Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast

Abstract

Sister chromatid cohesion in meiosis is established by cohesin complexes, including the Rec8 subunit. During meiosis I, sister chromatid cohesion is destroyed along the chromosome arms to release connections of recombined homologous chromosomes (homologues), whereas centromeric cohesion persists until it is finally destroyed at anaphase II. In fission yeast, as in mammals, distinct cohesin complexes are used depending on the chromosomal region; Rec8 forms a complex with Rec11 (equivalent to SA3) mainly along chromosome arms, while Psc3 (equivalent to SA1 and SA2) forms a complex mainly in the vicinity of the centromeres. Here we show that separase activation and resultant Rec8 cleavage are required for meiotic chromosome segregation in fission yeast. A non-cleavable form of Rec8 blocks disjunction of homologues at meiosis I. However, displacing non-cleavable Rec8 restrictively from the chromosome arm by genetically depleting Rec11 alleviated the blockage of homologue segregation, but not of sister segregation. We propose that the segregation of homologues at meiosis I and of sisters at meiosis II requires the cleavage of Rec8 along chromosome arms and at the centromeres, respectively.

Fig. 1. Rec8 degradation and meiotic nuclear division take place depending on separase activity. (A) Cells of a diploid cut2-HA strain (PY959) incubated in MM –N at 30°C for 5 h were fixed with formaldehyde and stained with anti-HA antibody, anti-tubulin antibody and DAPI. At this time, meiotic cells at all stages were present in the mixture. The stage of the meiotic cell cycle was judged by the DAPI and tubulin staining. (B) Cells of a diploid pat1-114/pat1-114 cut1-206/cut1-206 strain (PZ91, right) and its control cut1-206/cut1⁺ strain (PZ90, left) were synchronized in G₁ by culturing in MM –N for 15 h at 25°C. The cells were then incubated with 0.1 g/l NH₄Cl at 34°C to inactivate the Pat1 and Cut1 proteins simultaneously. The number of nuclei was counted by DAPI staining of samples collected every hour. Pictures of the cells at 8 h are shown. (C) Western blot analyses during a meiotic time courses with anti-Rec8 polyclonal antibodies (upper panel) and anti-tubulin antibody (lower panel) are shown. A probable cleavage product was detected at 5 h. Note that the migration of the Rad21/Rec8 subunit of cohesin is slower than expected (Birkenbihl and Subramani, 1995) (calculated molecular weight of Rec8 is 64.0 kDa). Bars, 5 µm.

Fig. 2. Rec8–RDRD is a non-cleavable form of Rec8 and inhibits meiotic nuclear division. (A) Sequence alignment of known and putative cohesin cleavage sites. The arrowhead indicates the cleavage position. Amino acid changes in three Rec8 mutants (RD1, RD2, RDRD) are also shown. (B) Cells of a rec8-RD1-GFP strain (PZ45, top right), a rec8-RD2-GFP strain (PZ50, bottom left), a rec8-RDRD-GFP strain (PZ47, bottom right) and a control rec8⁺-GFP strain (PZ44, top left) were subjected to synchronous meiosis by inactivating pat1-114. Meiotic nuclear divisions monitored by DAPI staining. (C) Western blot analysis during the same time course as (B) was performed with either anti-Rec8 polyclonal antibodies (upper panel) or anti-tubulin antibody TAT-1 (control; lower panel). In all strains except rec8-RDRD, a putative cleavage product was detected at 5–6 h when meiotic nuclear divisions occurred. Asterisks indicate antibody cross-reacting bands. (D) Spore formation and Rec8–GFP fluorescence were simultaneously observed in an h⁹⁰ rec8-RDRD-GFP strain (PY990, bottom) and a control rec8⁺-GFP strain (PY929, top). Bar, 5 µm. (E) Spore viability was measured by random spore analysis in the indicated strains, PY929 (rec8⁺), PY964 (RD1), PZ52 (RD2) and PY990 (RDRD). Four independent examinations were performed.

Fig. 3. Rec8–RDRD is non-cleavable even when wild-type Rec8 has been cleaved in the same cells. (A) Nuclear divisions during synchronous meiosis in PZ86 (pat1-114/pat1-114 rec8⁺-HA/rec8⁺-FLAG) and PZ89 (pat1-114/pat1-114 rec8⁺-HA/rec8-RDRD-FLAG, right) were assayed by DAPI staining. (B) Cells in (A) were subjected to western blot analysis by anti-HA 12CA5 (upper panel), anti-FLAG M2 (middle panel) or anti-tubulin TAT-1 (lower panel) antibody. (C) cut3-GFP signals were observed in the mei4-arrested h⁹⁰ cells of rec8⁺ (PZ137), rec8-RDRD (PZ138) and rec8Δ (PZ116). The percentage of cells containing more than two GFP dots, representing the split of sister cut3 sequences at either or both homologous chromosomes, is shown. Bar, 1 µm.

Fig. 4. Non-cleavable Rec8 completely inhibits the segregation of homologues. (A) Cells of an h⁹⁰ cen1-GFP rec8⁺ strain (PZ94, left), a rec8-RDRD strain (PZ97, middle) and a rec8-RDRD mes1 strain (PZ110, right) were incubated on sporulation-inducing medium SPA at 26.5°C for 1 day. The number of nuclei stained by DAPI was determined. (B) The segregation pattern of chromosome I was determined by observing cen1-GFP signals after completion of meiosis in the rec8⁺ (PZ94, left) and rec8-RDRD (PZ97, right) cells. All of the cen1-GFP dots packed into one nucleus indicate the non-disjunction of all the chromatids at both meiosis I and meiosis II. (C) Meiotic cells of PY929 (rec8⁺) and PY990 (rec8-RDRD) were fixed and stained with anti-tubulin TAT-1 (green) and DAPI (red). Representative cells are shown. Bars, 5 µm.

Fig. 5. Defect of recombination or arm cohesion restores disjunction of homologues, but not sisters, in rec8-RDRD cells. (A) h⁹⁰ cut3-GFP rec8-RDRD cells of either wild-type (PZ134), rec12Δ (PZ146) or rec11Δ (PZ140) background were incubated on sporulation-inducing medium SPA at 26.5°C for 1 day. The nuclear diffusing signal of chromatin-unassociated LacI-GFP-NLS and the dots signal of cut3-GFP were observed, and the segregation patterns of nuclei and cut3-GFP dots were classified. The corresponding cells with cen3-GFP instead of cut3-GFP are shown undergoing meiosis I and meiosis II by staining tubulin and DNA. Bar, 5 µm. (B) Using h⁹⁰ cut3-GFP rec8-RDRD cells as in (A), the percentage of cells containing three or four GFP dots, representing the split of sister cut3 sequences at either or both homologues, was measured. Representative cells are shown at the left. Arrowheads, paired GFP dots; barbed arrowheads, separated GFP dots. (C) h⁹⁰ cut3-GFP rec8-RDRD mes1 cells of either wild-type (PZ136), rec12Δ (PZ148) or rec11Δ (PZ142) were incubated on sporulation-inducing medium SPA at 26.5°C for 1 day. The number nuclei and cut3-GFP segregation pattern were examined. (D) An h⁺ rec8Δ cen2-GFP strain was mixed with an h^– rec8-RDRD strain, both carrying either wild type (PZ167 and PZ170), rec11Δ (PZ169 and PZ172), rec12Δ (PZ168 and PZ171) or rec11Δ psc3-2T (PZ992 and PZ962), and incubated on SPA at 26.5°C for 1 day. As a control, h^– rec8⁺ (PZ166) cells mixed with h⁺ rec8Δ cen2-GFP (PZ170) cells were examined similarly. Cells containing two cen2-GFP dots only in one nucleus (black bar) indicate non-disjunction of sister chromatids.

Fig. 6. During meiotic prophase, cohesion between sister chromatids is established by cohesin Rec8, which forms complexes with Psc3 mainly at the centromeres and with Rec11 mainly along the chromosome arms. Sister chromatid cohesion maintains tension between homologous chromosomes through chiasmata. Separase Cut1 is first activated during meiosis I, and cleaves Rec8 along chromosome arms to segregate homologous chromosomes. Centromeric Rec8 somehow escapes from cleavage by Cut1 during meiosis I, presumably until cleaved by re-activated Cut1 during meiosis II. The expression of non-cleavable Rec8 (RDRD) blocks disjunction of homologous chromosomes during meiosis I. Depletion of Rec8–RDRD from the arm regions by rec11Δ, however, restores disjunction of homologous chromosomes but not of sister chromatids, presumably because centromeric cohesion between sisters is still preserved by Psc3-associating Rec8–RDRD. Thus it is likely that Cut1-dependent cleavage of Rec8 triggers two successive chromosome segregations in meiosis I and II.