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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.

Show MeSH
hnt3Δ, mcm21Δ, emi1Δ, and emi2Δ mutants have spindle elongation phenotypes and components of the CPC become partially mislocalized in mcm21Δ mutant cells. (A) Quantitation of number of cells with fish hook spindles in single and double mutant cultures. A histogram illustrating the percentage of cells in the indicated mutant cultures with fish hook spindles is shown. Cells were synchronized with α factor for 90 min, released into fresh medium, and assessed for fish hook spindles after 60 min; images of at least 100 anaphase cells for each culture were captured and quantified. Error bars show the standard deviation from three independent experiments. (B, top) Ipl1p-GFP localization to the elongating spindle and spindle midzone is disrupted in 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. DIC images, fluorescent micrographs, and merged images are shown of representative single cells of wild-type (left) or mcm21Δ (right) strains expressing Ipl1p-GFP and RFP-Tub1p. Yellow arrowheads indicate the diffused localization of Ipl1p-GFP (for the mcm21Δ cells) and the spindle midzone. Bar, 5 µm. (B, bottom) Sli15p-GFP is mislocalized, and Bir1p-GFP localization is unaffected in mcm21Δ mutant cells. Mid-log phase cells were treated with nocodazole for 2.5 h and released into fresh medium before imaging. DIC images, fluorescent micrographs, and merged images of a representative wild-type or mcm21Δ anaphase cell expressing Sli5p-GFP/Bir1p-GFP and RFP-Tub1p are shown. Yellow arrowheads indicate the localization of Sli15p-GFP. (C and D) Quantitation of the defect in Ipl1p-GFP localization at the kinetochore during metaphase and the spindle midzone during anaphase in wild-type and mcm21Δ mutant cells. (C) For kinetochore localization, cells with spindles between <2 and 2–4 µm were quantified, as there was an increased mislocalization of Ipl1p-GFP/Sli15p-GFP during early anaphase. (D) For midzone localization, cells with spindles 4 µm and longer were assessed. Cells with Ipl1p-GFP signal throughout the spindle were also considered as enriched for midzone in the quantitation. n > 60–100 cells in each spindle length category.
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fig3: hnt3Δ, mcm21Δ, emi1Δ, and emi2Δ mutants have spindle elongation phenotypes and components of the CPC become partially mislocalized in mcm21Δ mutant cells. (A) Quantitation of number of cells with fish hook spindles in single and double mutant cultures. A histogram illustrating the percentage of cells in the indicated mutant cultures with fish hook spindles is shown. Cells were synchronized with α factor for 90 min, released into fresh medium, and assessed for fish hook spindles after 60 min; images of at least 100 anaphase cells for each culture were captured and quantified. Error bars show the standard deviation from three independent experiments. (B, top) Ipl1p-GFP localization to the elongating spindle and spindle midzone is disrupted in 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. DIC images, fluorescent micrographs, and merged images are shown of representative single cells of wild-type (left) or mcm21Δ (right) strains expressing Ipl1p-GFP and RFP-Tub1p. Yellow arrowheads indicate the diffused localization of Ipl1p-GFP (for the mcm21Δ cells) and the spindle midzone. Bar, 5 µm. (B, bottom) Sli15p-GFP is mislocalized, and Bir1p-GFP localization is unaffected in mcm21Δ mutant cells. Mid-log phase cells were treated with nocodazole for 2.5 h and released into fresh medium before imaging. DIC images, fluorescent micrographs, and merged images of a representative wild-type or mcm21Δ anaphase cell expressing Sli5p-GFP/Bir1p-GFP and RFP-Tub1p are shown. Yellow arrowheads indicate the localization of Sli15p-GFP. (C and D) Quantitation of the defect in Ipl1p-GFP localization at the kinetochore during metaphase and the spindle midzone during anaphase in wild-type and mcm21Δ mutant cells. (C) For kinetochore localization, cells with spindles between <2 and 2–4 µm were quantified, as there was an increased mislocalization of Ipl1p-GFP/Sli15p-GFP during early anaphase. (D) For midzone localization, cells with spindles 4 µm and longer were assessed. Cells with Ipl1p-GFP signal throughout the spindle were also considered as enriched for midzone in the quantitation. n > 60–100 cells in each spindle length category.

Mentions: We scored the fish hook spindle phenotype in a synchronized population of cells (Fig. 3 A) and by time-lapse imaging (Fig. S2 A, yellow arrows). Analysis of the kinetics of spindle elongation in mcm21Δ, hnt3Δ, emi1Δ, and emi2Δ mutant strains (Fig. S2, B–E and G) revealed a clear delay in anaphase spindle disassembly showing a fish hook spindle with varying degrees of defect. A small percentage of hnt3Δ bni1Δ double mutant cells also showed swapping of spindle poles between the mother and daughter cells after spindle disassembly (Fig. S2 A, red arrows), which is consistent with a loss of spindle pole polarity (Pereira et al., 2001) and consequent delay in cytokinesis (not depicted; Jensen et al., 2004). Time-lapse imaging also revealed cycles of partial elongation and collapse of the mitotic spindle without full elongation along the mother–bud axis in both mcm21Δ and hnt3Δ mutants (Fig. S2 A, blue arrows; and Fig. S2 F). Interestingly, we observed both the spindle instability and hyper-elongation phenotypes in the same cell (Fig. S2 A). We conclude that Mcm21p and Hnt3p may regulate both the stability of the growing anaphase spindle as well as spindle disassembly.


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)

hnt3Δ, mcm21Δ, emi1Δ, and emi2Δ mutants have spindle elongation phenotypes and components of the CPC become partially mislocalized in mcm21Δ mutant cells. (A) Quantitation of number of cells with fish hook spindles in single and double mutant cultures. A histogram illustrating the percentage of cells in the indicated mutant cultures with fish hook spindles is shown. Cells were synchronized with α factor for 90 min, released into fresh medium, and assessed for fish hook spindles after 60 min; images of at least 100 anaphase cells for each culture were captured and quantified. Error bars show the standard deviation from three independent experiments. (B, top) Ipl1p-GFP localization to the elongating spindle and spindle midzone is disrupted in 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. DIC images, fluorescent micrographs, and merged images are shown of representative single cells of wild-type (left) or mcm21Δ (right) strains expressing Ipl1p-GFP and RFP-Tub1p. Yellow arrowheads indicate the diffused localization of Ipl1p-GFP (for the mcm21Δ cells) and the spindle midzone. Bar, 5 µm. (B, bottom) Sli15p-GFP is mislocalized, and Bir1p-GFP localization is unaffected in mcm21Δ mutant cells. Mid-log phase cells were treated with nocodazole for 2.5 h and released into fresh medium before imaging. DIC images, fluorescent micrographs, and merged images of a representative wild-type or mcm21Δ anaphase cell expressing Sli5p-GFP/Bir1p-GFP and RFP-Tub1p are shown. Yellow arrowheads indicate the localization of Sli15p-GFP. (C and D) Quantitation of the defect in Ipl1p-GFP localization at the kinetochore during metaphase and the spindle midzone during anaphase in wild-type and mcm21Δ mutant cells. (C) For kinetochore localization, cells with spindles between <2 and 2–4 µm were quantified, as there was an increased mislocalization of Ipl1p-GFP/Sli15p-GFP during early anaphase. (D) For midzone localization, cells with spindles 4 µm and longer were assessed. Cells with Ipl1p-GFP signal throughout the spindle were also considered as enriched for midzone in the quantitation. n > 60–100 cells in each spindle length category.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812844&req=5

fig3: hnt3Δ, mcm21Δ, emi1Δ, and emi2Δ mutants have spindle elongation phenotypes and components of the CPC become partially mislocalized in mcm21Δ mutant cells. (A) Quantitation of number of cells with fish hook spindles in single and double mutant cultures. A histogram illustrating the percentage of cells in the indicated mutant cultures with fish hook spindles is shown. Cells were synchronized with α factor for 90 min, released into fresh medium, and assessed for fish hook spindles after 60 min; images of at least 100 anaphase cells for each culture were captured and quantified. Error bars show the standard deviation from three independent experiments. (B, top) Ipl1p-GFP localization to the elongating spindle and spindle midzone is disrupted in 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. DIC images, fluorescent micrographs, and merged images are shown of representative single cells of wild-type (left) or mcm21Δ (right) strains expressing Ipl1p-GFP and RFP-Tub1p. Yellow arrowheads indicate the diffused localization of Ipl1p-GFP (for the mcm21Δ cells) and the spindle midzone. Bar, 5 µm. (B, bottom) Sli15p-GFP is mislocalized, and Bir1p-GFP localization is unaffected in mcm21Δ mutant cells. Mid-log phase cells were treated with nocodazole for 2.5 h and released into fresh medium before imaging. DIC images, fluorescent micrographs, and merged images of a representative wild-type or mcm21Δ anaphase cell expressing Sli5p-GFP/Bir1p-GFP and RFP-Tub1p are shown. Yellow arrowheads indicate the localization of Sli15p-GFP. (C and D) Quantitation of the defect in Ipl1p-GFP localization at the kinetochore during metaphase and the spindle midzone during anaphase in wild-type and mcm21Δ mutant cells. (C) For kinetochore localization, cells with spindles between <2 and 2–4 µm were quantified, as there was an increased mislocalization of Ipl1p-GFP/Sli15p-GFP during early anaphase. (D) For midzone localization, cells with spindles 4 µm and longer were assessed. Cells with Ipl1p-GFP signal throughout the spindle were also considered as enriched for midzone in the quantitation. n > 60–100 cells in each spindle length category.
Mentions: We scored the fish hook spindle phenotype in a synchronized population of cells (Fig. 3 A) and by time-lapse imaging (Fig. S2 A, yellow arrows). Analysis of the kinetics of spindle elongation in mcm21Δ, hnt3Δ, emi1Δ, and emi2Δ mutant strains (Fig. S2, B–E and G) revealed a clear delay in anaphase spindle disassembly showing a fish hook spindle with varying degrees of defect. A small percentage of hnt3Δ bni1Δ double mutant cells also showed swapping of spindle poles between the mother and daughter cells after spindle disassembly (Fig. S2 A, red arrows), which is consistent with a loss of spindle pole polarity (Pereira et al., 2001) and consequent delay in cytokinesis (not depicted; Jensen et al., 2004). Time-lapse imaging also revealed cycles of partial elongation and collapse of the mitotic spindle without full elongation along the mother–bud axis in both mcm21Δ and hnt3Δ mutants (Fig. S2 A, blue arrows; and Fig. S2 F). Interestingly, we observed both the spindle instability and hyper-elongation phenotypes in the same cell (Fig. S2 A). We conclude that Mcm21p and Hnt3p may regulate both the stability of the growing anaphase spindle as well as spindle disassembly.

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