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First published online 28 February 2006
doi: 10.1242/jcs.02820

Journal of Cell Science 119, 1130-1143 (2006)
Published by The Company of Biologists 2006
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The Cdc14p phosphatase affects late cell-cycle events and morphogenesis in Candida albicans

Andrés Clemente-Blanco1, Alberto González-Novo2, Félix Machín3, David Caballero-Lima1, Luis Aragón3, Miguel Sánchez2, Carlos R. Vázquez de Aldana2, Javier Jiménez2 and Jaime Correa-Bordes1,*

1 Departamento de Microbiología, Facultad de Ciencias, Universidad de Extremadura, Avda Elvas SN, 06071, Badajoz, Spain
2 Instituto de Microbiología-Bioquímica, Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain
3 Cell Cycle Group, Clinical Sciences Centre, Medical Research Council, Imperial College London, W12 0NN, UK


Figure 1
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  		Fig. 1. Cdc14p levels are cell-cycle regulated. (A) Structural comparison of the S. cerevisiae Cdc14p (ScCdc14p) phosphatase with the C. albicans Cdc14p homolog (CaCdc14p). White box, PTP motif; black box, putative NLS; hatched box, PEST sequence; black dots, Cdc28 phosphorylation sites. (B,C) Small G1 cells carrying Cdc14p-HA (JC94) were isolated by elutriation and released into (B) YPD at 30°C (yeast growth) or (C) YPD + 5% FCS at 37°C (hyphal growth). Samples were collected at intervals of 15 minutes after release and were assayed by western blotting for Cdc14p-HA levels. (D) Mitotic extracts corresponding to sample at 120 minutes from experiment B (- FCS) or sample at 135 minutes from experiment C (+ FCS) were treated with (+) or without (-) {lambda} phosphatase ({lambda} PPTase) for 30 minutes. Samples were separated by SDS-PAGE and probed with anti-HA antibodies (12CA5). Anti-PSTAIRE antibody to detect Cdc28p was used as loading control. (E) Ratio of phosphorylated (Cdc14p-PO4) versus unphosphorylated (Cdc14p) forms of Cdc14p during yeast (black line) and hyphal growth (gray line). The ratio was calculated using Adobe Photoshop 7.0 software to quantify the intensity of the signals of the blots.

 

Figure 2
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  		Fig. 2. Localization of Cdc14p during yeast growth. (A) Time-lapse fluorescence microscopy of unbudded yeast cells transformed with a plasmid containing the pACT1-CDC14-GFP construct (JJ22). Cells were mounted on glass slides containing 2% agar and differential interference contrast (DIC) (rows 1, 3 and 5) or GFP (rows 2, 4 and 6), and pictures were taken every 15 minutes. Note the presence of the GFP signal at the bud neck after the nucleus has migrated to the daughter cell (arrow). (B) Images of a single cell that was simultaneously stained with Calcofluor White (CW) and GFP are shown, and the overlap of both images (Merge).

 

Figure 3
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  		Fig. 3. Localization of Cdc14p-YFP expressed from its endogenous promoter. (A) Cdc14p is not tethered to the nucleolus during interphase. Exponentially growing cells expressing CDC14-YFP NOP1-CFP (JC252) were prepared for microscopy. Images of Cdc14p-YFP, Nop1p-CFP (blue) plus DAPI (red) and the merge of the three channels plus DIC (Merge+DIC) are shown. Representative photographs with different cell-cycle stages are shown (see text). (B) Cdc14p is located at the SPB. Exponentially growing cells expressing CDC14-YFP/CDC14-YFP (JC189) were grown in YPD at 25°C and scored for Cdc14p-YFP at foci (n>100; representative photographs with faint and strong foci signal are shown). (C) Micrograph of a cell in anaphase from strain JC310 (CDC14-YFP TUB2-CFP). (D) Cdc14p is localized to the bud neck in a fraction (35% of binucleated cells) of large budded cells (JC189). (E) Quantification of the localization of Cdc14p at the SPB at different stages of the cell cycle from the experiment shown in B. Bars, 2 µm.

 

Figure 4
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  		Fig. 4. Localization of Cdc14p-GFP in hyphal cells. Cells expressing Cdc14p-GFP (JJ22) were grown in YPD plus 10% FCS at 37°C to induce hypha formation before images were taken. Images using DIC, GFP staining or Calcofluor White staining (CW) to visualize septa are shown. Note that Cdc14p is located in the nucleus of the apical cell (arrows). Lines: position of septa.

 

Figure 5
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  		Fig. 5. cdc14{Delta} cells have severe defects in cell separation. (A) Morphology of cdc14{Delta}/cdc14{Delta} mutant cells grown in YPD at 30°C (JC13). (B) Electron microscopy of wild-type (BWP17) and cdc14{Delta} mutant (JC13) cells. Wild-type (panels I and II) and cdc14{Delta} mutant (panels III and IV) cells were grown to mid-log phase before preparation for electron microscopy. The neck region of cdc14{Delta} mutants (IV) shows the presence of normal septum, in which the primary (ps) and secondary septa (ss) can be easily visualized. Note that cell separation has already started in the septum of the wild-type cell shown in II, and the cell wall has already been dissolved, but the primary septum is still present (white *). Bars: I and III, 0.5 µm; II and IV, 0.2 µm.

 

Figure 6
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  		Fig. 6. (A) Morphology of ace2{Delta}/ace2{Delta} mutant cells (JC99) grown in YPD at 30°C. (B) cdc14{Delta} cells show reduced expression of cell-wall-degrading enzymes. Polyadenylated RNA isolated from exponentially growing cells of the indicated strains was probed with specific probes for ACT1, CHT3, ENG1, DSE1 and ACE2 genes.

 

Figure 7
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  		Fig. 7. Localization of Ace2p in the daughter nucleus depends on Cdc14p. Exponentially growing cells expressing Ace2p-GFP in wild-type (A, SCAY1) or cdc14{Delta} mutant (B, JC198) cells were visualized using DIC and fluorescence (GFP) microscopy. Bars, 5 µm.

 

Figure 8
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  		Fig. 8. Localization of Cdc14p-YFP in hgc1{Delta} cells. Exponentially growing cells of strain JC194 (hgc1{Delta} CDC14-YFP/CDC14) were induced for hyphal growth in YPD plus 10% FCS for 120 minutes and cells were stained with Calcofluor White (CW). DIC, CW and YFP fluorescence images are shown. Arrows indicate position of the septum.

 

Figure 9
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  		Fig. 9. cdc14{Delta} cells show upregulated levels of B-cyclins. (A) Protein extracts from asynchronous cultures of the indicated strains grown in YPD were prepared and analyzed by western blot using 12CA5 antibodies to detect HA-tagged proteins. Triangle, Clbs-HA; diamond, a cross-reacting band used as loading control. (B) Flow cytometry analysis of asynchronous cultures of strains CDC14 and cdc14{Delta} grown in YPD or SC media at 30°C. (C) G1 cells from strains JC64 (CDC14/CDC14; CLB2HA/CLB2) and JC70 (cdc14{Delta}/cdc14{Delta}; CLB2HA/CLB2) were isolated by elutriation and released into YPD at 30°C. Samples were collected at intervals of 15 minutes after release, and protein extracts were prepared and assayed by western blotting for Clb2p-HA levels. Cdc28p (anti-PSTAIRE antibody) was used as loading control. Samples were also monitored for bud morphology and DAPI staining.

 

Figure 10
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  		Fig. 10. Cells lacking Cdc14p display impaired invasive and hyphal growth. (A) Cells of the indicated genotype were grown on YPD agar plates at 30°C for 7 days. Colonies were photographed before and after the plates had been washed. After washing, transverse sections (TS) were cut from the plates and photographed. Bar, 5 mm. (B) Colony morphology of the indicated strains grown on different hypha-inducing media after 5 days at 30°C. (C) Overnight cultures of strains JC190 (CDC14/CDC14) and JC12 (cdc14{Delta}/cdc14{Delta}) were diluted in pre-warmed YPD plus 5% serum at 37°C. At the indicated times, cells were removed and fixed to monitor response (% evagination) and the length of germ tubes. (D) Analysis of ECE1 and HWP1 expression by northern blot in CDC14/CDC14 and cdc14{Delta}/cdc14{Delta} strains at different times after cells had been transferred to YPD plus 5% FCS at 37°C.

 

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© The Company of Biologists Ltd 2006