Article Figures & Data
- FIG 1
Flow cytometry analysis of cell cycle progression during capsule enlargement. (A) Samples were taken for flow cytometry analysis at the time indicated after PI staining (see Materials and Methods). Micrographs of cells suspended in India ink were taken in parallel. Sab, Sabouraud medium. (B) Capsule sizes at the different time points.
- FIG 2
Capsule enlargement in the presence of cell cycle inhibitors. Bars show the means ± standard deviations (SD) of the capsule sizes measured from cultures incubated with different concentrations of sirolimus (rapamycin) (A) or benomyl (C). *, P < 0.05. (B and D) Growth curves of C. neoformans in capsule-inducing medium with the different concentrations of sirolimus (B) and benomyl (D). Cultures containing DMSO were used as controls.
- FIG 3
Capsule growth visualization by time-lapse microscopy. Capsule enlargement was observed based on the capsular “quellung” reaction produced by the 13F1 MAb. (A) Sequence of frames showing capsule and cell body enlargement. (B) The rate of capsule and cell body growth was determined by linear regression analysis of lengths as a function of time. The capsule enlargement speed was 8.8 nm/min, and the cell body enlargement speed was 3.6 nm/min. A plateau in capsule and cell body size is observed following yeast replication (boxed in gray).
- FIG 4
Morphogenesis of wt, cln1, and cln1::CLN1 strains from C. neoformans. (A and B) Distribution of total cell size, cell body size, and capsule diameter of C. neoformans cells grown in Sabouraud medium (A) and in 10% Sabouraud medium buffered at pH 7.3 with 50 mM MOPS (B).
- FIG 5
The cln1 mutant shows increased production of extracellular vesicles. (A) Comparative analysis of vesicle production based on an indirect sterol-based vesicle quantification of fractions obtained from the wt, mutant, and reconstituted C. neoformans strains, using a fluorometric assay kit. Error bars represent standard deviations of 3 independent experiments. (B) Comparative analysis of the sterol content in vesicle preparations using HPTLC. An arrow indicates the migration of an ergosterol standard (Std). The result is representative of 2 independent analyses showing similar results. (C) Contrast image of the area highlighted with the arrow in panel B to emphasize the differences in band intensity among strains.
- FIG 6
Relative abundance of proteins accumulated in the cln1 mutant under capsule-inducing conditions. The upper dendrogram shows the relationship among individual gels, while the left dendrogram shows the relationship among proteins. Hierarchical clustering analysis (HCA) was performed to obtain the values of abundance of every spot, which were then represented in a heat map. Different red spots show a positive relative abundance (cln1 mutant/H99 ratio), while different green spots show a negative relative abundance.
- FIG 7
In vivo model of the effect of cln1. (A) Survival curves of G. mellonella infected with 106 cells of C. neoformans strains at 30°C (left) and 37° C (right) (B) Capsule sizes of yeast cells recovered from G. mellonella after 3 days of infection at 30 and 37°C. (C) Phagocytosis was performed at a 2:1 ratio of yeast cells to macrophages (see Materials and Methods). Bars show the mean ± SD phagocytosis of C. neoformans cells after 2 h at 37°C. *, P <0.05.
- TABLE 1
List of proteins identified by mass spectrophotometrya
Spot Protein identification cln1 mutant/H99 ratio (fold change) ORF 1040 Transketolase 3.74 CNAG_07445 617 Heat shock proteín 90 3.59 CNAG_06150 2002 Wos2 (p21) 2.76 CNAG_07558 861 Malate synthase 2.54 2228 Phosphopyruvate hydrogenase 2.37 CNAG_03072 1427 Aldoketoreductase 1.94 CNAG_01257 974 Chaperone 1.92 CNAG_01750 1222 Ornithine carbamyltransferase 1.8 CNAG_02813 1062 Aminotransferase 1.79 CNAG_02853 860 Malate synthase 1.7 CNAG_05653 1149 Adenylsuccinate synthase 1.68 CNAG_02858 2227 Phosphogluconate dehydrogenase 1.67 CNAG_07561 598 Heat shock protein 1.59 CNAG_05199 915 UTP-glucose-1-phosphate uridyltransferase 1.51 CNAG_02748 1133 Methione adenosyltransferase −1.52 CNAG_00418 584 Heat shock protein 70 −1.55 CNAG_01727 532 Heat shock protein −1.69 CNAG_06443 1473 Budding-related protein −2.08 CNAG_04194 531 Meiosis-related protein −2.71 CNAG_00995 ↵a The first and second columns indicate the proteins. The third column shows the fold change in accumulation of the protein between wt and cln1 strains. The last column is the corresponding ORF for each protein when a BLAST protein-protein comparison was performed in the C. neoformans database of the Broad Institute
Supplemental Material
Movie S1
Real-time imaging of capsule growth. The movie shows time-lapse microscopy images at 5 frames per s of C. neoformans undergoing capsule enlargement followed by yeast budding. Image acquisition was performed at 5-min intervals over a total period of 395 min using a 63× DIC objective. The scale bar represents 5 µm. Download Movie S1, AVI file, 0.7 MB.
Copyright © 2014 García-Rodas et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Movie S2
The movie shows a representative cell of the H99 strain in Sabouraud liquid medium at 30°C in which the replication rate and G1 lapse were calculated. In this case, pictures were taken every 2 min. The interval between when the first bud emerges until that “daughter cell” buds again corresponds to the G1 lapse. In this case, the duration of the G1 phase is 61 min (28 pictures). Download Movie S2, AVI file, 2.5 MB.
Copyright © 2014 García-Rodas et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Movie S3
The movie shows a representative cell of the cln1 mutant strain in Sabouraud liquid medium at 30°C in which the replication rate and G1 lapse were calculated. In this case, the pictures were taken every 3 min. The interval between when the first bud emerges until that “daughter cell” buds again corresponds to the G1 lapse. In this case, the duration of the G1 phase is 98 min (31 pictures). Download Movie S3, AVI file, 4.3 MB.
Copyright © 2014 García-Rodas et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
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