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Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis.

Vizeacoumar FJ, van Dyk N, S Vizeacoumar F, Cheung V, Li J, Sydorskyy Y, Case N, Li Z, Datti A, Nislow C, Raught B, Zhang Z, Frey B, Bloom K, Boone C, Andrews BJ - J. Cell Biol. (2010)

Bottom Line: We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR).We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly.Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

ABSTRACT
We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.

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Sumoylation of Mcm21p regulates the localization of the CPC to the spindle midzone. (A and B) Ipl1p-GFP and Sli15p-GFP localization to the spindle midzone in wild-type, mcm21-32R, and mcm21Δ mutant cells. Mid-log phase cells were treated with nocodazole for 2.5 h at room temperature and released into fresh medium before imaging. Fluorescent micrographs of representative single cells of wild-type, mcm21Δ, or mcm21-32R strains expressing Ipl1p-GFP (A), Sli15p-GFP (B), and RFP-Tub1p are shown. Numbers at the top of each panel denote the spindle length (µm) range that each micrograph represents, and the numbers at the bottom represent the spindle length (µm) of the cell shown. (C and D) Quantitation of the defect in Ipl1p-GFP and Sli15p-GFP localization to the spindle midzone in wild-type, mcm21Δ mutant cells, and mcm21-32R mutant cells. Cells with spindles 4 µm and longer were used to quantify Ipl1p-GFP (C) or Sli15p-GFP (D) localization to the spindle midzone as indicated. n > 60–100 cells in each spindle length category. Cells with Ipl1p-GFP or Sli15p-GFP signal throughout the spindle were also counted as mid-zone enriched for the quantitation. (E) Localization of Ipl1p-GFP and Sli15p-GFP in ubc9-2 strains. Cells were grown at 34°C for 3 h and imaged immediately (34°C panels) or shifted to 26°C for 2 h before samples were imaged (26°C panels). DIC images, fluorescent micrographs, and merged images are shown of representative single cells. Bar, 5 µm. (F) Quantitation of the defect in spindle localization of Ipl1p-GFP and Sli15p-GFP for mutants represented in E. n > 100 cells.
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fig6: Sumoylation of Mcm21p regulates the localization of the CPC to the spindle midzone. (A and B) Ipl1p-GFP and Sli15p-GFP localization to the spindle midzone in wild-type, mcm21-32R, and mcm21Δ mutant cells. Mid-log phase cells were treated with nocodazole for 2.5 h at room temperature and released into fresh medium before imaging. Fluorescent micrographs of representative single cells of wild-type, mcm21Δ, or mcm21-32R strains expressing Ipl1p-GFP (A), Sli15p-GFP (B), and RFP-Tub1p are shown. Numbers at the top of each panel denote the spindle length (µm) range that each micrograph represents, and the numbers at the bottom represent the spindle length (µm) of the cell shown. (C and D) Quantitation of the defect in Ipl1p-GFP and Sli15p-GFP localization to the spindle midzone in wild-type, mcm21Δ mutant cells, and mcm21-32R mutant cells. Cells with spindles 4 µm and longer were used to quantify Ipl1p-GFP (C) or Sli15p-GFP (D) localization to the spindle midzone as indicated. n > 60–100 cells in each spindle length category. Cells with Ipl1p-GFP or Sli15p-GFP signal throughout the spindle were also counted as mid-zone enriched for the quantitation. (E) Localization of Ipl1p-GFP and Sli15p-GFP in ubc9-2 strains. Cells were grown at 34°C for 3 h and imaged immediately (34°C panels) or shifted to 26°C for 2 h before samples were imaged (26°C panels). DIC images, fluorescent micrographs, and merged images are shown of representative single cells. Bar, 5 µm. (F) Quantitation of the defect in spindle localization of Ipl1p-GFP and Sli15p-GFP for mutants represented in E. n > 100 cells.

Mentions: Unlike in the mcm21Δ deletion mutant, the kinetochore association of Ipl1p-GFP and Sli15p-GFP was not affected during metaphase in the mcm21-32R mutant, which suggests that Mcm21p-32R still retains the scaffolding function associated with wild-type Mcm21p at the kinetochore (Fig. 5, C–F). Nevertheless, like the mcm21Δ deletion mutant, the mcm21-32R mutant was defective for targeting Ipl1p-GFP and Sli15p-GFP to the spindle midzone, resulting in the formation of extended fish hook spindles (Fig. 6, A–D). Wild-type cells were able to target Ipl1p-GFP and Sli15p-GFP to the midzone, such that the spindle reached a maximum length of 6–8 µm before disassembly, whereas the mcm21Δ and the mcm21-32R mutant cells were defective in this localization and showed an extended fish hook spindle that often grew beyond 9 µm (Fig. 6, A–D). Invariably, every cell that formed a fish hook spindle did not localize Ipl1p-GFP to the midzone in both the mcm21Δ deletion mutant and the mcm21-32R mutant, which suggests that sumoylation of Mcm21p may signal the efficient relocation of the CPC to the spindle midzone.


Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis.

Vizeacoumar FJ, van Dyk N, S Vizeacoumar F, Cheung V, Li J, Sydorskyy Y, Case N, Li Z, Datti A, Nislow C, Raught B, Zhang Z, Frey B, Bloom K, Boone C, Andrews BJ - J. Cell Biol. (2010)

Sumoylation of Mcm21p regulates the localization of the CPC to the spindle midzone. (A and B) Ipl1p-GFP and Sli15p-GFP localization to the spindle midzone in wild-type, mcm21-32R, and mcm21Δ mutant cells. Mid-log phase cells were treated with nocodazole for 2.5 h at room temperature and released into fresh medium before imaging. Fluorescent micrographs of representative single cells of wild-type, mcm21Δ, or mcm21-32R strains expressing Ipl1p-GFP (A), Sli15p-GFP (B), and RFP-Tub1p are shown. Numbers at the top of each panel denote the spindle length (µm) range that each micrograph represents, and the numbers at the bottom represent the spindle length (µm) of the cell shown. (C and D) Quantitation of the defect in Ipl1p-GFP and Sli15p-GFP localization to the spindle midzone in wild-type, mcm21Δ mutant cells, and mcm21-32R mutant cells. Cells with spindles 4 µm and longer were used to quantify Ipl1p-GFP (C) or Sli15p-GFP (D) localization to the spindle midzone as indicated. n > 60–100 cells in each spindle length category. Cells with Ipl1p-GFP or Sli15p-GFP signal throughout the spindle were also counted as mid-zone enriched for the quantitation. (E) Localization of Ipl1p-GFP and Sli15p-GFP in ubc9-2 strains. Cells were grown at 34°C for 3 h and imaged immediately (34°C panels) or shifted to 26°C for 2 h before samples were imaged (26°C panels). DIC images, fluorescent micrographs, and merged images are shown of representative single cells. Bar, 5 µm. (F) Quantitation of the defect in spindle localization of Ipl1p-GFP and Sli15p-GFP for mutants represented in E. n > 100 cells.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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fig6: Sumoylation of Mcm21p regulates the localization of the CPC to the spindle midzone. (A and B) Ipl1p-GFP and Sli15p-GFP localization to the spindle midzone in wild-type, mcm21-32R, and mcm21Δ mutant cells. Mid-log phase cells were treated with nocodazole for 2.5 h at room temperature and released into fresh medium before imaging. Fluorescent micrographs of representative single cells of wild-type, mcm21Δ, or mcm21-32R strains expressing Ipl1p-GFP (A), Sli15p-GFP (B), and RFP-Tub1p are shown. Numbers at the top of each panel denote the spindle length (µm) range that each micrograph represents, and the numbers at the bottom represent the spindle length (µm) of the cell shown. (C and D) Quantitation of the defect in Ipl1p-GFP and Sli15p-GFP localization to the spindle midzone in wild-type, mcm21Δ mutant cells, and mcm21-32R mutant cells. Cells with spindles 4 µm and longer were used to quantify Ipl1p-GFP (C) or Sli15p-GFP (D) localization to the spindle midzone as indicated. n > 60–100 cells in each spindle length category. Cells with Ipl1p-GFP or Sli15p-GFP signal throughout the spindle were also counted as mid-zone enriched for the quantitation. (E) Localization of Ipl1p-GFP and Sli15p-GFP in ubc9-2 strains. Cells were grown at 34°C for 3 h and imaged immediately (34°C panels) or shifted to 26°C for 2 h before samples were imaged (26°C panels). DIC images, fluorescent micrographs, and merged images are shown of representative single cells. Bar, 5 µm. (F) Quantitation of the defect in spindle localization of Ipl1p-GFP and Sli15p-GFP for mutants represented in E. n > 100 cells.
Mentions: Unlike in the mcm21Δ deletion mutant, the kinetochore association of Ipl1p-GFP and Sli15p-GFP was not affected during metaphase in the mcm21-32R mutant, which suggests that Mcm21p-32R still retains the scaffolding function associated with wild-type Mcm21p at the kinetochore (Fig. 5, C–F). Nevertheless, like the mcm21Δ deletion mutant, the mcm21-32R mutant was defective for targeting Ipl1p-GFP and Sli15p-GFP to the spindle midzone, resulting in the formation of extended fish hook spindles (Fig. 6, A–D). Wild-type cells were able to target Ipl1p-GFP and Sli15p-GFP to the midzone, such that the spindle reached a maximum length of 6–8 µm before disassembly, whereas the mcm21Δ and the mcm21-32R mutant cells were defective in this localization and showed an extended fish hook spindle that often grew beyond 9 µm (Fig. 6, A–D). Invariably, every cell that formed a fish hook spindle did not localize Ipl1p-GFP to the midzone in both the mcm21Δ deletion mutant and the mcm21-32R mutant, which suggests that sumoylation of Mcm21p may signal the efficient relocation of the CPC to the spindle midzone.

Bottom Line: We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR).We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly.Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

ABSTRACT
We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.

Show MeSH