Meiosis I chromosome segregation is established through regulation of microtubule–kinetochore interactions
- Massachusetts Institute of Technology, United States
- University of California, San Francisco, United States
Figures
Figure 1
Characterization of premature cyclin expression and corresponding total CDK activity.
(A) Wild-type (A4962) and GAL4-ER (A19151) cells were induced to sporulate. At the indicated time points, an aliquot was removed and treated with estradiol (1 μM). The percentage of cells that had …
Figure 2 with 8 supplements
Premature expression of CLB1 or CLB3 causes sister kinetochore biorientation during prophase I and sister chromatid segregation in meiosis I.
Wild-type or CUP-CLB cells were induced to sporulate. After 2 hr 15 min, cyclins were induced by addition of CuSO4 (50 μM). Cells were either arrested during prophase I or released from an NDT80 …
Figure 2—figure supplement 1
Spindle pole body separation in CUP-CLB cells.
Wild-type or CUP-CLB cells also carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate. After 2 hr 15 min, cyclins were induced by addition of CuSO4 (50 μM). Cells were arrested during …
Figure 2—figure supplement 2
Homolog separation in CUP-CLB4 cells.
Wild-type (A22688), CUP-CLB4 (A32470) also carrying the GAL4-ER and GAL-NDT80 fusions or cdc20-mn (A15163) cells all carrying homozygous CENV GFP dots were induced to sporulate. After 2 hr 15 min, …
Figure 2—figure supplement 3
Chromosome III sister chromatid segregation in CUP-CLB3 cells.
Wild-type (A18185) and CUP-CLB3 (A22682) cells also carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate. After 3 hr, CLB3 was induced by addition of CuSO4 (50 μM). At 6 hr, cells …
Figure 2—figure supplement 4
Sister chromatid segregation in CUP-CLB3 cells using dual-color marked chromosomes.
Wild-type (A27476) and CUP-CLB3 (A27480) cells carrying the GAL4-ER and GAL-NDT80 fusions and CENV-LacO/LacI-GFP on one homolog of chromosome V (green) and CENV-tetO/tetR-RFP on the other homolog of …
Figure 2—figure supplement 5
Recombination in CUP-CLB3 cells.
Left panel: wild-type (A21104) and GAL-CLB3 (A21105) cells were induced to sporulate and estradiol (1 μM) was added 3 hr after transfer into sporulation medium. Genomic DNA was prepared and digested …
Figure 2—figure supplement 6
Localization of Rad51 in CUP-CLB3 cells.
Wild-type (A22864) and CUP-CLB3 (A22866) cells were induced to sporulate and CuSO4 (50 μM) was added 3 hr after transfer into sporulation medium. Localization of the double-strand break repair …
Figure 2—figure supplement 7
Localization of Zip1 in CUP-CLB3 cells.
Wild-type (A22836) and CUP-CLB3 (A22838) cells were induced to sporulate and CuSO4 (50 μM) was added 3 hr after transfer into sporulation medium. Localization of the synaptonemal complex component …
Figure 2—figure supplement 8
Preventing homologous recombination does not affect the phenotypes caused by premature CLB3 expression.
Wild-type (A19396), GAL-CLB3 (A19400), spo11∆ (A21193) and spo11∆ GAL-CLB3 (A21194) cells were induced to sporulate and estradiol (1 μM) was added 3 hr after transfer into sporulation medium. …
Figure 3 with 3 supplements
Premature CLB3 expression disrupts monopolin function.
(A) Lrs4-13myc (green) localization relative to Ndc10-6HA (red) was determined in spread nuclei from wild-type (A9217), CUP-CLB3 (A26278) and CUP-CLB4 (A29643) harboring a Cdc20 depletion allele (cdc…
Figure 3—figure supplement 1
Monopolin association with kinetochores is disrupted in CUP-CLB3 but not in CUP-CLB4 cells.
Wild-type (A7450), CUP-CLB3 (A28673) and CUP-CLB4 (A28674) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation …
Figure 3—figure supplement 2
Premature Clb3 expression does not interfere with Mam1 expression.
Wild-type (A7450), CUP-CLB3 (A28673) and CUP-CLB4 (A28674) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation …
Figure 3—figure supplement 3
Lrs4 phosphorylation is not disrupted in CUP-CLB4 cells.
Wild-type (A26277) and CUP-CLB4 (A29643) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Levels of Lrs4, …
Figure 4 with 11 supplements
CLB3 misexpression disrupts protection of centromeric cohesin.
Cyclin expression was induced after 2 hr 15 min (C) and (D), 2 hr 30 min (A), (B), (E), (F) and (H) or 3 hr (G) and (I) of sporulation. (A) Chromosomal association of Rec8-13myc was monitored by …
Figure 4—figure supplement 1
Chromosomal association of Rec8 in CUP-CLB3 cells.
Wild-type (A26547) and CUP-CLB3 (A26548) cells were induced to sporulate and CuSO4 (50 μM) was added 3 hr after transfer into sporulation medium. Rec8-3HA localization (red) was determined in spread …
Figure 4—figure supplement 2
Chromosomal association of Pds5 in CUP-CLB3 cells.
Wild-type (A28681) and CUP-CLB3 (A28682) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Pds5 …
Figure 4—figure supplement 3
CUP-CLB3 cells partially bypass the nuclear division delay of mam1∆ cells.
Wild-type (A22678), CUP-CLB3 (A22702), mam1∆ (A31340) and mam1∆ CUP-CLB3 (A31342) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate and CuSO4 (50 μM) was added 2 hr 15 min …
Figure 4—figure supplement 4
Meiotic progression of the cells analyzed for Rec8 cleavage in Figure 4C.
REC8-myc/REC8-HA (A29957), REC8-myc/rec8-29A-HA (A29961), REC8-myc/REC8-HA CUP-CLB3 (A29959) and REC8-myc/rec8-29A-HA CUP-CLB3 (A29963) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced …
Figure 4—figure supplement 5
Analysis of Rec8 cleavage in cells used for Figure 4D.
Wild-type (A22804), CUP-CLB3 (A29965), rec8-29A (A22803) and rec8-29A CUP-CLB3 (A29967) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate and CuSO4 (50 μM) was added 2 hr 15 …
Figure 4—figure supplement 6
Meiotic progression of the cells analyzed for Rec8 cleavage in Figure 4D.
Wild-type (A22804), CUP-CLB3 (A29965), rec8-29A (A22803) and rec8-29A CUP-CLB3 (A29967) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate and CuSO4 (50 μM) was added 2 hr 15 …
Figure 4—figure supplement 7
Chromosomal association of Sgo1 in CUP-CLB3 cells.
Wild-type (A28712) and CUP-CLB3 (A28713) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Sgo1-3V5 …
Figure 4—figure supplement 8
Localization of Rts1 in CUP-CLB3 cells.
Wild-type (A28331) and CUP-CLB3 (A28332) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Rts1-13myc …
Figure 4—figure supplement 9
Chromosomal association of Spo13 in CUP-CLB3 cells.
(Left panel) wild-type (A30856), CUP-CLB3 (A30858) and CUP-CLB4 (A30860) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate and CuSO4 (50 μM) was added 2 hr 15 min after …
Figure 4—figure supplement 10
Rts1 localization in binucleate CUP-CLB3 cells.
Wild-type (A28329) and CUP-CLB3 (A28330) cells were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Rts1-13myc (green) localization relative to …
Figure 4—figure supplement 11
Analysis of Rts1 localization in Rec8 phosphomimetic mutants.
Wild-type (A29645) and rec8-S136D S179D S197D T209D (A29647) cells were induced to sporulate and Rec8-3HA/rec8-4D-3HA or Rts1-3V5 localization relative to Ndc10-13myc was determined in spread nuclei …
Figure 5 with 9 supplements
Transient disruption of microtubule–kinetochore interactions suppresses the chromosome segregation defects in CUP-CLB3 cells.
(A) Wild-type (A10684) and GAL-CDC5 GAL-MAM1 (A26546) cells, carrying a MET-CDC20 allele and CENIV-GFP dots, were monitored for chromosome segregation in anaphase (see ‘Materials and methods’ for …
Figure 5—figure supplement 1
Sporulation efficiency of ndc80-1 mutants.
Wild-type (A22678) and ndc80-1 (A28221) cells were induced to sporulate at 25°C. 2 hr 30 min after transfer into sporulation medium, cells were shifted to the indicated temperature and sporulation …
Figure 5—figure supplement 2
Sister kinetochore coorientation in ndc80-1 cells under a continuous inactivation regime at 34°C during a metaphase I arrest.
Wild-type (A7118), CUP-CLB3 (A23074), ndc80-1 (A29690) and ndc80-1 CUP-CLB3 (A29692) cells also carrying the cdc20-mn allele, were induced to sporulate at 25°C. 2 hr 45 min after transfer into …
Figure 5—figure supplement 3
Sister kinetochore coorientation in ndc80-1 cells after transient inactivation regime at 34°C during a metaphase I arrest.
Wild-type (A20958), CUP-CLB3 (A23076), ndc80-1 (A29718) and ndc80-1 CUP-CLB3 (A29720) cells also carrying the GAL4-ER and GAL-NDT80 fusions and the cdc20-mn allele were induced to sporulate at 25°C. …
Figure 5—figure supplement 4
Meiosis I chromosome segregation in ndc80-1 cells after a transient inactivation regime at 34°C.
Wild-type (A22678), ndc80-1 (A28621), CUP-CLB3 (A22702) and CUP-CLB3 ndc80-1 (A28623) cells also carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate at 25°C. 2 hr 45 min after …
Figure 5—figure supplement 5
Transient disruption of microtubule–kinetochore interactions using dam1-1 allele restores meiosis I chromosome segregation in CUP-CLB3 cells.
Wild-type (A22678), dam1-1 (A28311), CUP-CLB3 (A22702) and CUP-CLB3 dam1-1 (A28341) cells also carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate at 25°C. 2 hr 45 min after …
Figure 5—figure supplement 6
Transient disruption of microtubule–kinetochore interactions by benomyl treatment restores meiosis I chromosome segregation in CUP-CLB3 cells.
Wild-type (A22678) and CUP-CLB3 (A22702) cells also carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate at 30°C. 2 hr 15 min after transfer into sporulation medium CuSO4 (50 μM) was …
Figure 5—figure supplement 7
Transient disruption of microtubule–kinetochore interactions during S phase/prophase I suppresses CUP-CLB3-induced meiosis I sister chromatid segregation in a spindle assembly checkpoint independent manner.
(Top panel) wild-type (A22678), mad3∆ (A30386), ndc80-1 (A28621), ndc80-1 mad3∆ (A30390), CUP-CLB3 (A22702), CUP-CLB3 mad3∆ (A30388), CUP-CLB3 ndc80-1 (A28623) and CUP-CLB3 ndc80-1 mad3∆ (A30392) …
Figure 5—figure supplement 8
Transient disruption of microtubule–kinetochore interactions during S phase/prophase I restores meiotic chromosome segregation in CUP-CLB3 cells in a spindle assembly checkpoint independent manner.
Wild-type (A22688), ndc80-1 (A28625), CUP-CLB3 (A22708), CUP-CLB3 ndc80-1 (A28627), mad3∆ (A30638), ndc80-1 mad3∆ (A30642), CUP-CLB3 mad3∆ (A30640) and CUP-CLB3 ndc80-1 mad3∆ (A30644) cells also …
Figure 5—figure supplement 9
Transient ndc80-1 inactivation does not alter in vitro Cdk1 activity.
Wild-type (A25508), ndc80-1 (A33203), CUP-CLB3 (A33201) and CUP-CLB3 ndc80-1 (A33205) cells also carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate at 25°C. 2 hr 45 min after …
Figure 6 with 11 supplements
Meiosis I sister chromatid segregation correlates with presence of outer kinetochore components.
(A) Schematic description of the experimental regime and segregation of sister chromatids using heterozygous CENV-GFP dots quantified in binucleates from wild-type (A22678) and CUP-CLB3 (A29406) …
Figure 6—figure supplement 1
Schematic representation of the kinetochore–microtubule interface.
Components of the kinetochore subcomplexes are grouped in color coded boxes. Note that the schematic representation is not drawn to scale.
Figure 6—figure supplement 2
Meiotic cluster analysis of kinetochore components.
Cluster analysis of kinetochore components from the indicated time points. Further details are in ‘Materials and methods’ and in Brar et al. (2012).
Figure 6—figure supplement 3
Meiotic expression of DASH complex subunits.
Ordered plot of mRNA-seq and ribosome footprinting data for the DASH complex components at indicated stages of sporulation. Dotted line indicates time of release from NDT80 block. See Brar et al. …
Figure 6—figure supplement 4
Meiotic expression of Ndc80 complex subunits.
Ordered plot of mRNA-seq and ribosome footprinting data for the Ndc80 complex at indicated stages. Dotted line indicates time of release from NDT80 block. See Brar et al. (2012) for details.
Figure 6—figure supplement 5
Meiotic expression of Mif2.
Ordered plot of the mRNA-seq and ribosome footprinting data for Mif2 at the indicated stages. Dotted line indicates time of release from the NDT80 block. See Brar et al. (2012) for details.
Figure 6—figure supplement 6
Meiotic expression of KNL-1 complex subunits.
Ordered plot of the mRNA-seq and ribosome footprinting data for KNL-1 complex subunits (Spc105 complex) at the indicated stages. Dotted line indicates time of release from the NDT80 block. See Brar …
Figure 6—figure supplement 7
Meiotic expression of Mis12 complex subunits.
Ordered plot of the mRNA-seq and ribosome footprinting data for Mis12 complex subunits at the indicated stages. Dotted line indicates time of release from NDT80 block. See Brar et al. (2012) for …
Figure 6—figure supplement 8
Meiotic expression of Ctf19 complex subunits.
Ordered plot for mRNA-seq and ribosome footprinting data for Ctf19 complex at indicated stages. Dotted line indicates time of release from the NDT80 block. See Brar et al. (2012) for details.
Figure 6—figure supplement 9
Meiotic expression of Cbf3 complex subunits.
Ordered plot of the mRNA-seq and ribosome footprinting data for Cbf3 complex subunits at the indicated stages. Dotted line indicates time of release from the NDT80 block. See Brar et al. (2012) for …
Figure 6—figure supplement 10
Meiotic expression of Histone subunits.
Ordered plot for mRNA-seq and ribosome footprinting data for the histones at indicated stages. Dotted line indicates time of release from NDT80 block See (Brar et al., 2012) for details.
Figure 6—figure supplement 11
Meiotic expression of Dam1 and Ask1.
DAM1-3V5 (A28898) cells carrying the GAL4-ER and GAL-NDT80 fusion and ASK1-13myc (A29161) carrying ndt80∆ were induced to sporulate and were either released from (left panel) or arrested in (right …
Figure 7
Characterization of NDC80 and HSK3 overexpression.
(A) CUP-NDC80-3V5 (A30342) and CUP-HSK3-3HA (A32060) cells also carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate. After 2 hr 30 min CuSO4 (50 μM) was added and cells were …
Figure 8
Expression of NDC80 and HSK3 in prophase I enhances Clb3-CDK-induced meiosis I sister chromatid segregation.
For (A)–(E), wild-type (A31945), CUP-CLB3 (A31947), CUP-NDC80 CUP-HSK3 (A31951), CUP-NDC80 CUP-CLB3 (A31949), CUP-NDC80 CUP-HSK3 CUP-CLB3 (A31953) and cdc20-mn (A31955) cells were induced to …
Figure 9
CUP-CLB3 CUP-NDC80 CUP-HSK3-induced meiosis I sister chromatid segregation is independent of the length of the prophase I arrest.
(A) Schematic description of the experimental regime used in (B) and (C). (B), (C) Wild-type (A22678), CUP-CLB3 (A22702) and CUP-CLB3 CUP-NDC80 CUP-HSK3 (A31857) cells also carrying the GAL4-ER and G…
Figure 10
Model for temporal regulation of microtubule–kinetochore interactions during meiosis.
(A) As prophase I progresses, the propensity of sister chromatids to biorient decreases and the ability to coorient sister chromatids increases. (B) Top panel: inhibiting Clb-CDK activity and outer …
Additional files
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Supplementary file 1
Strains used in this study.
- https://doi.org/10.7554/eLife.00117.055
