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. 1999 Jun 14;145(6):1233-49.
doi: 10.1083/jcb.145.6.1233.

A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast

A Yamamoto  1 , R R West, J R McIntosh, Y Hiraoka
Affiliations

A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast

A Yamamoto et al. J Cell Biol. .

Abstract

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.

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Figures

Figure 1
Figure 1
Cloning and disruption of dhc gene. (A) Restriction map and cloned genomic DNA fragments of dhc1+. Arrow, position of the ORF for Dhc1p; white boxes on the arrow, sites corresponding to four P-loops; shaded bar, the original 2.7-kb genomic DNA clone isolated from genomic DNA libraries; solid bars, clones obtained by recovering plasmids from the genome that had been integrated at the dhc1 locus. Names of clones are shown on the right. B, BamHI; C, ClaI; EV, EcoRV; Sc, SacI; Sl, SalI; Sm, SmaI; Xb, XbaI; Xh, XhoI. (B) A diagram of the disruption schemes used for the dhc1+ gene. Solid arrows, ORF; dotted lines, overlapping regions; open boxes, integrated markers; thin lines, plasmid DNA. Allele names of each disruption are shown on the left. Sp, SphI; RI, EcoRI.
Figure 9
Figure 9
A chromosome map of genetic markers and hybridization sites for DNA probes. Bars and open circles indicate three chromosomes and centromeres, respectively. The positions of genetic markers and hybridization sites for DNA probes are approximately indicated according to the maps presented by Kohli et al. (1977), Gygax and Thuriaux (1984), Hoheishel et al. (1993), and Mizukami et al. (1993). Open rectangles indicate hybridization sites for DNA probes. 146, 212, 256, 1228, and 0727 represent cosmids, cos146, cos212, cos256, cos1228, and ICRFc60D0727, respectively, used for DNA probes. rDNA indicates chromosomal regions encoding ribosomal RNA genes. A thin line at the bottom right indicates an approximate chromosome length corresponding to 1 Mb of genomic DNA.
Figure 2
Figure 2
Deduced amino acid sequence of the dhc1+ gene and its comparison to other dhc genes. (A) Deduced amino acid sequence of Dhc1p. The four sites corresponding to the P-loops of other DHCs are shown by boxes. (B) A dot-plot matrix comparing the predicted DHC of fission yeast with that of A. nidulans with a stringency of 7 in a window of 23. Numbers indicate amino acid numbers. (C) Sequence comparison of the four regions containing putative ATP-binding sites from several different organisms. The ATP-binding motifs are shown by shadowed letters (GXXXXGKS/T). Dots and hyphens indicate identical amino acids and spaces, respectively. GenBank/EMBL/DDBJ accession numbers of the heavy chain sequences of A. nidulans, Drosophila melanogaster, and S. cerevisiae are U03904, L23195, and L15626, respectively.
Figure 3
Figure 3
Mitotic cell-cycle progression and spindle behavior of wild-type and the dynein mutant. (A) Cell-cycle progression of wild-type and the mutant cells synchronized by hydroxyurea. Wild-type (upper graph) and the dynein mutant (lower graph) cells grown in YEA medium were arrested in S phase with hydroxyurea (Materials and Methods), and synchronously released from the arrest by removing the drug from the medium. After release from arrest, a portion of the cells were fixed at intervals and analyzed for cell types. More than 100 cells were examined at each time point. Percentage of cells containing one nucleus (closed diamonds) and those containing two nuclei, with (closed triangles) or without (open squares) a septum are shown. (B) Time-lapse series of wild-type (left) and dhc1-d2 (right) cells expressing GFP-tagged α-tubulin on a multicopy plasmid. Wild-type (strain CRL152) and mutant (strain CRL1521) cells bearing a GFP-tubulin plasmid, pDQ105 (Ding et al., 1998), were grown in EMM medium supplemented with appropriate amino acids for growth and 5 μM thiamine for the low-level expression of GFP-tubulin, and examined for spindle dynamics at 30°C. Representative series from wild-type and mutant cells are shown. Numbers indicate time in minutes.
Figure 4
Figure 4
Chromosome dynamics in the dynein mutant cells during meiosis. Chromosomal DNA of conjugated cells was monitored by staining with Hoechst 33342 (Materials and Methods). (A) Chromosome behavior of wild-type (strain CRL152) and the dhc1-d2 (strain CRL1521) mutant in single-nuclear zygotes. The left and right columns are time-lapse series of a single wild-type or of the dynein mutant cell, respectively. The numbers indicate time in minutes. (B) Sequential observation of chromosomal DNA in the dhc1-d2 (strain CRL1521) cell during meiosis. The dynamics of chromosomal DNA in a single cell was followed by time-lapse observation immediately after nuclear fusion. The numbers indicate time in minutes.
Figure 10
Figure 10
FISH analysis of centromere and telomere position in wild-type and the dynein mutant cells. (A) Centromere and telomere position in wild-type cells. Wild-type (strain CRL152) cells were induced into meiosis and processed for FISH using DNA probes against centromeric repetitive DNA sequences (a, d, and g) and a ribosomal DNA sequence, which is located near the telomeres of chromosome III (b, e, and h). Chromosomal DNA was stained with DAPI (c, f, and i). Single-nuclear zygotes are shown. (B) Centromere and telomere position in the dynein mutant cells. The dhc1-d2 mutant (strain CRL1521) cells were treated as in A. A two-nuclear zygote in karyogamy (a–c) and single-nuclear zygotes (d–l) are shown. a, d, g, and j, centromere staining; b, e, h, and k, ribosomal DNA staining; c, f, i, and l, DNA staining. (C) Simultaneous staining of the SPB and telomeres in the dynein mutant cells. The single-nuclear zygotes of the dynein mutant were processed for indirect immunofluorescence staining of the SPB using anti–γ-tubulin antibody, followed by FISH staining using DNA probes against telomere-associated sequences on both ends of chromosome I and II. Chromosomal DNA was stained with DAPI. Staining is shown in merged images: red, γ-tubulin; green, telomere; and blue, DNA staining.
Figure 5
Figure 5
Time course changes in a number of different cell types of azygotic diploid cells after nitrogen starvation. Diploid wild-type (strain AY162) and dhc1-d2 (strain AY163) cells were synchronously induced into meiosis by depleting nitrogen from the growth medium and examined at the times indicated. More than 100 cells were examined at each time point. Upper and lower graphs indicate time course of changes in cell types of wild-type and mutant cells, respectively. Closed squares, open squares, open circles, and closed circles indicate percentages of cells containing one round nucleus, two nuclei, deformed nucleus, and four nuclei, respectively.
Figure 6
Figure 6
Spore formation efficiency of wild-type and dhc1 mutant cells. Wild-type (strain CRL152) and dhc1 mutant (strains CRL1521 and CRL1522) cells were sporulated on the ME solid medium and scored for spore numbers in zygotic cells. More than 100 zygotes were examined for each strain. Bars indicate the population of cells that formed different numbers of spores.
Figure 7
Figure 7
Microtubule morphology in dhc1-d2 mutant during karyogamy and meiosis. dhc1-d2 (strain CRL1521) cells expressing GFP-tagged α-tubulin were induced into meiosis in the presence of 5 μM thiamine to repress the expression of the tubulin-GFP to low levels, as described by Ding et al. (1998). Chromosomal DNA was stained with a DNA specific dye, Hoechst 33342. A two-nuclear zygote in karyogamy (a and b), a single-nuclear zygote before meiotic division (c–f), and two-nuclear zygotes in first (g and h) and second (i and j) meiotic divisions are shown. a, c, e, g, and i, α-tubulin GFP; and b, d, f, h, and j, DNA signal of Hoechst 33342.
Figure 8
Figure 8
Localization of GFP-tagged dynein during karyogamy and meiosis. (A) Localization of dynein-GFP in living cells. Homothallic cells bearing the dynein-GFP fusion gene (strain CRL1526) were induced into meiosis and observed for localization of dynein-GFP. Chromosomal DNA was stained with Hoechst 33342. Cells undergoing karyogamy (a and b), the oscillatory nuclear movement (c–f), first meiotic division (g and h), and second meiotic division (i and j) are shown. a, c, e, g, and i, DNA staining; b, d, f, h, and j, GFP signal. The large arrows indicate the GFP dot at the leading edge of the moving nucleus where the SPB is expected. The small arrows indicate a GFP dot at a point where a GFP line contacts the cell cortex. (B) Time-lapse series of meiotic cells expressing dynein-GFP. Two zygotic cells undergoing the oscillatory nuclear movement are shown. Red and green indicate DNA staining and GFP signal, respectively. Numbers indicate time in seconds. Large and small arrowheads indicate the GFP dots at the leading edge of the moving nucleus and at the point where a GFP line contacts the cell cortex, respectively. (C) Immunofluorescence staining of dynein-GFP and microtubules in fixed cells. Cells induced to meiosis, as in A, were fixed and stained using antibodies against GFP (a and e) and α-tubulin (b and f). They were also stained with DAPI (c and g). Merged images are shown in d and h. Yellow/green, GFP; red, tubulin; and blue, DNA staining.
Figure 11
Figure 11
FISH analysis of nuclear position of chromosome-arm regions in wild-type and the dynein mutant cells. (A) Position of chromosomal regions in single-nuclear zygotes, to which cos146 DNA probe hybridized. a–f, wild-type (strain CRL152) cells; g–l, dhc1-d2 (strain CRL1521) cells; a, d, g, and j, FISH signals; b, e, h, and k, DNA staining; c, f, i, and l, merged images of FISH signals and DNA staining. (B) Position of chromosomal regions in single-nuclear zygotes, to which cos1228 DNA probe hybridized. a–f, wild-type (strain CRL152) cells; g–l, dhc1-d2 (strain CRL1521) cells; a, d, g, and j, FISH signals; b, e, h, and k, DNA staining; c, f, i, and l, merged images of FISH signals and DNA staining.
Figure 12
Figure 12
Model for a role of the oscillatory nuclear movement in homologous chromosome pairing. Behaviors of two pairs of homologous chromosomes (solid and shaded lines), the SPB (closed circles) and centromeres (open circles), before meiotic chromosome segregation are shown. During karyogamy, chromosomes are positioned opposite to each other, with telomeres at the leading edges of the approaching nuclei (a and b). The subsequent SPB-led nuclear movement changes the telomere-centromere orientation of a homologous set of chromosomes from an antiparallel to a parallel relationship, thus placing homologous regions of homologous chromosomes in proximity (c). The centromeres fall apart transiently before homologous centromeres pair (d), and then, pairing of centromeres results in the formation of a single centromere cluster, which may also facilitate pairing of other homologous regions (e). In the dynein mutant, homologous chromosomes fail to align properly due to the lack of the nuclear movement (f) and centromeres disperse as a consequence of inefficient pairing of centromeres (g). The small arrows indicate direction of chromosome movement.
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