Meiosis II spindle disassembly requires two distinct pathways

Abstract

During exit from meiosis II, cells undergo several structural rearrangements, including disassembly of the meiosis II spindles and cytokinesis. Each of these changes is regulated to ensure that they occur at the proper time. Previous studies have demonstrated that both SPS1, which encodes a STE20-family GCKIII kinase, and AMA1, which encodes a meiosis-specific activator of the Anaphase Promoting Complex, are required for both meiosis II spindle disassembly and cytokinesis in the budding yeast Saccharomyces cerevisiae. We examine the relationship between meiosis II spindle disassembly and cytokinesis and find that the meiosis II spindle disassembly failure in sps1Δ and ama1∆ cells is not the cause of the cytokinesis defect. We also see that the spindle disassembly defects in sps1Δ and ama1∆ cells are phenotypically distinct. We examined known microtubule-associated proteins Ase1, Cin8, and Bim1, and found that AMA1 is required for the proper loss of Ase1 and Cin8 on meiosis II spindles while SPS1 is required for Bim1 loss in meiosis II. Taken together, these data indicate that SPS1 and AMA1 promote distinct aspects of meiosis II spindle disassembly, and that both pathways are required for the successful completion of meiosis.

FIGURE 1:

The spindle and PSM during meiosis. The spindle and the PSM were visualized during meiosis in wild-type cells (LH1145), which contains the genomically integrated SPB marker SPC42-mRUBY (SPC42-yomRUBY2), tubulin marker GFP^ENVY-TUB1 (GFP^ENVY-TUB1+3′UTR:LEU2), and PSM marker B20 (ura3:B20:URA3). Images were acquired using a confocal microscope and are maximum intensity projections of 4 µm z-stacks.

FIGURE 2:

Spindle disassembly does not suppress the hyperelongated PSM defect in sps1∆ and ama1∆ cells. Wild type (LH1146), sps1∆ (LH1147), and ama1∆ (LH1148) cells were sporulated and treated with a cocktail of microtubule depolymerizing drugs (benomyl and nocodazole; see Materials and Methods). PSMs were visualized using the genomically integrated K20 marker (his3:SPO20^51-91-mKATE2:HIS3); tubulin was visualized using GFP^ENVY-TUB1. PSMs were analyzed every 10 min for 40 min after drug-induced microtubule depolymerization. Example confocal images show typical microtubule depolymerization and resultant PSM morphology. Images are maximum intensity projections of 4 µm z-stacks. Cells of interest are indicated by dashed circle when more than one cell is within the frame.

FIGURE 3:

sps1Δ and ama1Δ cells exhibit distinct spindle disassembly defects. Wild type (LH1146), ama1∆ (LH1148), sps1∆ (LH1147), and ama1∆ sps1∆ (LH1149) cells were sporulated and analyzed during and immediately following anaphase II. (A) Representative images of wild-type (LH1146) and ama1∆ (LH1148) cells. (B) Average anaphase II spindle lengths were measured by measuring GFP^ENVY-TUB1. Black bar indicates the median anaphase II spindle length. (C) The number of tubulin foci seen in post-anaphase II cells. Data from each of the three biological replicates were colored differently (day 1 in blue, day 2 in red, and day 3 in green). The magenta line indicates the median number of tubulin foci. (D) The length of tubulin fragments was measured in post anaphase II cells. Data from each of the three biological replicates was colored differently (day 1 in blue, 2 in red, and 3 in green). Median length is indicated by magenta line. (E) Representative images of sps1 (LH1147) cells. (F) Representative images of ama1∆ sps1∆ (LH1149) cells. Panels A, E, and F are widefield images. Images are maximum intensity projections of 2.5 µm z-stacks. PSMs were visualized using the genomically integrated K20 marker, in red; tubulin was visualized using GFP^ENVY-TUB1, in green.

FIGURE 4:

Ase1, Cin8, and Bim1 are expressed during meiosis and are present at the meiosis II spindle. (A) Ase1 expression was examined during sporulation using Ase1-myc (LH1161). (B) Ase1 localization during meiosis was examined using Ase1-GFP^Envy (LH1150); spindles were labeled using yomRuby3-Tub1 and PSMs were labeled using the B20 marker. (C) Cin8 expression was examined during sporulation using Cin8-myc (LH1165). (D) Cin8 localization during meiosis was examined using Cin8-yomRuby3 (LH1157); spindles were labeled using GFP^Envy-Tub1. (E) Bim1 expression was examined during sporulation using a Bim1-myc/+ heterozygous strain (LH1170). (F) Bim1 localization during meiosis was examined using Bim1-yomRuby3 (LH1153); spindles were labeled using GFP^Envy-Tub1. For immunoblots (A, C, and E), Pgk1 was used as a loading control; untagged control strain (LH177). The size of the molecular weight (MW) standards are indicated next to the appropriate band. Images (B, D, and F) were acquired using a widefield microscope and are maximum intensity projections of 2.5 µm z-stacks.

FIGURE 5:

Removal of Ase1 from the meiosis II spindle requires AMA1. (A) The localization of Ase1-GFP^Envy was examined in wild-type (LH1150), sps1∆ (LH1151), ama1∆ (LH1152), and ama1∆ sps1∆ (LH1153) cells. Tubulin was visualized using yomRuby3-Tub1. (B) Quantification of cells that have Ase1 colocalizing with microtubules. One way ANOVA [F(3, 11) = 785, p < 1 × 10⁶] followed by Tukey’s HSD post hoc test (α = 0.01), letters denote statistically distinct groups among post-anaphase II cells. (C) Immunoblots of Ase1-myc expression in wild type (LH1161), ama1∆ (LH1162), sps1∆ (LH1163) and ama1∆ sps1∆ (LH1164) at 6 and 8 h after transfer to sporulation media; Pgk1 is used as the loading control. The size of the MW standards are indicated next to the appropriate band.

FIGURE 6:

Removal of Cin8 from the meiosis II spindle requires AMA1. (A) The localization of Cin8-yomRuby3 was examined in wild-type (LH1157), sps1∆ (LH1158), ama1∆ (LH1159), and ama1∆ sps1∆ (LH1160) cells. Tubulin was visualized using GFP^Envy-Tub1. (B) Quantification of cells that have Cin8 colocalizing with microtubules. One way ANOVA [F(3, 8) = 505, p < 1 × 10⁹] followed by Tukey’s HSD post hoc test (α = 0.01), letters denote statistically distinct groups among post-anaphase II cells. (C) Immunoblots of Cin8-myc expression in wild type (LH1165), ama1∆ (LH1167), and sps1∆ (LH1166) at 6 and 8 h after transfer to sporulation media; Pgk1 is used as the loading control. The size of the MW standards are indicated next to the appropriate band.

FIGURE 7:

Removal of Bim1 from the meiosis II spindle requires SPS1 but not AMA1. (A) The localization of Bim1-yomRuby3 was examined in wild type (LH1154), sps1∆ (LH1155), and ama1∆ (LH1156). Tubulin was visualized using GFP^Envy-Tub1, PSMs were visualized using B20. (B) The localization of Bim1-yomRuby3 was examined in ama1∆ sps1∆ (LH1171) cells. Tubulin was visualized using GFP^Envy-Tub1, PSMs were visualized using B20. (C) Quantification of cells that have Bim1 colocalizing with microtubules. For post-anaphase II open PSMs, letters denote statistically distinct groups. One way ANOVA [F(3, 8) = 16.4, p < 1 × 10⁴] followed by Tukey’s HSD post hoc test (α = 0.04). Note that sps1∆ ama1∆ double mutants do not close their PSMs (Paulissen et al., 2016), which is why the post-anaphase II, closed PSM category is not applicable (NA). (D) Immunoblots of Bim1-myc/+ expression in untagged (LH177), wild type (LH1170), ama1∆ (LH1168), and sps1∆ (LH1169) at 6 and 8 h after transfer to sporulation media; Pgk1 is used as the loading control. The size of the MW standards are indicated next to the appropriate band.