of cells were taken as shown. Cdc13 was immunoprecipitated from protein extracts with the anti-Cdc13 6F 10/11 antibody. Western blot analysis was carried out with anti-Cdc13 6F 10/11 and anti-PSTAIR antibodies. (B) The interaction between Cdc2 and Cdc13 in cdc37-184, cdc25-22 and cdc37+ strains was analysed. Strains were cultured at 28 and 36°C over a 3 hour time course and samples of cells were taken hourly. Cdc13 was immunoprecipitated from protein extracts with the anti-Cdc13 6F 10/11 antibody. Western blot analysis was carried out with anti-Cdc13 6F 10/11 and anti-PSTAIR antibodies. (C) Immunoprecipitation experiments to detect a biochemical interaction between Cdc2 and Cdc37 in native S. pombe protein extracts from cdc37+, cdc37-681 and cdc37-184 cells cultured at both 28 and 36°C. Immunoprecipitates with the anti-S. pombe Cdc37 and anti-rat IgG (control) antibodies were run on SDS-PAGE and analysed by western blot to determine whether Cdc2 precipitates with Cdc37 from native S. pombe protein extracts.

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