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First published online 24 May 2005
doi: 10.1242/jcs.02393

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The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans

Molecular and Cell Biology Laboratory, Institute of Molecular and Cell Biology, 61 Bioplis Drive, Singapore 138673, Republic of Singapore

View larger version (14K): [in a new window]   Fig. 1. Schematic of the domain structure of CaBni1p and CaBnr1p. The functional domains of CaBni1p and CaBnr1p and their boundaries were identified and defined using the SMART program and alignment with ScBni1p and ScBnr1p sequences. The amino acid (aa) identity (%) of the corresponding domains between CaBni1p and ScBni1p and between CaBnr1p and ScBnr1p is given below each domain.

View larger version (120K): [in a new window]   Fig. 2. GFP-CaBni1p localization in C. albicans. (A) During yeast growth. Elutriated G1 yeast cells (strain WYL5) were released into GMM at 30°C and collected at 30-minute intervals for microscopic observation. From left to right, 30, 60, 90, 120 minutes. Images were captured using differential interference phase contrast (phase) and GFP settings. (B) During hyphal growth. The elutriated G1 yeast cells were released into GMM supplemented with 10% serum at 37°C and collected at 30-min intervals. Left to right, 30, 60, 90, 120 minutes. The yeast and hyphal cells in each figure were obtained using the same conditions unless otherwise indicated.

View larger version (142K): [in a new window]   Fig. 3. The Cabni1 mutant exhibited morphological defects during both yeast and hyphal growth. The yeast cells (A) of the wild-type (CaBNI1, strain WYL4) and the mutant (Cabni1, strain WYL3) were grown in YPD at 30°C to exponential phase. The hyphal cells (B) were induced in YPD containing 10% serum at 37°C for 2 and 4 hours. (C) Cabni1 mutant exhibited random budding. The bud scars were visualized by Calcofluor staining. (D) Cells were grown on solid medium containing 0.05 mM ammonium sulphate for 3 days at 37°C. (E) Strains WYL23 (Cabnr1 CaBNI1) and WYL25 (Cabnr1 pMET3CaBNI1) were streaked onto two GMM plates, one containing 2.5 mM each of methionine (Met) and cysteine (Cys) and the other not. The plates were incubated at 30°C for 3 days. (F) WYL25 yeast cells were inoculated into liquid GMM supplemented with or without 2.5 mM methionine and cysteine and grown at 30°C for 5 hours (top). Then serum was added to each culture to a final concentration of 10% and the cells were incubated at 37°C for 3 hours (bottom). Bars, 5 µm.

View larger version (129K): [in a new window]   Fig. 4. Cabni1 cells showed actin patch mislocalization. The exponentially growing yeast (A) and hyphal (B) cells of the CaBNI1 (WYL4) and Cabni1 (WYL3) were stained using rhodamine-phalloidin to visualize actin. The arrows indicate actin cables. (C) Localization of GFP-CaBnr1p (strain WYL19) and CaSpa2p-GFP (strain WYL20) in Cabni1 yeast (top) and hyphal (bottom) cells. Though the signals were faint, GFP-CaBnr1p was unambiguously detected at the bud neck but never seen at the bud tip. The arrows indicate GFP-CaBnr1p localization at the neck. The pattern of CaSpa2p-GFP localization is representative of CaBud6p-GFP (not shown). (D) Actin staining. Strain WYL25 (Cabnr1 pMET3-CaBNI1) yeast cells were grown in GMM at 30°C to log phase (a) or grown in GMM containing 2.5 mM each of methionine and cysteine at 30°C for 5 hours (b); 10% serum was added to the culture shown in b for hyphal induction at 37°C for 3 hours (c). Bars, 5 µm.

View larger version (77K): [in a new window]   Fig. 5. Cabni1 produced multinucleate yeast cells. Approximately 4% of Cabni1 (strain WYL3) yeast cells from an overnight culture were multinucleate. Some of these cells were also often significantly enlarged. Strain WYL4 was used as the wild type (CaBNI1). Nuclei were stained with DAPI.

View larger version (77K): [in a new window]   Fig. 6. The Cabni1 mutant is defective in spindle and cytoplasmic MT localization and orientation, particularly during yeast growth. The MTs were visualized by expressing a CaTub2p-GFP fusion protein in both the wild-type (CaBNI1, strain WYL15) and Cabni1 (strain WYL16) strains. (A) Large-budded yeast cells (right panels phase contrast optics). (B) Hyphal cells (top panels phase contrast optics).

View larger version (91K): [in a new window]   Fig. 7. CaKar9p is delocalized in the Cabni1 mutant. One copy of the CaKAR9 gene in both the wild-type (CaBNI1, strain WYL17) and Cabni1 (strain WYL21) strains was tagged with GFP under the control of the MET3 promoter. (A) Exponential phase yeast cells. (B) Hyphal cells. (C) The cortical localization of CaKar9p depends on actin and microtubules. Random yeast cells and 2-hour hyphal cells of strain WYL17 were treated with either 200 µM LAT-A or 40 µM nocodazole (NOC) for 30 min. Bars, 5 µm.

View larger version (56K): [in a new window]   Fig. 8. Subcellular localization of domain deletion mutants of CaBni1p. (A) A schematic description of the domain deletion mutants of CaBni1p. The boundaries of each domain are the same as described in Fig. 1. (B) The localization patterns of GFP-CaBni1p containing a deletion in FH1 (FH1, strain WYL11) or FH2 (FH2, WYL12) in yeast (Y) and hyphal (H) cells. The GFP fluorescence pattern of FH1 is representative of those in CC (WYL9), SBD (WYL10) and BBD (WYL14) mutants (not shown).

View larger version (72K): [in a new window]   Fig. 9. The Cabni1 mutant exhibited reduced virulence. (A) The survival rates of mice (BALB/c female mice, 7- to 8-weeks old, 10 mice per group) infected with CaBNI1 (WYL4) or Cabni1 (WYL3) strains. The cells were grown in GMM at 30°C to exponential phase (OD600=0.8), harvested and resuspended in phosphate-buffered saline (pH 8) at 5x106 cells per ml. Each animal was injected with 200 µl of the cell suspension through the tail vein. At the end of day 2, two animals from each group were sacrificed for histological examination of the kidneys. (B) The corresponding cortical regions of kidney sections of mice infected with CaBNI1 or Cabni1 are shown. The arrows indicate C. albicans cells.

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