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Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability.

Choy JS, O'Toole E, Schuster BM, Crisp MJ, Karpova TS, McNally JG, Winey M, Gardner MK, Basrai MA - Mol. Biol. Cell (2013)

Bottom Line: We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities.The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants.Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.

ABSTRACT
How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.

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Model convolution simulation, fluorescence microscopy, electron tomography, and FRAP reveal increased altered iMT and kMT assembly in spc98/+ mutants. (A, B) Model convolution simulation of Tub1-GFP distributions for wild-type and spc98/+ mutant cells. The simulations predict fewer kMTs (gold) and more iMTs (light blue) in spc98/+ mutants than in wild-type cells. (C) The distance between KT clusters and SPBs was measured in anaphase wild-type (red curve) and spc98/+ mutant cells expressing Spc110-mCherry to mark the spindle poles, Tub1-GFP to label microtubules, and Nuf2-GFP to mark kinetochores (blue curve). The plot indicates that spc98/+ mutants contain shorter kMTs than do wild-type cells; n = 42 for spc98/+ mutants, and n = 57 for wild-type cells. Average spindle length is 7.4 ± 1.5 μm in wild-type cells (mean ± SD) and 7.8 ± 2.3 μm in spc98/+ mutants. (D) Electron tomography revealed that spc98/+ spindles contained abnormal SPB positioning and spindle curvature (Figures 2D and 3A). More iMTs (green and pink) are present and have a much longer region of interdigitation than is typically found in wild-type cells. See also Supplemental Figure S2 and Supplemental Videos S1–S3. Bar, 200 nm. (E) FRAP (left) was performed on mid-anaphase spindles in spc98/+ mutant (n = 7) and wild-type cells (n = 4). A rectangular region (1.2 × 0.6 μm) over the mother–bud neck was photobleached, and the rate at which fluorescence was recovered normalized to total spindle fluorescence is plotted. In both strains the starting and ending spindle lengths are comparable (right).
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Figure 4: Model convolution simulation, fluorescence microscopy, electron tomography, and FRAP reveal increased altered iMT and kMT assembly in spc98/+ mutants. (A, B) Model convolution simulation of Tub1-GFP distributions for wild-type and spc98/+ mutant cells. The simulations predict fewer kMTs (gold) and more iMTs (light blue) in spc98/+ mutants than in wild-type cells. (C) The distance between KT clusters and SPBs was measured in anaphase wild-type (red curve) and spc98/+ mutant cells expressing Spc110-mCherry to mark the spindle poles, Tub1-GFP to label microtubules, and Nuf2-GFP to mark kinetochores (blue curve). The plot indicates that spc98/+ mutants contain shorter kMTs than do wild-type cells; n = 42 for spc98/+ mutants, and n = 57 for wild-type cells. Average spindle length is 7.4 ± 1.5 μm in wild-type cells (mean ± SD) and 7.8 ± 2.3 μm in spc98/+ mutants. (D) Electron tomography revealed that spc98/+ spindles contained abnormal SPB positioning and spindle curvature (Figures 2D and 3A). More iMTs (green and pink) are present and have a much longer region of interdigitation than is typically found in wild-type cells. See also Supplemental Figure S2 and Supplemental Videos S1–S3. Bar, 200 nm. (E) FRAP (left) was performed on mid-anaphase spindles in spc98/+ mutant (n = 7) and wild-type cells (n = 4). A rectangular region (1.2 × 0.6 μm) over the mother–bud neck was photobleached, and the rate at which fluorescence was recovered normalized to total spindle fluorescence is plotted. In both strains the starting and ending spindle lengths are comparable (right).

Mentions: Interpolar MTs, which emanate from opposite poles, overlap, and interdigitate at the midzone, facilitate spindle elongation during anaphase (Winey et al., 1995; Goshima and Scholey, 2010). Changes in the length distribution of iMTs affect the length of spindles and, in some cases, the rate of spindle elongation (Goshima and Scholey, 2010; Avunie-Masala et al., 2011). As a first approximation of the length distribution of iMTs, we measured the levels of Tub1–green fluorescent protein (GFP) in metaphase and anaphase spindles in wild-type and spc98/+ mutants. During metaphase, spc98/+ mutants had a distribution of Tub1-GFP that was similar to that of wild-type cells (Supplemental Figure S1B). In contrast, anaphase spindles in spc98/+ mutants contained less Tub1-GFP near the spindle poles and more at the midzone than wild-type cells, suggesting that the length distribution of spindle microtubules during anaphase is disrupted in spc98/+ mutants (Figure 3A). To determine whether the increase in Tub1-GFP at the midzone might be a result of greater polymerization of microtubules in spc98/+ mutants compared with wild-type cells, we performed fluorescence recovery after photobleaching (FRAP) experiments. This allowed us to determine the exchange of Tub1-GFP and assembly at the region where photobleaching occurs. Because we were most interested in iMT assembly at the midzone, our experimental design was similar to FRAP experiments reported by Maddox et al. (2000), in which we photobleached mid-anaphase spindles at the mother–daughter bud neck region. We analyzed only anaphase spindles in spc98/+ mutants and wild-type cells that started with a length of 5.3–6.2 μm and ended with a length of 7.9–8.2 μm (Figure 4E). By doing so, we could ensure that the measurements were being made on comparable anaphase spindles. We observed that fluorescence was nearly fully recovered by ∼210 s in spc98/+ mutants (Figure 4E). Near-complete recovery in wild-type cells, however, was not reached until ∼270 s (Figure 4E). Taken together, these results indicate that spc98/+ mutants have greater iMT assembly compared with wild-type cells.


Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability.

Choy JS, O'Toole E, Schuster BM, Crisp MJ, Karpova TS, McNally JG, Winey M, Gardner MK, Basrai MA - Mol. Biol. Cell (2013)

Model convolution simulation, fluorescence microscopy, electron tomography, and FRAP reveal increased altered iMT and kMT assembly in spc98/+ mutants. (A, B) Model convolution simulation of Tub1-GFP distributions for wild-type and spc98/+ mutant cells. The simulations predict fewer kMTs (gold) and more iMTs (light blue) in spc98/+ mutants than in wild-type cells. (C) The distance between KT clusters and SPBs was measured in anaphase wild-type (red curve) and spc98/+ mutant cells expressing Spc110-mCherry to mark the spindle poles, Tub1-GFP to label microtubules, and Nuf2-GFP to mark kinetochores (blue curve). The plot indicates that spc98/+ mutants contain shorter kMTs than do wild-type cells; n = 42 for spc98/+ mutants, and n = 57 for wild-type cells. Average spindle length is 7.4 ± 1.5 μm in wild-type cells (mean ± SD) and 7.8 ± 2.3 μm in spc98/+ mutants. (D) Electron tomography revealed that spc98/+ spindles contained abnormal SPB positioning and spindle curvature (Figures 2D and 3A). More iMTs (green and pink) are present and have a much longer region of interdigitation than is typically found in wild-type cells. See also Supplemental Figure S2 and Supplemental Videos S1–S3. Bar, 200 nm. (E) FRAP (left) was performed on mid-anaphase spindles in spc98/+ mutant (n = 7) and wild-type cells (n = 4). A rectangular region (1.2 × 0.6 μm) over the mother–bud neck was photobleached, and the rate at which fluorescence was recovered normalized to total spindle fluorescence is plotted. In both strains the starting and ending spindle lengths are comparable (right).
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Figure 4: Model convolution simulation, fluorescence microscopy, electron tomography, and FRAP reveal increased altered iMT and kMT assembly in spc98/+ mutants. (A, B) Model convolution simulation of Tub1-GFP distributions for wild-type and spc98/+ mutant cells. The simulations predict fewer kMTs (gold) and more iMTs (light blue) in spc98/+ mutants than in wild-type cells. (C) The distance between KT clusters and SPBs was measured in anaphase wild-type (red curve) and spc98/+ mutant cells expressing Spc110-mCherry to mark the spindle poles, Tub1-GFP to label microtubules, and Nuf2-GFP to mark kinetochores (blue curve). The plot indicates that spc98/+ mutants contain shorter kMTs than do wild-type cells; n = 42 for spc98/+ mutants, and n = 57 for wild-type cells. Average spindle length is 7.4 ± 1.5 μm in wild-type cells (mean ± SD) and 7.8 ± 2.3 μm in spc98/+ mutants. (D) Electron tomography revealed that spc98/+ spindles contained abnormal SPB positioning and spindle curvature (Figures 2D and 3A). More iMTs (green and pink) are present and have a much longer region of interdigitation than is typically found in wild-type cells. See also Supplemental Figure S2 and Supplemental Videos S1–S3. Bar, 200 nm. (E) FRAP (left) was performed on mid-anaphase spindles in spc98/+ mutant (n = 7) and wild-type cells (n = 4). A rectangular region (1.2 × 0.6 μm) over the mother–bud neck was photobleached, and the rate at which fluorescence was recovered normalized to total spindle fluorescence is plotted. In both strains the starting and ending spindle lengths are comparable (right).
Mentions: Interpolar MTs, which emanate from opposite poles, overlap, and interdigitate at the midzone, facilitate spindle elongation during anaphase (Winey et al., 1995; Goshima and Scholey, 2010). Changes in the length distribution of iMTs affect the length of spindles and, in some cases, the rate of spindle elongation (Goshima and Scholey, 2010; Avunie-Masala et al., 2011). As a first approximation of the length distribution of iMTs, we measured the levels of Tub1–green fluorescent protein (GFP) in metaphase and anaphase spindles in wild-type and spc98/+ mutants. During metaphase, spc98/+ mutants had a distribution of Tub1-GFP that was similar to that of wild-type cells (Supplemental Figure S1B). In contrast, anaphase spindles in spc98/+ mutants contained less Tub1-GFP near the spindle poles and more at the midzone than wild-type cells, suggesting that the length distribution of spindle microtubules during anaphase is disrupted in spc98/+ mutants (Figure 3A). To determine whether the increase in Tub1-GFP at the midzone might be a result of greater polymerization of microtubules in spc98/+ mutants compared with wild-type cells, we performed fluorescence recovery after photobleaching (FRAP) experiments. This allowed us to determine the exchange of Tub1-GFP and assembly at the region where photobleaching occurs. Because we were most interested in iMT assembly at the midzone, our experimental design was similar to FRAP experiments reported by Maddox et al. (2000), in which we photobleached mid-anaphase spindles at the mother–daughter bud neck region. We analyzed only anaphase spindles in spc98/+ mutants and wild-type cells that started with a length of 5.3–6.2 μm and ended with a length of 7.9–8.2 μm (Figure 4E). By doing so, we could ensure that the measurements were being made on comparable anaphase spindles. We observed that fluorescence was nearly fully recovered by ∼210 s in spc98/+ mutants (Figure 4E). Near-complete recovery in wild-type cells, however, was not reached until ∼270 s (Figure 4E). Taken together, these results indicate that spc98/+ mutants have greater iMT assembly compared with wild-type cells.

Bottom Line: We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities.The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants.Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.

ABSTRACT
How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.

Show MeSH
Related in: MedlinePlus