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. 2003 May;14(5):2163-80.
doi: 10.1091/mbc.02-05-0076. Epub 2003 Feb 21.

Depletion of a polo-like kinase in Candida albicans activates cyclase-dependent hyphal-like growth

Catherine Bachewich  1 , David Y Thomas, Malcolm Whiteway
Affiliations

Depletion of a polo-like kinase in Candida albicans activates cyclase-dependent hyphal-like growth

Catherine Bachewich et al. Mol Biol Cell. 2003 May.

Abstract

Morphogenesis in the fungal pathogen Candida albicans is an important virulence-determining factor, as a dimorphic switch between yeast and hyphal growth forms can increase pathogenesis. We identified CaCDC5, a cell cycle regulatory polo-like kinase (PLK) in C. albicans and demonstrate that shutting off its expression induced cell cycle defects and dramatic changes in morphology. Cells lacking CaCdc5p were blocked early in nuclear division with very short spindles and unseparated chromatin. GFP-tagged CaCdc5p localized to unseparated spindle pole bodies, the spindle, and chromatin, consistent with a role in spindle elongation at an earlier point in the cell cycle than that described for the homologue Cdc5p in yeast. Strikingly, the cell cycle defects were accompanied by the formation of hyphal-like filaments under yeast growth conditions. Filament growth was determinate, as the filaments started to die after 24 h. The filaments resembled serum-induced hyphae with respect to morphology, organization of cytoplasmic microtubules, localization of nuclei, and expression of hyphal-specific components. Filament formation required CaCDC35, but not EFG1 or CPH1. Similar defects in spindle elongation and a corresponding induction of filaments occurred when yeast cells were exposed to hydroxyurea. Because CaCdc5p does not appear to act as a direct repressor of hyphal growth, the data suggest that a target of CaCdc5p function is associated with hyphal-like development. Thus, an internal, cell cycle-related cue can activate hyphal regulatory networks in Candida.

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Figures

Figure 1.
Figure 1.
(A) Northern analysis of CaCDC5 expression under control of the Candida PCK1 promotor. Strains CB104 (cacdc5Δ::hisG/cacdc5Δ:: HIS1/PCK1::CaCDC5), CB102 (cacdc5Δ::hisG/CaCDC5 PCK1::CaCDC5) and RM1000 (CaCDC5/CaCDC5) were grown in SD medium for 1, 2, 4, or 7 h, or in SS medium. Total RNA, 20 μg, was analyzed with a CaCDC5-specific probe, followed by an ACT1 probe to compare RNA loading. CaCDC5 is overexpressed in SS medium and repressed in SD medium at 1–7 h of incubation, although some leakiness is present at 7 h. (B) Northern analysis of CaCDC5 expression in cells grown in YPD at 30°C without serum or at 37°C with serum for 30 and 60 min.
Figure 2.
Figure 2.
Repressing CaCDC5 expression results in the formation of filaments under conditions favoring yeast growth. (A) Strains CB104 (cacdc5Δ::hisG/cacdc5Δ:: HIS1/PCK1::CaCDC5) and CB102 (cacdc5Δ::hisG/CaCDC5 PCK1:: CaCDC5) were incubated for 24 h on solid SD or SS medium. Bar, 30 μm. (B) Time course analysis of the formation of filaments in liquid SD medium. Strains CB104 and RM1000 were grown in SS medium (0 h), washed, diluted into SD medium, and fixed in 70% ethanol after 3, 9, and 24 h of incubation at 30°C. Bars, 10 μm. (C) Filament formation upon repressing CaCDC5 with the MET promotor. Strains CB108 (cacdc5Δ::hisG/MET:: CaCDC5) and CB109 (cacdc5Δ::hisG/MET::) were grown in SD medium lacking methionine for promotor induction, then diluted to an OD600 of 0.25 in SD medium containing methionine and cysteine to repress the promotor for 24 h. Bar, 10 μm. (D) Hyphal growth in strain RM1000 incubated in the presence of 10% serum at 37°C for 4 h. Bar, 10 μm.
Figure 3.
Figure 3.
(A) Nuclear division and septation are impaired upon repressing CaCDC5. Strains CB104 (cacdc5Δ::hisG/cacdc5Δ::HIS1/PCK1:: CaCDC5) and RM1000 (CaCDC5/CaCDC5) were grown in SS medium, washed, transferred to SD medium, and then fixed and stained with DAPI and calcofluor at various time points. Septa (small arrowheads) and fragmented DNA (arrow) were observed after 5 h. Note the absence of nuclei in the mother yeast cell after 5 h. (B) Nuclei and septa in serum-induced hyphae grown at 37°C from strain RM1000 after 1.5 and 3 h. Bar, 10 μm.
Figure 4.
Figure 4.
Spindle elongation is blocked in CaCDC5-repressed filaments. (A) Tub1p-GFP was visualized in strains CB110 (CaCDC5/CaCDC5, TUB1-GFP) and CB112 (cacdc5::hisGΔ/cacdc5Δ:: HIS1/PCK1::CaCDC5, TUB1-GFP) grown in SD medium for 3, 5, and 24 h. Bar, 10 μm. (B) Paired images of immunolocalized α-tubulin and DAPI-stained DNA in strains CB104 (cacdc5Δ::hisG/cacdc5Δ:: HIS1/PCK1::CaCDC5) and RM1000 (CaCDC5/CaCDC5) grown in SD medium for 3 h. Note the cytoplasmic microtubules in the filaments. The short spindle in strain CB104 is indicated by an arrow. Bar, 5 μm. (C) Microtubule organization in serum-induced hyphae. Cells of strain CB110 (CaCDC5/CaCDC5, TUB1-GFP) were incubated in SD medium containing 10% fetal calf serum at 37°C for 2 h to induce hyphal formation and visualize microtubules. Bar, 10 μm.
Figure 5.
Figure 5.
CaCdc5p localizes to the spindle pole bodies, spindle, DNA, and bud neck. Strain CB115 (A–C) containing PCK1:: CaCDC5-GFP was grown in S medium containing 2% casaminoacids for overexpression. Strain CB116 (CaCDC5-GFP-URA3) (D–I) demonstrates similar localization patterns. Note the staining of a spindle-like structure overlaying chromatin (arrow in E) and spindle pole bodies and chromatin in small budded cells (arrow heads in E and G). Bars, 10 μm
Figure 6.
Figure 6.
Hydroxyurea induces filament formation under yeast growth conditions and impairs spindle elongation. (A) Strain SC5314 was grown in YPD medium overnight and then diluted to an OD600 of 0.4 in fresh YPD containing 200 mM HU. Cells were fixed at 6 h and stained with DAPI and calcofluor. (B) Strain CB110 (CaCDC5/CaCDC5, TUB1-GFP) was incubated in 200 mM HU for 3, 7, and 24 h. Note the similarity to filaments of CaCDC5-repressed cells (Figures 2B, 3A, and 4A). Bar, 10 μm. (C) FACS analysis demonstrating S phase and G2 phase blocks in HU-treated and CaCDC5-repressed cells, respectively. Cells of strains CB104 (cacdc5::hisGΔ/cacdc5Δ::HIS1/PCK1::CaCDC5) and RM1000 (CaCDC5/CaCDC5) were transferred from SS to SD medium, collected at the indicated time points, and processed for FACS analysis. Strain SC5314 was grown in YPD containing 200 mM HU and processed for FACS analysis. Note the fragmentation of DNA at later time points of 9 and 6 h.
Figure 7.
Figure 7.
Filaments produced by repression of CaCDC5 or exposure to HU are similar to hyphae in expression of a serum-induced antigen and RNA. (A) Serum-induced hyphae from strain SC5314, filaments from strain CB104 (cacdc5Δ::hisG/cacdc5Δ:: HIS1/PCK1::CaCDC5) depleted of CaCdc5p for 7 h, and filaments from strain SC5314 incubated in HU for 24 h were fixed and processed for immunolocalization of MAb16B1-F10. Bar, 10 μm. (B) Northern analysis demonstrating the filament-induced expression of RNA normally induced by the hyphal-generating conditions of serum and high temperature. Total RNA, 20 μg, from strain SC5314 incubated in YPD with 200 mM HU for 6 h, SC5314 without HU, CB104 (cacdc5::hisGΔ/cacdc5Δ:: HIS1/PCK1::CaCDC5) grown in SD medium for 24, 7, and 4 h, and CB102 (cacdc5Δ::hisG/CaCDC5 PCK1:: CaCDC5) grown in SD medium for 4 h was hybridized with probes specific for HWP1, DDR48, and ACT1 as a loading control.
Figure 8.
Figure 8.
Filamentous growth in cells depleted of CaCdc5p or exposed to HU require CaCdc35p, but not Efg1p and Cph1p. Cells from strains CB108 (cacdc5Δ::hisG/MET:: CaCDC5-URA3), CB305 (cacdc5Δ::hisG/MET:: CaCDC5-URA3 cph1Δ::hisG/cph1Δ::hisG efg1Δ::hisG/efg1Δ::hisG) and CB303 (cacdc5Δ::hisG/MET:: CaCDC5-URA3 cdc35Δ::hisG/cdc35Δ::hisG) were incubated in SD medium–methionine for overexpression of MET:: CaCDC5, or SD medium + methionine and cysteine to repress MET:: CaCDC5 expression for 7 h. Cells from strains SC5314 (+/+), HLC54 (cph1Δ::hisG/cph1Δ::hisG efg1Δ::hisG/efg1Δ::hisG -URA3-hisG) and CR216 (cdc35Δ::hisG/cdc35Δ::hisG-URA3-hisG) were grown in SD medium, diluted to an OD600 of 0.4 in fresh medium containing 200 mM HU, and incubated for 7 h. Cells were fixed in 70% EtOH before collection of images. Bar, 10 μm.
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