Characterization of Pds5 protein level and localization to chromosomes during meiosis. (A) Immunoblot analysis of Pds5 in wild-type (WT) and P_CLB2PDS5 cells during meiosis. Yeast cultures were induced to enter meiosis synchronously. Protein extracts were prepared at the indicated times. Pds5 was detected with a polyclonal anti-Pds5 antibody. β-Tubulin served as a loading control. (B) Meiotic nuclear division in wild-type and P_CLB2PDS5 cells. Cell aliquots were withdrawn at the indicated times, fixed with 4% paraformaldehyde, stained by DAPI, and visualized by fluorescence microscopy. Only minimal nuclear division was observed in P_CLB2PDS5 cells after 12 h of induction. (C) Pds5 and Rec8 colocalization to meiotic chromosomes is shown. Yeast nuclear spreads were prepared from synchronous meiotic cultures. Pds5 and Rec8-3HA were detected with anti-Pds5 and anti-HA antibodies (12CA5), respectively. Representative images from prophase I and anaphase I are shown. (D) ChIP assay of Pds5 and Rec8 binding to cohesin-associated regions at centromere 1 (CEN1), centromere 3 (CEN3), a cohesin site at the MAT locus (CARC3), and a cohesin site on chromosome XII (CARL2). Cells were arrested at pachytene by ndt80Δ after an 8-h induction. ChIP was performed as described previously by Yu and Koshland (2005) with anti-Pds5 and anti-HA antibodies. Note that Rec8 and Pds5 are not enriched at CARL2, which serves as a negative control. Error bars indicate SD.

Pds5 mediates chromosome morphological changes during meiosis. (A) Chromosome morphology at meiotic prophase I. Yeast cells were induced to enter synchronous meiosis for 5 (wild type [WT]) and 7 h (P_CLB2PDS5), and nuclear spreads were prepared for immunofluorescence microscopy. Rec8-3HA and Sgo1-9MYC were detected with anti-HA (12CA5) and anti-MYC (9E10) antibodies, respectively. The chromosome number in P_CLB2PDS5 cells appears to be twice that in wild-type cells. Red, DNA stained by DAPI; green, Rec8-3HA. (B) ChIP assay of Rec8 association with the chromosome. ChIP was performed as in Fig. 1 C. The locations of CEN3 and CARC3 are depicted on chromosome III. S. cerevisiae genome database coordinates are shown on the x axis. Error bars indicate SD. (C) Chromosome axis revealed by Red1 staining. Meiotic nuclear spreads were prepared as in A. Red1 (green) and Rec8-3HA (red) were detected with anti-Red1 and anti-HA antibodies, respectively.

Pds5 is required for homologue pairing and limits chromosome compaction during meiosis. (A) Monitoring homologue pairing and sister chromatid cohesion during meiosis. CEN5 on both chromosome V homologues was marked by tetO/tetR-GFP and visualized by fluorescence microscopy. Wild-type and P_CLB2PDS5 cells were arrested at pachytene with ndt80Δ. Four cell types were observed in P_CLB2PDS5 cells: type I, a single GFP spot, indicating that homologues are paired; type II, two GFP spots, indicating that homologues fail to pair but sister chromatids stay together; type III, three GFP spots, indicating that homologues fail to pair and one pair of sister chromatids separate; and type IV, four GFP spots, indicating that homologues fail to pair and sister chromatid cohesion is lost on both homologues. (B) Quantitative measurement of homologue pairing and sister chromatid cohesion. At least 200 cells were scored for each strain. (C) Measurement of the axial length of chromosome V. Meiotic nuclear spreads were prepared. Chromosome V was identified by CEN5-GFP signal, and the entire length of the chromosome was determined by measurement of the Rec8-3HA staining (detected as in A). (D) The long arm of chromosome IV was marked by GFP at two loci (CEN4 and TEL4) with the lacO/lacI-GFP system. Only one homologue of chromosome IV was marked in these cells. (E) The length of chromosome IV arm was determined by measurement of the distance between two GFP spots. Cells were induced to enter synchronous meiosis, and aliquots were withdrawn at the indicated times for preparation of immunofluorescence. Error bars indicate SD. (F) Representative images from three time points are shown. WT, wild type.

SC formation during meiosis. (A) Immunofluorescence analysis of SC formation in wild-type (WT) and P_CLB2PDS5 cells. Yeast cells were induced to undergo synchronous meiosis, and nuclear spreads were prepared for immunofluorescence microscopy as in Fig. 2 A. Zip1 (green) and Rec8-3HA (red) were detected with anti-Zip1 and anti-HA antibodies, respectively. Note that Zip1 still localizes to chromosomes in P_CLB2PDS5 cells despite the absence of homologue synapsis. (B, left) EM of SC formation in wild-type and P_CLB2PDS5 cells. Yeast nuclear spreads were stained with silver nitrate and visualized by EM. (middle) Magnified views of boxed regions are shown. (top right) A twofold enlargement of regions of interest is shown. (bottom right) Diagrams of sister chromatids are shown in black and gray. Note that short stretches of LEs are formed in P_CLB2PDS5 cells despite the absence of homologue synapsis. (C) Distribution of chromosome axial length from wild type (black bars) and P_CLB2PDS5 (gray bars). Eight cells from each stain were scored. (D) Immunofluorescence analysis of SC formation in haploid yeast cells. Haploids were induced to undergo synchronous meiosis for 8 h and processed as described in A. These haploid strains could enter meiosis because the SIR2 gene had been deleted, resulting in activation of both mating types. Rec8-3HA (red) and Zip1 (green) were detected as described in A. The arrow shows the polycomplex formed by aggregation of SC components.

SC formation depends on SPO11 but SC does not contribute to cohesion in P_CLB2PDS5 cells. (A) SC formation requires Spo11 activity. Yeast cells were induced to undergo synchronous meiosis, and nuclear spreads were prepared for immunofluorescence microscopy as in Fig. 2 A. Zip1 (green) and Rec8-3HA (red) were detected with anti-Zip1 and anti-HA antibodies, respectively. In the absence of Spo11 activity, Zip1 localizes to the polycomplex (PC; arrows). (B) Chromosome morphology in the absence of Zip1. Yeast cells were induced to undergo synchronous meiosis, and nuclear spreads were prepared for immunofluorescence microscopy as in Fig. 2 A. Red, Rec8-3HA; green, Sgo1-9Myc. (C) Sister chromatid cohesion assayed with CEN5-GFP in wild-type (WT), P_CLB2PDS5, zip1Δ, and P_CLB2PDS5 zip1Δ cells. Only one homologue of chromosome V was marked by tetO/tetR-GFP. Yeast cells were induced to undergo synchronous meiosis, aliquots were withdrawn at the indicated times, fixed with 4% formaldehyde, and visualized by fluorescence microscopy. At least 100 cells were counted at each time point.

Pds5 interacts with cohesin in meiotic chromosome morphogenesis. (A–D) Yeast cultures were induced to undergo synchronous meiosis. Protein extracts were prepared at the indicated times for immunoblotting (A) and meiotic nuclear spread for immunofluorescence (B–D). (A) Ectopic production of Mcd1 during meiosis. To induce P_CUP1MCD1 expression, we added 60 µM (final concentration) CuSO₄ to the sporulation medium after induction of meiosis. Mcd1 was detected with a polyclonal anti-Mcd1 antibody. β-Tubulin served as a loading control. WT, wild type. (B) Cohesin is required for Pds5 localization to chromosomes. Pds5 (green) was detected with anti-Pds5 antibody, microtubules (blue) by a monoclonal anti–α-tubulin antibody, and DNA (red) with DAPI. (C) Localization of ectopically expressed Mcd1 during meiosis. Mcd1 bound to meiotic chromosomes, but the SC component Zip1 (green) was present in the polycomplex (PC; arrows). (D) Quantitative analysis of chromosome V compaction in rec8Δ P_CUP1MCD1 and rec8Δ P_CUP1MCD1 P_CLB2PDS5 cells. SD is shown in parentheses.

Pds5 is necessary for meiotic recombination. (A) A physical assay of DSB formation and processing at the YCR047c/YCR048w locus. Yeast cells were induced to undergo synchronous meiosis, and DNA samples were extracted at the indicated time points. DSBs were detected by Southern blotting. Two prominent DSB sites at this location are depicted (DSB1 and DSB2). (B) Quantitative measurement of DSB formation in wild-type (WT) and P_CLB2PDS5 cells. The ratio of the intensity of DSB1 to that of the parental band is shown on the y axis. (C) Rad51 focus formation during meiosis. Yeast cells were induced to enter meiosis as in A. Yeast nuclear spreads were prepared for immunofluorescence. Rad51 was detected with an anti-Rad51 antibody and microtubules with an anti–α-tubulin antibody. Red, DNA stained by DAPI; green, Rad51; blue, microtubules. Bar, 4 µm. (D and E) Quantification of Rad51 foci in cells with aster microtubules and short bipolar spindles is shown. Note that Rad51 foci persist in P_CLB2PDS5 cells with a bipolar spindle. 20 cells from each strain were scored. (F) Cell viability was assayed by return to growth. (E) Yeast cells were induced to enter meiosis, and aliquots were withdrawn at the indicated times and plated on both YPD and Arg minus plates. Colony-forming units are defined as 1 at time 0. (G) Meiotic recombination at the ARG4 locus. Two heteroalleles of ARG4 (arg4-Bgl and arg4-Nsp) are present in the strains assayed. Recombination between the heteroalleles generates a wild-type ARG4 allele, which is detected as an Arg-positive colony. The ratio of colonies formed on Arg minus plates to those on YPD plates determines the recombination rate.