Monopolar spindle attachment of sister chromatids is ensured by two distinct mechanisms at the first meiotic division in fission yeast

Abstract

At meiosis I, sister chromatids attach to the same spindle pole (i.e. monopolar attachment). Mechanisms establishing monopolar attachment remain largely unknown. In the fission yeast Schizosaccharomyces pombe, monopolar attachment is established in haploid cells, indicating that homologous chromosomes are dispensable for its establishment. This monopolar attachment requires both mating pheromone signaling and inactivation of Pat1 kinase (a key negative regulator of meiosis). It also requires the meiotic cohesin factor Rec8 but not the recombination factor Rec12. In contrast, in diploid cells, monopolar attachment is established by Pat1 inactivation alone, and does not require mating pheromone signaling. Furthermore, monopolar attachment requires Rec12 in addition to Rec8. These results indicate that monopolar attachment of sister chromatids can be established by two distinct mechanisms in S.pombe, one that is pheromone dependent and recombination independent, and a second that is pheromone independent and recombination dependent. We propose that co-operation of these two mechanisms generates the high fidelity of monopolar attachment.

Fig. 1. Haploid meiosis induced by the mat genes. (A) Changes in nuclear morphology (a) and DNA content (b) of haploid cells containing transcriptionally active mating type genes of the P and M types (strain AY1931) after nitrogen starvation. (B) Microtubule and nuclear morphology (a) and behavior of the single spindle (b) in haploid meiotic cells (strain CRL4221). Microtubules were visualized by GFP-tagged α-tubulin (see Materials and methods): (a) microtubules (green) and chromosomal DNA (red). (b) photos were taken every 2.9 min. Numbers indicate time in minutes. (C) Chromosomal morphology and visualized chromosomal loci at the first (a–f) or second (g and h) division: (a) and (d): chromosomal DNA stained with DAPI; (b) and (e) the visualized chromosomal loci; (c) and (f)–(h) merged images of chromosomal DNA (red) and visualized chromosomal locus (green). Arrows indicate three individual chromosomes. (D) Frequencies of co-segregated sister loci. P and M are the mating type genes at the mat1 locus or at the ectopic locus. N, number of cells examined. Strains used for analyses were as follows: 1, AY1391; 2, AY1931; 3, AY2082; 4, AY2072.

Fig. 2. Haploid meiosis induced by Pat1 inactivation. (A) Changes in nuclear morphology (a) and DNA content (b) of pat1 haploid cells (strain AY191-6C) after a temperature shift. (B) Microtubule and nuclear morphology (a) and behavior of the single spindle (b) in pat1 haploid cells (strain CRL246 or CRL298): (a) microtubules (green) and chromosomal DNA (red); (b) photos were taken every 2 min. Numbers indicate time in minutes. (C) Chromosomal morphology and the visualized chromosomal loci at the first (a–f) or second (g and h) division; (a) and (d) chromosomal DNA stained with DAPI; (b) and (e) visualized chromosomal loci; (c) and (f)–(h) merged images of chromosomal DNA (red) and visualized chromosomal locus (green). (D) Frequencies of co-segregated various sister loci. Strains used for analyses were as follows: 1, CRL245; 2, AY191-6C.

Fig. 3. Effect of i-type or c-type mat genes or mating pheromone signaling on pat1-induced haploid meiosis. Meiosis induced in haploid pat1 cells containing transcriptionally active i-type (A) or c-type (B) mat genes of both types (strain CRL2411 or CRL2412). Changes in nuclear morphology (a) and DNA content (b) after a temperature shift. rDNA locus (green) and chromosomal DNA (red) after the first division (c). (C) Sister locus behavior at meiosis I in haploid pat1 cells in the presence (d–f) or absence (a–c) of mating pheromone response: (a and d) chromosomal DNA stained by DAPI; (b and e) the GFP-visualized lys1⁺ locus; (c and f) merged images of chromosomal DNA (red) and visualized chromosomal locus (green). (D) Frequencies of co-segregated sister loci: none, no ectopic mat gene; M(i+c), mat-Mi and mat-Mc; P(i+c), mat-Pi and mat-Pc. Strains used for analyses were as follows: 1, CRL2411; 2, CRL2412; 3, AY1396; 4, AY1395; 5, AY1902; 6, AY1901; 7 and 8, AY195-1C.

Fig. 4. Roles of Rec8 and Rec12 at meiosis I in haploid cells. (A) Sister locus behavior in cells lacking Rec8 (a, strain AY2081) or Rec12 (b, strain AY2071). Red and green indicate chromosomal DNA and GFP-visualized locus, respectively. (B) Frequencies of co-segregated sister loci. Strains used for analyses were as follows: 1, AY2081; 2, AY2071.

Fig. 5. Rec8 behavior in haploid meiosis. (A) Localization of GFP-tagged Rec8 in haploid cells undergoing meiosis. Left and right panels show mat gene-induced meiosis (strain AY2211) and pat1-induced meiosis (strain AY221-8A), respectively: (a) before nitrogen starvation (left panels) or shifting temperature (right panels); (b) meiotic prophase; (c) and (d) meiosis I; (e) after meiosis II. (B) Colocalization of Rec8–GFP (green) with Mis12–HA (red) in mitotically growing cells (strain AY311–19C). Chromosomal DNA is shown in white. (C) Live observation of Rec8–GFP at meiosis I in haploid pat1 cells (strain AY221-8A). Numbers indicate time in minutes. Red and green indicate chromosomal DNA and Rec8–GFP, respectively. (D) Chromosome spread of haploid meiotic cells: (a) mat gene-induced meiosis (strain AY2211); (b) pat1-induced meiosis (strain AY221-8A). (E) Changes in Rec8–GFP expression and localization during haploid meiosis: (a–c) mat gene-induced meiosis (strain AY2211); (d–f) pat1-induced meiosis (strain AY309-9C). Numbers indicate times after nitrogen starvation or a temperature shift. (a and d) DNA contents of the cells analyzed by FACS; (b and e) transcripts of Rec8–GFP (upper rows) detected by northern blotting. Cam1 was used as an internal control (lower rows); (c and f), Rec8–GFP localization (upper panels) and chromosomal DNA morphology (lower panels).

Fig. 6. Diploid meiosis induced by Pat1 inactivation. (A) Changes in nuclear morphology (a) and DNA content (b) of diploid pat1 cells (strain AY167d) after a temperature shift. (B) Behavior of the sister loci at the first (a) or second (b) division (strain AY167d). The lys1⁺ locus on one of the homologous chromosomes is visualized. (C) Behavior of the chromosomal loci on both homologous chromosomes at meiosis I (strain AY190d). The lys1⁺ loci on both homologous chromosomes are visualized. Photos indicate a cell showing 2:2 (a) or 3:1 (b) segregation patterns. Numbers indicate the populations of the cells. (D) Behavior of the sister loci at meiosis I in diploid pat1 cells lacking Rec8 (strain 210d, a) or Rec12 (strain AY2001, b). (E) Behavior of the sister loci at meiosis I in a diploid pat1 cell expressing both mat-Pc and mat-Mc (strain AY2463d). Red and green, respectively, indicate chromosomal DNA and the GFP-visualized chromosomal locus. (F) The populations of co-segregated sister loci. Closed or open bars indicate the populations of co-segregated lys1⁺ or ade6⁺ sister loci, respectively. The type of meiosis; meiosis induced in diploid wild-type cells (Wt meiosis) or diploid pat1 cells homozygous for the mat1 locus (pat1 meiosis). Other factors: a mutation or an ectopic mat1 gene. Strains used for analyses were as follows: 1, AY167-1D × CRL173; 2, AY241-11C × CRL173; 3, AY167d; 4, AY2101; 5, AY215d; 6, AY2001; 7, AY210d; 8, AY2463d.