The effect of nocodazole treatment on monokaryotic fruiting.

<p>JEC-21 cells were cultured in YPD broth plus different concentrations of nocodazole at 30°C for 24 hrs, then dropped onto filament agar and incubated at 25°C for 5 days. (A) Qualitative agar assay for monokaryotic fruiting. Nocodazole concentrations consisted of a.) 0 µM, b.) 0.075 µM, c.) 0.15 µM, d.) 0.30 µM, e.) 0.60 µM, f.) 1.20 µM. Hyphal production begins at nocodazole concentrations ≥0.15 µM. Bar = 0.5 mm. (B) Quantitative agar assay for monokaryotic fruiting. Concentrations of nocodazole are indicated. The percent of colonies undergoing monokaryotic fruiting was calculated from the total colonies that grew out on the agar plates. Plates were assayed in triplicate.</p

The effect of seed culture temperature on morphology.

<p>(A) Wet mount of cells scraped from filament agar after 24 h growth showing hyphae growing from an enlarged cell. (B) Wet mount of cells from YPD agar after 24 h growth at 30°C showing a homogenous population of normal-sized (2–5 µm) yeast cells. (C) Wet mount of cells scraped from YPD agar after 24 h growth at 37°C showing a mix of enlarged (filled white arrowhead) and normal-sized cells (open arrowhead). All images were obtained with DIC microscopy at 400× magnification. Bar = 10 µm. (D) Results of micromanipulation of enlarged and normal-sized cells from a 24 h, 37°C YPD agar culture scored for morphology after germinating on filament agar.</p

Monokaryotic fruiting on filament agar.

<p>(A) Individual colonies visualized at 2× on a spread plate after 4 days growth at 25°C from a seed culture (JEC-21) inoculum grown at 37°C for 24 h on YPD. Bar = 1 mm. (B) Individual cells under the same seed culture and plating conditions as in (A), but visualized after 4 h at 200× magnification. Bar = 10 µm. (C–F) Monokaryotic fruiting in all serotypes of <i>C. neoformans</i>. (C) WSA-79 (serotype D, also known as <i>C. neoformans</i> variety <i>neoformans</i>), (D) WSA-522 (serotype A, also known as <i>C. neoformans</i> variety <i>grubii</i>), (E) WSA-533 (serotype B, also known as <i>C. neoformans</i> variety <i>gattii</i>, or <i>C. gattii</i>), (F) WSA-2507 (serotype C, also known as <i>C. neoformans</i> variety <i>gattii</i>, or <i>C. gattii</i>). Colonies visualized at 200×. Bar = 10 µm.</p

Exclusion of <i>α</i>-<i>α</i> cell fusion as a mechanism for monokaryotic fruiting.

<p>(A) Crosses between <i>MATα</i> strains with complementary auxotrophic markers do not yield fusants (WSA-1226×WSA-70, or WSA-1226×WSA-1) unless the mating is assisted with a <i>MATa</i> (WSA-68) helper strain (WSA-1226×WSA-70×WSA-68, or WSA-1226×WSA-1×WSA-68). However, the three <i>MATα</i> strains are capable of monokaryotic fruiting (insets). (B) To rule out an undetectable fusion event as an explanation for the results seen in (A), a fusion defective strain (WSA-2126) was included in the assay. This strain, which is a <i>cpk1</i> mutant, did not yield fusants with WSA-591 alone or with a <i>MATa</i> (WSA-65) helper. Complementation of WSA-2126 with the <i>CPK1</i> gene (WSA-3145) did not yield fusants with WSA-591 unless WSA-65 was included as a helper. However, all three <i>MATα</i> strains were capable of monokaryotic fruiting, including the <i>cpk1</i> mutant (inserts) ruling out <i>α</i>-<i>α</i> cell fusion as the mechanism for monokaryotic fruiting.</p

Size comparison of sorted G1 and G2 phase cells.

<p>JEC-21 cells were grown on YPD agar at 37°C for 24 hrs, then were fixed and stained with propidium iodide. Cells were then sorted according to DNA content and then visualized by DIC microscopy at 400× in order to compare their sizes. A). Sorted G1 cell fraction. B). Sorted G2 cell fraction. Microscopy showed that the G2 fraction contained cells approximately 4× the size of cells in the G1 fraction. Bar = 5 µm.</p

The relationship between cell size, cell cycle, and monokaryotic fruiting ability.

<p>(A) Cell cycle analysis of sorted JEC-21 cells revealed that the smaller cells were almost exclusively (90.57%) G1 phase cells and yeast-like in morphology when plated onto filament agar. (B) In contrast to the results observed for smaller cells, the population of enlarged cells recovered by sorting revealed that enlarged cells were primarily G2 phase (88.61%), and contained a sub population of cells (∼40–50%) able to undergo monokaryotic fruiting when these cells were plated onto filament agar. Approximately 20,000 cells were sorted for each cell size. Bar = 1.0 mm.</p