Figure 1.
Cell size correlates with DNA content.
(A) Flow cytometry data plotted as cell size (FSC) versus DNA content (FL1-A) for cultures grown in the absence (top row) or presence (bottom row) of FLC for times indicated. (B) Bar graphs represent percent of cells that fell into colored regions defined in scatterplots above as determined using Gaussian fitting (see Materials and Methods).
Figure 2.
Abnormally large cells are viable.
(A) Violin plots (area indicates frequency along the y-axis) overlaid with box plots (gray) showing the distribution of cell sizes (see Materials and Methods). “No drug” control cultures (blue) show no significant changes in cell size distribution over time. FLC-exposed cultures (red) show a significant cell size increase at 8 and 12 h of FLC exposure (two-tailed t test, p value <0.05). (B) Violin plots, as above, showing size distribution of metabolically active (cyan, alive) and inactive (magenta, dead) cells as determined by FUN1 staining interpreted as shown in the micrograph insets. (C) Abnormally large cells with multiple nucleoli (detected with Nop1-GFP, green) continued to grow, produced new buds, and underwent unusual nucleolar segregation patterns. Inset in first panel is an unbudded cell from a “no drug” control shown on the same size scale. Scale bar, 5 µm.
Figure 3.
Bud emergence is delayed after 4 h of FLC exposure.
(A) Individual frames from time-lapse images of Nop1-RFP/Tub1-GFP–expressing cells in the absence (top two rows) and presence (4 h, bottom two rows) of FLC. BE, bud emergence; SA, spindle assembly; AO, anaphase onset; SEP, sister separation; SEG, sister segregation across bud neck; SD, spindle disassembly. Illustrations in rows 3 and 4 show relative timing of events. Numbers in fluorescent images denote time (min) of FLC exposure. Scale bars, 5 µm. (B) Cartoon illustrating relative timing of cell cycle events in the absence (left) and presence (right) of FLC. Lower left panel, average time between bud emergence (BE, purple) and anaphase onset (AO, gray) events. Bar graph, average time between BE and AO (red), AO and SEG (green), SEG and SD (blue), and SD and BE (purple). Error bars are 1 standard deviation from mean based on n = 25 cells observed on 2 d. Asterisks denote statistically significant differences (t test, p value <0.05). (C) Proportion of cells with normal pre-START and pre-anaphase phenotypes (blue) and aberrant START (unbudded cells with two SPBs, yellow) phenotypes. Cells not represented here were in mitosis. “No drug” control cultures showed no significant deviation from t = 0 numbers (unpublished data).
Figure 4.
Trimeras become prevalent after 8 h of FLC exposure and remain viable.
(A) Time-lapse microscopy of control (top row) and FLC-exposed cells expressing Nop1-GFP. Numbers indicate time (min) of FLC exposure. (B) Relative proportion of cells with indicated morphologies (indicated in color-outlined images) at different times (t, time) of FLC exposure. “No drug” control cultures showed no significant deviation from t = 0 numbers (unpublished data). Colors in pie charts correspond to color of image outline. Scale bars, 5 µm. At least 300 cells from each of two different strains were analyzed for each time point. (C) Colony formation assay following cell micromanipulation. Following 12 h pre-exposure to 10 µg/ml FLC, single budded cells (red) and trimeras (purple) were transferred to plates with indicated FLC concentration. The number of cells analyzed for single budded cells on 0, 2, and 10 µg/ml FLC plates were 52, 53, and 48, respectively. The number of trimeras analyzed on 0, 2, and 10 µg/ml FLC plates were 78, 65, and 48, respectively. Error bars indicate standard error, and statistical significance was determined using a Fisher's exact test.
Figure 5.
Nuclei within trimeras undergo mitotic collapse to form tetraploids.
(A) Time-lapse images of trimera formation and mitotic dynamics. Numbers in fluorescent images denote time in minutes. Also indicated is the percent of trimeras (n = 19) that formed four individual nucleoli (top sequence) and that formed three nucleoli (lower sequence) Scale bars, 5 µm. (B) Violin plots of nuclear DNA content (Hhf1-GFP fluorescence) and SPB number (Tub4-mCherry foci). Colors correspond to measurements of individual nuclei within cells as illustrated below plots: trimeras with two nuclei (purple, n = 56 nuclei), trimeras with four nuclei (pink, n = 24), and trimeras with three nucleoli, one large (green, n = 11) and two small (cyan, n = 22). Gray regions define average fluorescence intensity of 2N and 4N nuclei ±1 standard deviation based on log-phase “no drug” control cells.
Figure 6.
Unequal segregation occurs in nuclei with more than one spindle.
(A) Time-lapse microscopy of nuclear segregation patterns in tetraploid/diakaryotic cells. Type I segregation pattern (top two rows, 54% of events), both spindles elongated across the bud neck. Type II (bottom two rows, 46% of events), only one spindle elongated across the bud neck. Numbers denote time (min) of FLC exposure. Scale bars, 5 µm. Total of 13 cells analyzed. (B) Histone H4 (Hhf1)-GFP fluorescence intensity scatter plots. Sister nuclei are plotted relative to each other. Postanaphase cells containing a total of two SPBs (cyan) clustered around 1∶1, indicative of equal segregation (gray region, contains 95% of points from “no drug” cells). Postanaphase cells containing more than two SPBs (magenta) diverged significantly from 1∶1 at 12 h (two-tailed t test, p value <0.05).
Figure 7.
Trimeras give rise to aneuploid progeny.
Representative diploid, tetraploid, diploid/tetraploid mixed, and near diploid flow cytometry profiles showing DNA content of populations of cells derived from trimeras (n = 40) and their progeny (n = 134). Black arrows denote diploid peak pair; white arrows denote near-diploid peak pair. Profile outlines correspond to bars in bar graph showing the percent of trimeras (left) and trimera progeny (right) that exhibited each profile type.
Figure 8.
Percent of ConA-Texas Red/Eno1-GFP cells showing unbudded (blue), budded (red), trimera-like (purple), and hyphal (green) phenotypes within untreated (left, n = 195) and FLC-treated (right, n = 309) mouse host 48 h after injection. Error bars are 1 standard deviation. Scale bar, 5 µm.
Figure 9.
Trimera formation is not a general stress response.
Cells exposed to triazoles ketoconazole, voriconazole, and itraconazole formed trimeras at frequencies similar to FLC. Cells that were exposed to caspofungin, an echinocandidn, also produced many trimera-like and multimera-like cells (upper and lower panels). Exposure to toxin 5-FOA as well as heat shock did not result in a significant number of trimeras. No trimeras were detectable following exposure to 2-DOG (unpublished data). Percentages in upper right corner of DAPI image denote frequency of trimera formation in 300–400 cells.
Figure 10.
A model for aneuploid formation in C. albicans cells exposed to FLC.
Proposed model for aneuploid formation in C. albicans cells exposed to FLC. Nuclear membrane, thin black line; nucleolus, red; spindle, green.