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Cell wall protection by the Candida albicans class I chitin synthases.

Preechasuth K, Anderson JC, Peck SC, Brown AJ, Gow NA, Lenardon MD - Fungal Genet. Biol. (2015)

Bottom Line: Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed.Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation.Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses.

View Article: PubMed Central - PubMed

Affiliation: Aberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. Electronic address: kanya.p@cmu.ac.th.

No MeSH data available.


Related in: MedlinePlus

Localization of Chs2-YFP in yeast and hyphal cells grown in the presence of caspofungin and CaCl2 and CFW. A–F. Yeast cells of the CHS2-YFP/chs2Δ0 strain were grown in YPD (A and B) YPD with a sub-MIC concentration of caspofungin (C and D) or YPD with CaCl2/CFW (E and F) at 30 °C for 5 h before imaging. G–I. Hyphae of the CHS2-YFP/chs2Δ0 strain were grown on 20% FCS agar pads (G) with a sub-MIC concentration of caspofungin (H) or CaCl2/CFW (I) at 37 °C for 4 h before imaging. White arrow heads point to septa, and yellow arrow heads point to the tips of buds and hyphae. Scale bars are 5 μm. J. The average fluorescence intensity of Chs2-YFP spots at septa of yeast and hyphae treated with a sub-MIC concentration of caspofungin or CaCl2/CFW quantified using ImageJ. Error bars are SEM. n = 40–60 spots for each measurement. ∗ p < 0.05 by ANOVA. K. Yeast cells of the chs2S222A-YFP strain were grown in YPD (Untreated) or YPD with CaCl2 and CFW (+CaCl2/CFW) at 30 °C for 5 h before imaging. White arrow heads point to Chs2S222A-YFP at septa, and yellow arrow heads point to the tips of buds. Scale bars are 5 μm. L. The average fluorescence intensity of Chs2S222A-YFP spots at septa of yeast cells treated with a sub-MIC concentration of caspofungin and CaCl2/CFW quantified using ImageJ. Error bars are SEM. n = 20–40 spots for each measurement. ∗ P < 0.05 by t-test.
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f0020: Localization of Chs2-YFP in yeast and hyphal cells grown in the presence of caspofungin and CaCl2 and CFW. A–F. Yeast cells of the CHS2-YFP/chs2Δ0 strain were grown in YPD (A and B) YPD with a sub-MIC concentration of caspofungin (C and D) or YPD with CaCl2/CFW (E and F) at 30 °C for 5 h before imaging. G–I. Hyphae of the CHS2-YFP/chs2Δ0 strain were grown on 20% FCS agar pads (G) with a sub-MIC concentration of caspofungin (H) or CaCl2/CFW (I) at 37 °C for 4 h before imaging. White arrow heads point to septa, and yellow arrow heads point to the tips of buds and hyphae. Scale bars are 5 μm. J. The average fluorescence intensity of Chs2-YFP spots at septa of yeast and hyphae treated with a sub-MIC concentration of caspofungin or CaCl2/CFW quantified using ImageJ. Error bars are SEM. n = 40–60 spots for each measurement. ∗ p < 0.05 by ANOVA. K. Yeast cells of the chs2S222A-YFP strain were grown in YPD (Untreated) or YPD with CaCl2 and CFW (+CaCl2/CFW) at 30 °C for 5 h before imaging. White arrow heads point to Chs2S222A-YFP at septa, and yellow arrow heads point to the tips of buds. Scale bars are 5 μm. L. The average fluorescence intensity of Chs2S222A-YFP spots at septa of yeast cells treated with a sub-MIC concentration of caspofungin and CaCl2/CFW quantified using ImageJ. Error bars are SEM. n = 20–40 spots for each measurement. ∗ P < 0.05 by t-test.

Mentions: It has been shown that phosphorylation of Chs3 on S139 is important for the localization of Chs3-YFP to sites of polarized growth (Lenardon et al., 2010a). We therefore tested whether phosphorylation was involved in regulating the localization of Chs2. To do this, strains expressing C-terminally YFP-tagged phospho-mutant versions of Chs2 (chs2S222A-YFP and chs2S222E-YFP; Table 1) were constructed and the localization of Chs2S222A-YFP and Chs2S222E-YFP was examined by fluorescence microscopy. In single snap-shot images, Chs2S222A-YFP was observed at septa of yeast cells as a bar and as a spot (Fig. 3A–D) and at the septa of hyphal cells (Fig. 3E–H) in the same configurations as Chs2-YFP (Fig. 2A–G). However, Chs2S222A-YFP was not observed at hyphal tips (Fig. 3G and H). Chs2S222E-YFP was also observed in the same configuration as Chs2-YFP at the septa of yeast (Fig. 3I–L) and hyphal cells (Fig. 4M and N), as well as at hyphal tips (Fig. 3O and P). These results indicate that either phosphorylation on S222 is required for Chs2 to be localized to hyphal tips, or that the amount of Chs2S222A-YFP at hyphal tips was reduced to a non-detectable level in this mutant.


Cell wall protection by the Candida albicans class I chitin synthases.

Preechasuth K, Anderson JC, Peck SC, Brown AJ, Gow NA, Lenardon MD - Fungal Genet. Biol. (2015)

Localization of Chs2-YFP in yeast and hyphal cells grown in the presence of caspofungin and CaCl2 and CFW. A–F. Yeast cells of the CHS2-YFP/chs2Δ0 strain were grown in YPD (A and B) YPD with a sub-MIC concentration of caspofungin (C and D) or YPD with CaCl2/CFW (E and F) at 30 °C for 5 h before imaging. G–I. Hyphae of the CHS2-YFP/chs2Δ0 strain were grown on 20% FCS agar pads (G) with a sub-MIC concentration of caspofungin (H) or CaCl2/CFW (I) at 37 °C for 4 h before imaging. White arrow heads point to septa, and yellow arrow heads point to the tips of buds and hyphae. Scale bars are 5 μm. J. The average fluorescence intensity of Chs2-YFP spots at septa of yeast and hyphae treated with a sub-MIC concentration of caspofungin or CaCl2/CFW quantified using ImageJ. Error bars are SEM. n = 40–60 spots for each measurement. ∗ p < 0.05 by ANOVA. K. Yeast cells of the chs2S222A-YFP strain were grown in YPD (Untreated) or YPD with CaCl2 and CFW (+CaCl2/CFW) at 30 °C for 5 h before imaging. White arrow heads point to Chs2S222A-YFP at septa, and yellow arrow heads point to the tips of buds. Scale bars are 5 μm. L. The average fluorescence intensity of Chs2S222A-YFP spots at septa of yeast cells treated with a sub-MIC concentration of caspofungin and CaCl2/CFW quantified using ImageJ. Error bars are SEM. n = 20–40 spots for each measurement. ∗ P < 0.05 by t-test.
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f0020: Localization of Chs2-YFP in yeast and hyphal cells grown in the presence of caspofungin and CaCl2 and CFW. A–F. Yeast cells of the CHS2-YFP/chs2Δ0 strain were grown in YPD (A and B) YPD with a sub-MIC concentration of caspofungin (C and D) or YPD with CaCl2/CFW (E and F) at 30 °C for 5 h before imaging. G–I. Hyphae of the CHS2-YFP/chs2Δ0 strain were grown on 20% FCS agar pads (G) with a sub-MIC concentration of caspofungin (H) or CaCl2/CFW (I) at 37 °C for 4 h before imaging. White arrow heads point to septa, and yellow arrow heads point to the tips of buds and hyphae. Scale bars are 5 μm. J. The average fluorescence intensity of Chs2-YFP spots at septa of yeast and hyphae treated with a sub-MIC concentration of caspofungin or CaCl2/CFW quantified using ImageJ. Error bars are SEM. n = 40–60 spots for each measurement. ∗ p < 0.05 by ANOVA. K. Yeast cells of the chs2S222A-YFP strain were grown in YPD (Untreated) or YPD with CaCl2 and CFW (+CaCl2/CFW) at 30 °C for 5 h before imaging. White arrow heads point to Chs2S222A-YFP at septa, and yellow arrow heads point to the tips of buds. Scale bars are 5 μm. L. The average fluorescence intensity of Chs2S222A-YFP spots at septa of yeast cells treated with a sub-MIC concentration of caspofungin and CaCl2/CFW quantified using ImageJ. Error bars are SEM. n = 20–40 spots for each measurement. ∗ P < 0.05 by t-test.
Mentions: It has been shown that phosphorylation of Chs3 on S139 is important for the localization of Chs3-YFP to sites of polarized growth (Lenardon et al., 2010a). We therefore tested whether phosphorylation was involved in regulating the localization of Chs2. To do this, strains expressing C-terminally YFP-tagged phospho-mutant versions of Chs2 (chs2S222A-YFP and chs2S222E-YFP; Table 1) were constructed and the localization of Chs2S222A-YFP and Chs2S222E-YFP was examined by fluorescence microscopy. In single snap-shot images, Chs2S222A-YFP was observed at septa of yeast cells as a bar and as a spot (Fig. 3A–D) and at the septa of hyphal cells (Fig. 3E–H) in the same configurations as Chs2-YFP (Fig. 2A–G). However, Chs2S222A-YFP was not observed at hyphal tips (Fig. 3G and H). Chs2S222E-YFP was also observed in the same configuration as Chs2-YFP at the septa of yeast (Fig. 3I–L) and hyphal cells (Fig. 4M and N), as well as at hyphal tips (Fig. 3O and P). These results indicate that either phosphorylation on S222 is required for Chs2 to be localized to hyphal tips, or that the amount of Chs2S222A-YFP at hyphal tips was reduced to a non-detectable level in this mutant.

Bottom Line: Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed.Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation.Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses.

View Article: PubMed Central - PubMed

Affiliation: Aberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. Electronic address: kanya.p@cmu.ac.th.

No MeSH data available.


Related in: MedlinePlus