Figure 1.
A forward genetic screen for meiotic mutants reveals RIE1 as an entry factor. (A) Schematic overview of RBP meiotic entry screen. Representative fluorescence images are shown for wild type (B2381) and a meiotic entry mutant (rim4Δ, B2585). Zip1-GFP is shown in green, Spc42-mCherry is shown in magenta. Scale bar, 2 µm. (B) Strains homozygous for Zip1-GFP, Spc42-mCherry, ndt80Δ, and deletion of a gene encoding an RBP-containing protein were induced to sporulate at 30°C. At 6 h, when cells had arrested in G2 due to the lack of NDT80, cells were collected, fixed with formaldehyde, and nuclear Zip1 signal was assessed by fluorescence microscopy with DAPI staining to assess position of the nucleus. The y axis shows the percentage of Zip1-positive cells as defined by a fivefold nuclear GFP signal over the background. Shown are screen results of three biological replicates where each point represents an independent experiment. Orange bars indicate RRM mutants, and purple bars indicate genes encoding RBPs without an RRM. The results of two wild type control strains (B2381) are shown in teal. (C)rie1Δ mutants exhibit a severe entry phenotype. Shown are representative examples of images taken at 6 h for wild type (B2381) and rie1Δ (B2397) cells. Zip1-GFP is shown in green, Spc42-mCherry is shown in magenta (left panels) or red (right panels), and DAPI in blue. Scale bar, 2 µm. (D–F) Strains harboring fluorescently labeled tubulin (pTUB1-GFP-TUB1) and Spc42 (SPC42-mCherry) and either RIE1 (wild type, B1451) or rie1∆ (B1514) were induced to sporulate at 30°C. After 2.5 h of growth, the cells were loaded onto a microfluidics chip (Cell Asic), points were established, and cells were imaged every 7 min starting at 3.5 h. (D) Percentage of cells entering meiosis was recorded. Each point represents the percentage entry in a field of view (n = 12 fields [minimum five cells] for each genotype). (E and F) Single-cell analysis (for cells that entered meiosis, n = 25 for each genotype) of (E) time of metaphase I onset and (F) time in metaphase I, anaphase I, metaphase II, and anaphase II. Mean is indicated by a black bar and statistical significance (*P < 0.05) was determined by a two-sided student’s t test (data distribution was tested for normality).

A forward genetic screen for meiotic mutants reveals RIE1 as an entry factor. (A) Schematic overview of RBP meiotic entry screen. Representative fluorescence images are shown for wild type (B2381) and a meiotic entry mutant (rim4Δ, B2585). Zip1-GFP is shown in green, Spc42-mCherry is shown in magenta. Scale bar, 2 µm. (B) Strains homozygous for Zip1-GFP, Spc42-mCherry, ndt80Δ, and deletion of a gene encoding an RBP-containing protein were induced to sporulate at 30°C. At 6 h, when cells had arrested in G2 due to the lack of NDT80, cells were collected, fixed with formaldehyde, and nuclear Zip1 signal was assessed by fluorescence microscopy with DAPI staining to assess position of the nucleus. The y axis shows the percentage of Zip1-positive cells as defined by a fivefold nuclear GFP signal over the background. Shown are screen results of three biological replicates where each point represents an independent experiment. Orange bars indicate RRM mutants, and purple bars indicate genes encoding RBPs without an RRM. The results of two wild type control strains (B2381) are shown in teal. (C)rie1Δ mutants exhibit a severe entry phenotype. Shown are representative examples of images taken at 6 h for wild type (B2381) and rie1Δ (B2397) cells. Zip1-GFP is shown in green, Spc42-mCherry is shown in magenta (left panels) or red (right panels), and DAPI in blue. Scale bar, 2 µm. (D–F) Strains harboring fluorescently labeled tubulin (pTUB1-GFP-TUB1) and Spc42 (SPC42-mCherry) and either RIE1 (wild type, B1451) or rie1∆ (B1514) were induced to sporulate at 30°C. After 2.5 h of growth, the cells were loaded onto a microfluidics chip (Cell Asic), points were established, and cells were imaged every 7 min starting at 3.5 h. (D) Percentage of cells entering meiosis was recorded. Each point represents the percentage entry in a field of view (n = 12 fields [minimum five cells] for each genotype). (E and F) Single-cell analysis (for cells that entered meiosis, n = 25 for each genotype) of (E) time of metaphase I onset and (F) time in metaphase I, anaphase I, metaphase II, and anaphase II. Mean is indicated by a black bar and statistical significance (*P < 0.05) was determined by a two-sided student’s t test (data distribution was tested for normality).

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