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1.
Figure 2

Figure 2. Lack of Clp1 corrects merotelic attachments by reducing spindle elongation forces. From: A role for metaphase spindle elongation forces in correction of merotelic kinetochore attachments.

(A–B) The spindle length at the metaphase/anaphase transition was determined by ROC analysis (see Experimental Procedures for details) and compared between the indicated strains. Bars represent the mean +/− SD (n=3; 100 cells each). The differences between all pairwise combinations in each graph were statistically significant by unpaired t test (p<0.05), except for between WT and clp1Δ in (B) (p=0.072). (C) Strains expressing the SPB marker Cdc11-GFP and carrying GFP-marked centromere 2 (Cen2-GFP) were arrested at metaphase by overexpressing Mad2 from the thiamine repressible nmt1 promoter for 17h and fixed. The distance between Cen2-GFP dots was measured. Bars represent the mean +/− SD (n=3; >280 cells each). (D) A comparison of average inter-kinetochore distances obtained from panel (C). The values for the swi6Δ clp1Δ and swi6Δ klp9Δ showed statistically very significant differences compared to the swi6Δ parent by an unpaired t test (p<0.0001). The bars represent the mean +/− the standard error of the mean (n=3; >280 cells each). See also Figure S2.

Sung Hugh Choi, et al. Curr Biol. ;22(3):225-230.
2.
Figure 3

Figure 3. Correction of merotelic attachments can be modulated through manipulation of the activities of metaphase spindle elongation motors. From: A role for metaphase spindle elongation forces in correction of merotelic kinetochore attachments.

(A) Spindle lengths of the indicated strains at the metaphase/anaphase transition are shown (The differences between all pairwise combinations of the indicated strains were statistically significant by unpaired t test (p<0.05), except for between WT and mde4Δ klp9Δ or swi6Δ klp9Δ.). (B) Spindle lengths at the metaphase/anaphase transition was determined for wild-type cells before (0h) and after (17h) induction of expression of Klp9, Klp9 (S/A), or no protein (control) for 17h from the nmt1 promoter. Bars represent the mean +/− SD (n=3; 100 cells each). (C) The effect of klp9 on the frequency of lagging or mis-segregated chromosomes in the indicated strains is shown. (D) The effect of overexpression of Klp9 (pREP2-klp9), non-phosphorylatable Klp9 (pREP2-klp9(S/A)), or no protein (pREP2) for 17h using the nmt1 promoter in the indicated strains is shown. Bars represent the mean +/− SD (n=3; 110 cells each). (E) The frequency of lagging chromosomes in mde4Δ and swi6Δ mutants in the presence or absence of cut7-24 mutation is shown. (F) Spindle lengths at the metaphase/anaphase transition for the indicated strains were shown. Cells in parts E and F were grown at 25°C, the permissive temperature for the cut7-24 mutation. The differences between all pairwise combinations of strains were statistically significant by unpaired t test (p<0.05), except for between WT and cut7-24 (p=0.827), and WT and cut7-24 swi6Δ (p=0.435). See also Figure S3.

Sung Hugh Choi, et al. Curr Biol. ;22(3):225-230.
3.
Figure 4

Figure 4. Merotelic correction in human cells can be controlled through manipulation of spindle elongation motors. From: A role for metaphase spindle elongation forces in correction of merotelic kinetochore attachments.

(A) RPE-1 cells stable expressing centrin-GFP were treated with nocodazole for 7h and released into media with 15μM monastrol for 40 minutes, and fixed. (Top) An example cell stained for kinetochores (CENP-B), tubulin, and DNA. (Bottom) Frequency of lagging chromosomes in anaphase. Bars represent the mean +/− SD (n=3; 100 cells each). Arrow indicates a single lagging chromosome. Only single chromosomes (as judged by CENP-B staining) were scored. (B) Comparisons of the metaphase spindle length and interkinetochore distances are shown. RPE1 centrin-GFP cells were treated with 15μM Monastrol for 16h, then 5μM MG132 for 1h and fixed. (Top) An example cell stained for kinetochores (CENP-B), tubulin, and DNA. Arrowheads indicate centrosomes, and arrows indicates paired sister kinetochores. (Bottom) Metaphase spindle length and interkinetochore distance was determined by measuring the distance between centrosomes and paired sister kinetochores respectively from cells arrested at metaphase. Bars represent the mean +/− SD (n=3; 80 cells each). (C) CaCo-2 cells were treated with 15μM Monastrol for 16h, and fixed. (Top) An example of cells stained for kinetochores (CREST), tubulin, and DNA. (Bottom left) The frequency of lagging chromosomes is shown. Only single chromosomes (as judged by CREST staining) were scored. Bars represent the mean +/− SD (n=3; 50 cells each). (Bottom right) A comparison of the metaphase spindle length is shown. Cells were treated with 15μM Monastrol for 16h, then 5μM MG132 for 1h and fixed. Metaphase spindle length was measured by distance between centrosomes from cells arrested at metaphase. Bars represent the mean +/− SD (n=3; 40 cells each). (D) A hypothetical model depicting how merotelic attachment could be corrected by manipulation of motor activities on interpolar microtubules. Phospho-regulation of Klp9 by Cdk1 and Clp1, or the indicated mutations in motor molecules, affects tension applied on sister kinetochores. In turn, tension may regulate the stability of merotelic kinetochores either directly or indirectly by affecting their exposure to the kinase Aurora B. See also Figure S4.

Sung Hugh Choi, et al. Curr Biol. ;22(3):225-230.
4.
Figure 1

Figure 1. Aurora and Cdk1 kinases affect correction of merotelic attachments. From: A role for metaphase spindle elongation forces in correction of merotelic kinetochore attachments.

(A) The diagram depicts both normal (bipolar) and aberrant (merotelic) attachment of sister kinetochores to microtubules in metaphase (top) and the resulting segregation pattern of each type of attachment in anaphase (bottom). (B) Genetic interactions between mde4Δ, swi6Δ and mutations in the chromosome passenger complex are shown (top). The mde4Δ and swi6Δ mutants were crossed with the chromosome passenger complex mutants, Aurora kinase (ark1-T7), Survivin (cut17-275) and INCENP (pic1-13myc). Genetic interactions are shown as synthetic lethality (S.L) and weak growth (+/−). (bottom) swi6Δ ark1-T7 double mutant cells show a synthetic growth defect compared to either single mutant. The indicated strains were grown in YE at 25°C, and then serial dilutions were spotted on YE plates and incubated at the indicated temperatures for 3–5 days. (C) The frequency of lagging or unevenly segregated chromosomes was scored for asynchronously growing anaphase and telophase mde4Δ and wild-type cells that mildly express Ark1-GFP under leaky expression of the nmt1 promoter in the presence of thiamine. Bars represent the mean +/− SD (n=3; 100 cells each). (D–F)The frequency of lagging or mis-segregated chromosomes was determined for the following asynchronously growing cells: (D) mde4Δ and swi6Δ mutants in the presence or absence of clp1, (E) mde4Δ and swi6Δ mutants with and without low levels of clp1-NLS expression (i.e., leaky expression from the nmt1 promoter in the presence of thiamine), and (F) mde4Δ, mde4Δ clp1Δ and wild-type cells that mildly overexpress cyclin B (pCdc13) using a genomic clone on a multicopy plasmid. Cells were fixed with methanol and DNA was stained with DAPI. Bars represent the mean +/− SD (n=3; 100 cells each). See also Figure S1 and Movie S1.

Sung Hugh Choi, et al. Curr Biol. ;22(3):225-230.

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