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First published online 15 May 2007
doi: 10.1242/jcs.002931

Journal of Cell Science 120, 1898-1907 (2007)
Published by The Company of Biologists 2007
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Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation

Chang-Run Li, Raymond Teck-Ho Lee, Yan-Ming Wang, Xin-De Zheng and Yue Wang*

Candida albicans Molecular and Cell Biology Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, 138673, Singapore


Figure 1
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  		Fig. 1. CaSEC3 is a true homologue of ScSEC3. (A) CaSEC3 can partially rescue the temperature sensitivity of Scsec3{Delta} mutants. Cells of S. cerevisiae NY2449 (SEC3), NY2448 (Scsec3{Delta}), and WYL68 (derived by transforming NY2448 with CaSEC3 under control of the ScSEC3 promoter on a CEN plasmid) were first grown overnight in synthetic medium, diluted, and streaked onto YPD plates for incubation at 22, 30 and 37°C for 2 days. (B) Casec3{Delta} cells accumulated secretory vesicles in the cytoplasm. Cells of SC5314 (SEC3) and WYL28 (sec3{Delta}) were first grown at 30°C overnight in GMM and then diluted into fresh YPD medium for further growth at 37°C for 3 hours before electron microscopy. Bar, 0.5 µm. (C) Sec3p-GFP localized to sites of cell growth. Synchronized G1 cells of WYL30 that expresses Sec3p-GFP were grown in GMM at 30°C to exponential phase (left panel). Hyphal cells (right panel) were induced in GMM supplemented with 10% serum at 37°C, and samples were collected at the indicated times for microscopy. Bar, 5 µm.

 

Figure 2
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  		Fig. 2. Growth defects of Casec3{Delta} cells. (A) sec3{Delta} cells exhibited temperature and rich medium sensitivity. SC5314 (SEC3) and WYL28 (sec3{Delta}) cells were grown at 30°C overnight in GMM, diluted and inoculated onto GMM or YPD agar plates for incubation at 30, 37 and 42°C for 2 days. (B) The same overnight cultures were diluted into liquid GMM or YPD medium and grown at 30°C with vigorous shaking. Growth was monitored by taking OD600 at intervals. Doubling times were calculated according to the average OD values measured during the exponential phase of growth from three independent experiments. Data are means ± s.d. of three experiments. (C) Morphological defects of sec3{Delta} cells. SC5314 (SEC3) and WYL28 (sec3{Delta}) were grown in YPD at 30 and 37°C (top) or grown in YPD containing 10% serum at 37°C for hyphal induction (bottom). Cell images were captured using differential-interference-phase contrast (DIC). Arrowheads indicate the junctions between the germ tube and the swollen daughter cell. Bars, 5 µm.

 

Figure 3
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  		Fig. 3. Septin rings mark the site where sec3{Delta} cells start to lose apical tip growth under hyphal-induction conditions. (A) Cdc3p-GFP localization in wild-type (WYL38) yeast and hyphal cells. (B) Cdc3p-GFP localization in sec3{Delta} cells (WYL44) grown under yeast growth (left panel) or hyphal-induction conditions (center and right panels). An aliquot of cells at 3 hours of induction was also stained with Calcofluor White to visualize the cell wall (right panel). Bars, 5 µm.

 

Figure 4
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  		Fig. 4. Cellular localizations of Sec15p-GFP and GFP-Sec4p in sec3{Delta} cells. (A) Sec15p-GFP localization in WYL41 (SEC3) and WYL42 (sec3{Delta}) cells grown as yeast (upper panels) or hyphae (lower panels). (B) GFP-Sec4p localization in WYL39 (SEC3) and WYL40 (sec3{Delta}) cells grown as yeast (upper panels) or hyphae (lower panels). Bars, 5 µm.

 

Figure 5
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  		Fig. 5. Effect of SEC3 deletion on Cdc42p cellular localization. GFP-Cdc42p localization was examined in WYL35 (SEC3) and WYL36 (sec3{Delta}) cells under yeast (top) and hyphal (bottom) growth conditions. Bars, 5 µm.

 

Figure 6
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  		Fig. 6. Effects of SEC3 deletion on actin (A) and Spa2p (B) localization. (A) Cells of SC5314 (SEC3) and WYL28 (sec3{Delta}) were grown under yeast (top) and hyphal (bottom) growth conditions and stained with rhodamine-phalloidin. (B) Cells of WYZ8 (SEC3 SPA2-GFP) and WYL43 (sec3{Delta}, SPA2-GFP) were grown under yeast (top) and hyphal (bottom) growth conditions. Bars, 5 µm.

 

Figure 7
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  		Fig. 7. Genetic interactions between septin genes and SEC3. (A) Sec3p-GFP localizations in WYL30 (wild type), WYL50 (cdc10{Delta}) and WYL55 (cdc11{Delta}) cells under yeast growth conditions. (B) Deletion of CDC10 or CDC11 restored hyphal growth in sec3{Delta} cells. Cells of WYL55 (sec3{Delta} cdc10{Delta}) and WYL58 (sec3{Delta} cdc11{Delta}) were induced for hyphal growth for 4 hours, and stained with DAPI and Calcofluor White to visualize nuclei and cell walls. (C) Cdc3p-GFP exhibited normal localization in sec3{Delta} cdc10{Delta} and sec3{Delta} cdc11{Delta} hyphal cells. Cells of WYL56 and WYL59 were induced for 3 hours. (D) The sec3{Delta} cdc10{Delta} (WYL55) and sec3{Delta} cdc11{Delta} (WYL58) mutants remain temperature sensitive. Cells of SC5314 (1), sec3{Delta} (2), sec3{Delta} cdc10{Delta} (3), and sec3{Delta} cdc11{Delta} (4) were inoculated onto GMM plates and incubated at 30°C or 42°C for 2 days. (E) Genetic interactions of SEC3 with GIN4 and CLA4. Cells of WYL61 (cla4{Delta}), WYL63 (sec3{Delta} cla4{Delta}), WYL65 (gin4{Delta}) and WYL67 (sec3{Delta} gin4{Delta}) were grown in GMM containing 1 mM each of methionine and cysteine at 30°C for 3 hours (yeast growth condition); then an aliquot was inoculated into the same medium supplemented with 20% serum and incubated at 37°C for 2 hours of hyphal induction. Arrows indicate dead cells. Bars, 5 µm.

 

Figure 8
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  		Fig. 8. Cdc3p-GFP co-immunoprecipitated with Myc-Sec3p and Myc-Sec5p. Cell lysates were prepared from four strains: WYL47 (CDC3-GFP Myc-SEC3), WYL48 (CDC3-GFP Myc-SEC5), WYL49 (sec3{Delta} CDC3-GFP Myc-SEC5) and WYL38 (SEC3 CDC3-GFP). Myc-tagged proteins were precipitated by using anti-Myc beads and separated by SDS-PAGE. Cdc3p-GFP in the anti-Myc precipitates was detected by anti-GFP western blotting. WYL38 was included as a negative control and processed in the same way as other strains. 2 ml cell lysate was prepared from a 200-ml culture as described in Materials and Methods and used for co-immunoprecipitation; 30 µl of the 2-ml lysate was loaded in lanes labeled as cell lysate, and all the precipitate from the 2-ml lysate of each strain was loaded onto the gel.

 

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© The Company of Biologists Ltd 2007