Article Figures & Data
Figures
- Figure 1. Cdc53 depletion induces abnormal synaptonemal complex (SC).
(A) Expression of Cdc53. Lysates obtained from wild-type (NKY1551, only at 0 h) and CDC53mn (ZHY94) cells at various time points during meiosis were analyzed by Western blotting using anti-HA (HA-Cdc53, upper) or anti-tubulin (lower) antibodies. “X” indicates a non-specific band reacted with anti-HA. (B) Meiotic cell cycle progression. The entry into meiosis I and II in wild-type and CDC53mn cells were analyzed by DAPI staining. The number of DAPI bodies in a cell was counted. A cell with 2, 3, and 4, and 3 and 4 DAPI bodies was defined as a cell that passed through meiosis I and meiosis II, respectively. The graph shows the percentages of cells that completed MI or MII at the indicated time points. More than 200 cells were counted at each time point. The representative results (n = 3) are shown; see the same results shown in Figs 3A and 6A. Wild type, MI, blue closed circles; wild type, MII, blue open circles; CDC53mn MI, red closed circles; CDC53mn MII, red open circles. (C) Tubulin staining in the CDC53mn mutant. Whole wild-type (5 h) and CDC53mn cells (8 h) were fixed and stained with anti-tubulin (green) and DAPI (blue). Representative images are shown. Bar = 2 μm. (D) Classification of tubulin/DAPI staining at each time point of meiosis in wild-type (left) and CDC53mn mutant (right) cells. Dot, short line, and long line tubulin-staining with single DAPI mass were defined as prophase I, metaphase I, and anaphase I, respectively, and are shown in different colors. Short and long tubulin staining were defined as metaphase II and anaphase II, respectively. At each time point, more than 100 cells were counted. The representative results are shown (n = 2). (E) Zip1 and Rad51 staining. Nuclear spreads from wild-type and CDC53mn mutant cells were stained with anti-Zip1 (green), anti-Rad51 (red), and DAPI (blue), and then categorized into different cell cycle stages. SCs of wild-type cells are shown in leptotene (Class I), zygotene (Class II), and pachytene (Class III) stages. Class II and II contained less than 10 and more than 10 Zip1 lines, respectively. SCs of CDC53mn mutants are shown in leptotene and zygotene-like stages (E). Representative images are shown. Zygotene-like CDC53mn cells contain polycomplexes (PCs), as shown by an arrow. Bars = 1 μm. (F) Plots show each class of SC (wild type, left; CDC53mn mutant, right) at the indicated time points during meiosis. Class I (dots; blue bars), Zip1 dots; Class II (short lines; green bars), partial Zip1 linear; Class III (long lines; red bars), linear Zip1 staining. At each time point, more than 100 cells were counted. The representative results (n = 3) are shown; see the same results shown in Figs 3H and 5C. (G) The kinetics of Zip1 PC formation is shown for each strain. Spreads with Zip1 PC were counted. Wild type, blue; CDC53mn, red. Representative results are shown (n = 3); see the same results shown in Fig 5D. (H) Kinetics of Rad51 assembly/disassembly. The number of Rad51-positive cells (with more than five foci) was counted at each time point. At each time point, more than 100 cells were counted. Representative results are shown (n = 3); see the same results shown in Figs 2J, 3D, and 5D. Wild type, blue; CDC53mn, red.
Source data are available for this figure.
Source Data for Figure 1[LSA-2020-00933_SdataF1.xlsx]
- Figure S1. CDC53mn mutant shows meiosis I arrest.
(A) The lysates from the meiotic cells with CDC53-3XFLAG gene (ZHY183) were analyzed by Western blotting using anti-Flag (upper) and anti-tubulin (lower) antibodies. The representative results are shown (n = 2). (B) Meiotic DNA replication in CDC53mn. DNA contents of wild-type and CDC53mn cells were examined at various time points (right) by FACS analysis. The representative results are shown (n = 2). The FACS profile of CDC53mn is shown in Fig S4A. (C) The progression of meiosis I. Wild-type (NKY1551, blue), CDC53mn (ZHY94, red), spo11-Y135F (MSY1737, purple), and spo11-Y135F CDC53mn (ZHY272, green) strains were analyzed by DAPI staining. The representative results are shown (n = 2). (D) The expression of various proteins in the CDC53mn mutant. Lysates obtained from wild-type and CDC53mn single cells during meiosis were analyzed by Western blotting using anti-Sic1 (upper), anti-Cdc6 (second), anti-Cdc5 (polo-like kinase; third), or anti-tubulin (lower) antibodies. The representative results are shown (n = 2). (E) CHEF gel analysis of double-strand break repair during meiosis. Chromosomal DNAs from wild-type (NKY1551) and CDC53mn cells (ZHY94) (left panel), and pch2 (ZHY350) and pch2 CDC53mn double mutant (ZHY351) (right panel) cells were separated by CHEF, and gels were stained with ethidium bromide. The position of fragmented chromosomes (50,000–800,000) is shown as a line and dotted line on the right size. Representative results are shown (n = 3).
- Figure 2. Cdc53-depletion mutant is proficient in meiotic recombination.
(A) A schematic diagram of the HIS4-LEU2 recombination hotspot. Restriction sites for PstI, XhoI, BamHI, and MluI are shown. Diagnostic fragments for analysis on double-strand break (DSB) and crossover (CO) are shown at the bottom. The sizes of each fragment (kilo-bases) are presented within parentheses. (B, C) DSB repair at the HIS4-LEU2 locus was analyzed by Southern blotting (B) and quantified (C). Genomic DNA was prepared and digested with PstI. Error bars (SD) are based on three independent experiments. Wild type, NKY1551, blue circles; CDC53mn, ZHY94, red circles. (D, E) CO formation at the HIS4-LEU2 locus was analyzed by Southern blotting (D) and quantified (E). Ratios of the R1 band to P1 were calculated. Genomic DNA was digested with XhoI. Error bars (SD) are based on three independent time courses. (F) A schematic diagram of the URA3-ARG4 recombination hotspot. Restriction sites for XhoI and EcoRI are shown. Diagnostic fragments for analysis of parent, DSB, and crossover (CO) fragments are shown at the bottom. The sizes of each fragment (kilo-bases) are presented within parentheses. (G, H) CO formation in the CDC53mn mutant was verified by Southern blotting. (G, H) Ectopic CO formation at the URA3-ARG4 recombination locus was analyzed by Southern blotting (G) and quantified (H). Genomic DNA was digested with XhoI. Error bars (SD) are based on three independent cultures. Wild type, MJL2442, blue circles; CDC53mn, ASY1202, red circles. (I) The localization of Zip3 and Msh5 in the CDC53mn mutant. Chromosome spreads from wild type (4 h, NKY1551) and CDC53mn mutant (8 h, ZHY94) were stained with anti-Zip3 or anti-Msh5 antibodies (green) together with anti-Zip1 (red). The representative images are shown. Bar = 1 μm. (J) The assembly of Msh4-Msh5 in the CDC53mn mutant. The percentages of cells positive for Msh4, Msh5, or Rad51 foci (more than five foci per nucleus) were counted at each time point. At least 100 nuclei were counted at each time point. Wild type, blue triangles and circles; CDC53mn mutant, red triangles and circles. Triangles and circles are for Rad51 and Msh4 (right) or Msh5 (left), respectively. The representative results are shown (n = 2). (K) The number of Msh5 foci per spread was counted at 4 h in the wild type (NKY1551) and at 6 h in the CDC53mn mutant (ZHY94). Twenty spreads were counted. The mean and SD are shown in the plot.
Source data are available for this figure.
Source Data for Figure 2[LSA-2020-00933_SdataF2.xlsx]
- Figure S2. CDC53 depletion induces abnormal ZMM assembly.
(A) The assembly of various ZMM proteins (Zip2, Mer3, Spo22, and Msh4) in the CDC53mn mutant. Chromosome spreads from the wild type (4 h) and CDC53mn mutant (8 h) were stained with anti-Zip2, anti-Mer3, anti-Spo22, or anti-Msh4 antibodies (green) together with anti-Zip1 (red). Wild type, NKY1551; CDC53mn, ZHY94. Bar = 1 μm. (B) Western blot analysis of Cdc5 kinase (top) and tubulin (bottom) in various strains. The representative results are shown (n = 2). Wild type, NKY1551; ndt80, ZHY516; CDC53mn, ZHY94; ndt80 CDC53mn, ZHY522. (C) The chromosomal compaction was measured using cells with GFP-marked CenIV and TelIV. The representative images of the wild type and CDC53mn mutant are shown. Bar = 1 μm.
- Figure 3. Cdc53 and Zip3 work independently in synaptonemal complex formation and meiotic recombination.
(A) The cell cycle progression of various mutants. The entry into meiosis I in the wild-type (NKY1551, blue), CDC53mn (ZHY94, red), zip3 (MSY2889, green), zip3 CDC53mn mutant (ZHY259, brown) cells were analyzed by DAPI staining/counting as described in Fig 1B. Representative results are shown (n = 3). (B, C) Double-strand break repair at the HIS4-LEU2 locus in various strains was analyzed as described above. Blots (B) and quantifications (C) are shown. Error bars (SD) are based on three independent cultures. (D) Rad51 staining in various mutants was analyzed as described above. (G) Typical staining patterns of each mutant are shown in (G). At least 100 spreads were counted at each time point. Wild type, blue; CDC53mn, red; zip3, green; zip3 CDC53mn, brown. (E, F) CO formation in various strains was analyzed at the HIS4-LEU2 locus, as described above. Blots (E) and quantifications (F) are shown. Error bars (SD) are based on three independent experiments. (G, H) The chromosome spreads from wild-type, CDC53mn, zip3, zip3 CDC53mn cells were stained with anti-Zip1 (green) as well as anti-Rad51 (red) antibodies, and the staining pattern for Zip1 was classified into classes and plotted (H) as shown in Fig 1F. The representative results are shown (n = 2). Bar = 1 μm. (I) The chromosome axis formation was analyzed by staining the chromosome spreads from various strains with anti-Red1 (green) and anti-Rec8 (red) antibodies. Wild type, 4 h; CDC53mn, 8 h; zip3, 8 h; zip3 CDC53mn, 8 h. Bar = 1 μm.
Source data are available for this figure.
Source Data for Figure 3[LSA-2020-00933_SdataF3.xlsx]
- Figure S3. The absence of NDT80 partially suppresses synaptonemal complex assembly defect in the CDC53mn.
(A) The expression of Cdc5 polo-like kinases in various strains. Lysates from wild-type, CDC53mn, ndt80, and ndt80 CDC53mn cells were analyzed by Western blotting using anti-Cdc5 (upper) and anti-tubulin (lower) antibodies. Cdc5 is under the control of Ndt80. The representative results are shown (n = 2). Wild type, NKY1551; ndt80, ZHY516; CDC53mn, ZHY94; ndt80 CDC53mn, ZHY522. (B) Synaptonemal complex formation in CDC53mn ndt80 cells. Chromosome spreads from wild-type, CDC53mn, ndt80, and ndt80 CDC53mn cells were stained with anti-Zip1 (red) and anti-Rec8 (green) antibodies. Typical images of wild-type (4 h), CDC53mn (8 h), ndt80 (8 h), and ndt80 CDC53mn (8 h) cells are shown. Bar = 1 μm. (C) Zip1-staining in each strain was classified and plotted at each time point. More than 100 nuclei were counted. Class I (blue bars), Zip1 dots; Class II (green bars), partial Zip1 linear lines; Class III (red bars), linear Zip1 staining. The representative results are shown (n = 2). (D) Cells containing Zip1 polycomplexes were counted at each time point and plotted. The representative results are shown (n = 2). Wild type, blue: CDC53mn, red; ndt80 brown; ndt80 CDC53mn, green. (E) Rec8 staining (red) in the wild type, CDC53mn, and gmc2 mutants.
- Figure 4. Cdc53 depletion induces altered axis formation.
(A) Hop1 staining in the wild type (NKY1551) and CDC53mn mutant (ZHY94). Chromosome spreads in each strain were stained with anti-Hop1 (green) and anti-Zip1 (red) antibodies. For the wild type, pachytene chromosomes with long Zip1 lines are shown with a few Hop1 foci. The representative images are shown. Bar = 1 μm. (B) The kinetics of Hop1 assembly/disassembly. The number of Hop1-positive cells was counted at each time point. More than 50 spreads were counted. The representative results are shown (n = 2). Wild type, blue circles; CDC53mn, red circles. (C, D) The nuclear spreads from the wild type and CDC53mn mutant were stained with anti-Red1 (green), anti-Rec8 (red), and DAPI (blue) and then categorized. Synaptonemal complexes of wild-type cells are shown as dots, short lines, and long lines. Synaptonemal complexes in the CDC53mn mutants are shown as dots and short lines. (D) Staining of Rec8/Red1 in two adjacent chromosome spreads of the mutant at 3 h is shown. The top is positive for both Rec8 and Red1. The bottom is positive for Red1 but negative for Rec8. Bar = 1 μm. (E) Kinetics of each class of Red1 (upper two graphs) and Rec8 (lower two graphs) in the wild type and CDC53mn mutant at the indicated times during meiosis. Each stained image was classified into dots (blue bars), short lines (green bars), and long lines (red bars) and then counted. At each time point, more than 50 spreads were counted. Error bars (SD) indicate SD (n = 4). (F) The chromosomal compaction was measured using cells with GFP-marked CenIV and TelIV. The representative images of the wild type and CDC53mn mutant are shown. Bar = 1 μm. (G) The distance between the two GFP signals was measured using NIH Image J and plotted. Wild type, ZHY749 (blue); CDC53mn, ZHY750 (red). Means (n = 166) and SD are shown. The P-value was calculated using the Mann-Whitney U test.
Source data are available for this figure.
Source Data for Figure 4[LSA-2020-00933_SdataF4.xlsx]
- Figure 5. The pch2 mutation suppresses synaptonemal complex defects induced by Cdc53 depletion.
(A) The chromosome spreads from various strains were stained with anti-Zip1 (red) and anti-Rec8 (green) antibodies as well as DAPI (white), and images were captured using DeltaVision epifluorescence microscope and deconvoluted as described in the Materials and Methods section. The representative images are shown. Wild type, NKY1551; CDC53mn, ZHY94; zip3, MSY2889; pch2, ZHY350; pch2 CDC53mn, ZHY351; zip3 CDC53mn, ZHY259. Bar = 1 μm. (B) The chromosome spreads from various strains were stained with anti-Rec8 (green) and anti-Zip1 (red) antibodies. Wild type, 4 h; CDC53mn, 8 h; pch2, 6 h; pch2 CDC53mn, 8 h. Bar = 1 μm. (C) Zip1-stained cells of each strain were classified and plotted at each time point. More than 100 nuclei were counted, as shown in Fig 1F. Class I (blue bars), Zip1 dots; Class II (green bars), partial Zip1 linear staining; Class III (red bars), linear Zip1 staining. N.A., not available. The representative results are shown (n = 2). (D) The cells containing Zip1 polycomplexes were counted at each time point and plotted. Wild type, blue; CDC53mn, red; pch2, orange; pch2 CDC53mn, purple. The representative results are shown (n = 2).
Source data are available for this figure.
Source Data for Figure 5[LSA-2020-00933_SdataF5.xlsx]
- Figure S4. The pch2 CDC53mn double mutant is arrested at the mid-pachytene stage.
(A) Meiotic DNA replication in the CDC53mn (ZHY94) and pch2 CDC53mn mutants (ZHY351) was analyzed by FACS. The representative results are shown (n = 2). (B) The expression of various proteins in the CDC53mn mutant. Lysates obtained from wild-type and CDC53mn single cells during meiosis were analyzed by Western blotting using anti-Sic1 (upper), anti-Cdc6 (second), anti-Cdc5 (polo-like kinase; third), or anti-tubulin (lower) antibodies. The representative results are shown (n = 2). (C) The expression of Hop1 in the CDC53mn mutant. Lysates obtained from wild-type (NKY1551) and CDC53mn (ZHY94) cells during meiosis were analyzed by Western blotting using anti-Hop1 antibody. The representative results are shown (n = 2). (D) The pairing of CENXV. Chromosome spreads from strains homozygous for LacI-GFP-Cen-XV at 0 h and 5 or 8 h were stained with anti-GFP antibody (green) and DAPI (blue) and examined under an epifluorescence microscope. Cells with either single or two GFP spots were counted. More than 100 cells were analyzed at each time point. The percentage of cells with a single GFP spot is shown in the main text. Wild type, ZHY770; pch2 CDC53mn, ZHY 772. Bar = 2 μm.
- Figure 6. The pch2 CDC53mn mutant is defective in meiotic recombination.
(A) The entry into meiosis I of various strains was analyzed by DAPI staining. Wild type, NKY1551; CDC53mn, ZHY94; pch2, ZHY350; pch2 CDC53mn, ZHY351. (B, C) Double-strand break repair at the HIS4-LEU2 locus in various strains was analyzed as described above. Blots (B) and quantifications (C) are shown. Error bars (SD) are based on three independent cultures. (D) The cells containing Rad51 foci were counted at each time point and plotted, as described above. The representative results are shown (n = 2). Wild type, blue: CDC53mn, red; pch2, orange; pch2 CDC53mn, purple. (E, F) CO formation in various strains was analyzed at the HIS4-LEU2 locus as described above. Blots (E) and quantifications (F) are shown. Error bars (SD) are based on three independent time courses. (G) Hop1/Zip1 staining in various mutants. Chromosome spreads in each strain were stained with anti-Hop1 (green) and anti-Zip1 (green) antibodies. (H) The expression of Pch2-HA during meiosis. Cell lysates from PCH2-3HA and CDC53mn PCH2-3HA cells were analyzed by Western blotting using anti-HA (top) and anti-tubulin (bottom) antibodies. The representative results are shown (n = 2). (I) The chromosome spreads from various strains were stained with anti-Pch2 (green) and anti-Zip1 (red) antibodies. Wild type, 4 h; CDC53mn, 8 h. Arrows indicate the “polycomplex” and arrowheads indicate the nucleolus. Bar = 1 μm.
Source data are available for this figure.
Source Data for Figure 6[LSA-2020-00933_SdataF6.xlsx]
- Figure 7. The cdc4-3 is defective in synaptonemal complex-assembly.
(A, B) The chromosome spreads from wild-type and cdc4-3 mutant cells incubated in SPM at 32°C were stained with anti-Zip1 (green, upper panels) and anti-Rad51 (red, upper panels), and with anti-Red1 (green, bottom panels) and anti-Rec8 (red, bottom panels), respectively, as described above. Wild type (NKY1551), 5 h; cdc4-3 (ZHY522), 8 h. (B) Kinetics of Rad51 focus-positive cells are shown in (B). The representative results are shown (n = 2). Bar = 1 μm. (C, D, E) The chromosome spreads from various strains at 32°C were stained with anti-Zip1 (red) and anti-Rad51 (green) antibodies. Typical staining patterns are shown in (C). Wild type (NKY1551), 5 h; cdc4-3 (ZHY522) 8 h; pch2 (ZHY350) 8 h; cdc4-3 pch2 (ZHY580) 8 h. Bar = 1 μm. Kinetics of poly-complex (D), as well as Zip-classes (E), were analyzed as described above. The representative results are shown (n = 2). (F) Entry into meiosis I in different strains was analyzed by DAPI staining as described above. The representative results are shown (n = 2).
Source data are available for this figure.
Source Data for Figure 7[LSA-2020-00933_SdataF7.xlsx]
- Figure 8. A model showing the regulation of synaptonemal complex (SC) formation by SCF and Pch2.
Refer to the text for more details. The SCFCdc4 promotes SC formation by down-regulating Pch2 and protein “X,” both of which may negatively regulate proper axis assembly for SC formation. SCFCdc4 may promote the ubiquitylation of protein “X” for degradation to promote SC assembly.
Supplementary Materials
Table S1 Strain list.
