Fig 1.
Zip1’s N terminal twenty residues are critical for MutSγ crossing over.
A) Line illustrations represent Zip1’s N terminal region (approximately residues 1–200); red brackets highlight residues that are deleted in the altered versions of Zip1 analyzed in this study (precise deletion information is indicated at the left of each line). B) Cartoons illustrate the seven genetic intervals that were utilized to assess meiotic crossover recombination in this study; strains carried two distinguishable alleles (genetic markers) corresponding to five loci whose position span the length of chromosome III, and four loci whose positions encompass more than half of chromosome VIII. Crossover recombination is measured by examining the frequency of chromatids for which adjacent genetic markers have become unlinked from one another during meiosis; a higher frequency of crossover recombination results in a larger map distance between genetic markers that define an interval. (C, D) Bar graphs plot the sum of map distances across all seven intervals in each strain, normalized to the control value. Note that the control for normalization in (C) is the pch2 value, while wild type is the control in (D). Random spore analysis was used to calculate the map distances shown in the hatched bars graphed in (C), while tetrad analysis was used to calculate the map distances illustrated by the solid bars in (C, D); see Materials and methods for map distance calculation procedures. Exact values corresponding to % of control map distances are indicated in gray color above each bar in (C, D). Map distances for individual intervals are reported in Table 1 (pch2 strains) and Table 2 (PCH2 strains). pch2 strains used in (C) are: AM3724 (ZIP1 MSH4), AM4025 (msh4), AM4023 (zip1), AM4026 (zip1 msh4), AM3725 (zip1[Δ2–163]). Strains used in (D) are: K842 (wt), K852 (msh4), AM3684 (zip1[Δ2–20]), K1000 (zip1[Δ2–20] msh4), MP43 (zip1[Δ2–9]), MP46 (zip1[Δ2–9] msh4), SYC107 (zip1[Δ10–14]), SYC149 zip1[Δ10–14] msh4, AF8 (zip1[Δ15–20]), K914 (zip1[Δ15–20] msh4), AF6 (zip1[Δ21–163]), and SYC151 (zip1[Δ21–163] msh4). Data for wild-type, msh4, zip1[Δ21–163], and zip1[Δ21–163] msh4 strains were reported previously [24]. Zip1’s first twenty amino acid residues are illustrated below the graph in (D), with colors corresponding to the residues removed in each of three zip1 mutant alleles: red = zip1[Δ2–9], blue = zip1[Δ10–14], green = zip1[Δ15–20].
Table 1.
Genetic map distances in mutant strains; pch2 background.
Table 2.
Genetic map distances in mutant strains; PCH2 background.
Fig 2.
SC assembly requires Zip1 residues 15–20 but not residues 2–14.
Panels show representative surface-spread mid-meiotic prophase nuclei from wild-type (top row), and various zip1 internal deletion or residue-substitution alleles (genotypes indicated at left; strains are K320 (ZIP1), AM4064 (zip1[Δ2–9]), AM4194 (zip1[Δ10–14]), AM4069 (zip1[10–14→A]), SYC79 (zip1[Δ15–20]), and K981 (zip1[Δ15–20→A]). Note that strains carrying a short internal deletion of Zip1 residues 10–14 or 15–20 exhibit a similar phenotype, respectively, as strains in which Zip1 residues 10–14 or 15–20 are replaced with alanine. All strains carry an ndt80 null allele, which allows meiotic cultures to accumulate at mid-late prophase stages when full-length SCs are normally present. Mid-meiotic prophase chromosomes are stained with DAPI to label DNA (white), anti-Zip1 (green), and anti-MYC to label Ecm11 (magenta). The merge between Zip1 and Ecm11 channels is shown in the final column. Quantitation of the number and cumulative length of SC linear assemblies in wild type as well as each internal deletion zip1 mutant strain is given in Fig 4. Arrows point to polycomplex structures. Arrowhead indicates a large focus or pair of foci that measures at 0.7 μm and thus may have been included in the assessment of “linear” SC structures (see Fig 4). Scale bar, 1 μm.
Fig 3.
Zip1[Δ2–9] assembles with proper orientation within SCs.
Structured illumination was used to probe the organization Zip1 and Ecm11 within linear assemblies in wild type (K1268; top panels) and zip1[Δ2–9] (AM4064; bottom panels) strains. Antibodies against the C terminal 264 residues of the Zip1 protein (green) were applied in conjunction with antibodies against a mixture of Ecm11 and Gmc2 partial proteins (magenta; see Methods for antibody information). In surface-spread meiotic nuclei containing wild-type SC (top panels), C terminal Zip1 antibody label often displays a wide ribbon in which parallel tracts are sometimes visible with the resolution power of structured illumination (~120 nm) [13]. Such parallel tracts of Zip1 (see zoomed insets) flank a central narrow band of Ecm11-Gmc2 protein, and reflect Zip1’s orientation within the mature SC, where Zip1’s C termini interface with lengthwise-aligned homologous chromosome axes and Zip1’s N termini orient closer to the SC central element (comprised of Ecm11 and Gmc2). DNA is labeled by DAPI in the experiment but not shown in the figure. Bottom panels show a representative spread where wild-type Zip1 organization is apparent within the SCs built by the Zip1[Δ2–9] protein. Scale bar, 1 μm.
Fig 4.
SC assembly requires Zip1 residues 15–20 but not residues 2–14.
Each circle in the scatterplots in the far left column represents the number of linear assemblies of Zip1 (blue), Ecm11 (orange) or coincident Ecm11 and Zip1 (purple) detected in surface spread meiotic nuclei of wild-type, zip1 or zip3 mutant strains (genotype indicated at the far right; strain names listed in Fig 2 legend), at 15, 18, 21 or 24 hours after placement into sporulation medium (time points indicated on the x axis). 50 nuclei were examined for each strain at every individual time point, except for wild-type strains and zip3 mutant strains at the 15 hour time point, where 125 and 100 nuclei were examined, respectively. Assemblies of SC proteins were considered to be linear if they measured 0.7 μm or greater in length, although some large or adjacent foci potentially were included in these calculations (see arrowhead in Fig 2 and Fig 2 legend). Individual circles in the middle column of scatterplots indicate the cumulative length of the linear assemblies of Zip1 (blue), Ecm11 (orange) or coincident Ecm11 and Zip1 (purple, “SC”) detected in the surface spread nuclei of indicated strains. Dark and light grey bars indicate the mean, and standard error of the mean, respectively. Bar graphs at far right indicate the percentage of nuclei in these datasets that exhibited a Zip1 polycomplex aggregate (see arrow in Fig 2 for an example). The vast majority of polycomplex structures detected contained both Zip1 and Ecm11 proteins. An asterisk indicates zero polycomplex structures detectable in any nuclei at the indicated time point. Raw data for Fig 4 plots is provided in S5 Table.
Fig 5.
SC in meiotic nuclei from zip1[Δ2–9], zip1[Δ10–14], and zip3 strains assembles predominantly between homologs.
A) Representative surface-spread meiotic nuclei from wild-type (AM4458 or AM4462), zip1[Δ10–14] (AM4446), or zip3 (AM4453), strains homozygous for an ndt80 null allele, a centromere IV-associated lacO array, and GFP-LacI. Surface-spread nuclei are labeled with DAPI-labeled DNA (light blue in top images), anti-Gmc2 (magenta), and anti-GFP (green). Meiotic nuclei were examined 23 hours after placement into sporulation medium. Only those nuclei exhibiting approximately 30% minimum SC assembly were assessed for distance between GFP foci. GFP foci were considered paired if they were positioned within 0.5 micron of one another. While most meiotic nuclei examined from all strains exhibit paired GFP signals, occasionally unpaired GFP signals were observed in zip1[Δ2–9] (AM4440 or AM4441), zip1[Δ10–14] (AM4446), or zip3 (AM4453) mutants; examples are shown in the two lower right panels (zip3 strains). Scale bar, 1 μm. B) The table lists the number of paired versus unpaired GFP-LacI foci observed in at least fifty meiotic nuclei from ZIP1 ZIP3 (AM4458 or AM4462), zip1[Δ2–9] (AM4440 or AM4441), zip1[Δ10–14] (AM4446), or zip3 (AM4453) strains.
Fig 6.
Zip1[Δ15–20] protein occasionally assembles fragile-appearing structures built of coincident Zip1 and Ecm11.
While the vast majority of meiotic nuclei from zip1[Δ15–20] strains (SYC97) exhibit only abundant and varying-sized foci of Zip1 and Ecm11 (Figs 2 and 4), frail-looking linear assemblies of Zip1 (green) were occasionally observed accompanying similar types of Ecm11 linear assemblies (magenta) on surface-spread meiotic chromosomes (labeled with DAPI, white). The abnormal-looking linear assemblies appear wavy, and often taper at their ends. These assemblies (as well as instances of adjacent large focal deposits of Zip1 and Ecm11) were included in the linear assembly measurements reported in Fig 4. The top row presents the singular rare nucleus, out of the more than 100 nuclei examined, in which these frail linear assemblies were most abundant. We note that unlike the robust linear assemblies of coincident Ecm11 and Zip1 observed in zip1[Δ2–9] or zip1[Δ10–14] strains, these diffuse linear assemblies do not appear to join lengthwise-aligned chromosomes. Scale bar, 1 μm.
Fig 7.
SC comprised of Zip1[Δ2–9] or Zip1[Δ10–14] protein initiates at both centromeric and non-centromeric chromosomal sites.
A) Representative surface-spread meiotic nuclei from wild-type (AM4203; top row), zip3 (AM4204; second row), zip1[Δ2–9] (AM4231; third row) or zip1[Δ10–14] (AM4175; bottom row) strains producing the centromere-associated Ctf19-MYC protein. Surface-spread nuclei are labeled with DAPI-labeled DNA (white), anti-Zip1 (magenta), and anti-MYC (green). While all strains at this early stage of synapsis exhibit a large number of short Zip1 assemblies associated with a centromere, fewer examples of ≤1 μm Zip1 assemblies without an associated centromere were observed in zip3 strains, relative to wild-type, zip1[Δ2–9] or zip1[Δ10–14] strains. Scale bar, 1 μm. Quantitation of the number of ≤1 μm Zip1 assemblies with or without an associated Ctf19-MYC signal is shown in the bar graphs in (B), where light shading represents the percentage of total ≤1 μm Zip1 assemblies in each strain that are unassociated with a centromere signal. In wild-type strains, 23 out of 36 ≤1 μm Zip1 assemblies (in 10 surface-spread nuclei) were associated with a centromere; In zip3 strains, 29 out of 35 ≤1 μm Zip1 assemblies (in 11 nuclei) were associated with a centromere; In zip1[Δ2–9] strains, 35 out of 73 ≤1 μm Zip1 assemblies (in 21 nuclei) were associated with a centromere; In zip1[Δ10–14] strains, 27 out of 52 ≤1 μm Zip1 assemblies (in 11 nuclei) were associated with a centromere. A Fishers Exact Test found no significant difference between the proportion of centromere-associated Zip1 stretches in wild-type versus zip3 in this data set (two-tailed P value = 0.107), but did find a significant difference between zip1[Δ2–9], and zip3 (two-tailed P value = 0.0007), and between zip1[Δ10–14] and zip3 (two-tailed P value = 0.0033).
Fig 8.
Zip1 residues 15–20 promote, while residues 2–14 limit, SUMOylation of the SC central element protein Ecm11.
A) A representative Western blot using an anti-MYC antibody reveals unSUMOylated, mono-SUMOylated, poly-SUMOylated and hyper-SUMOylated forms of Ecm11-MYC in meiotic extracts prepared from ZIP1 ZIP3, zip3, or various zip1 mutant strains (protein alterations caused by each zip1 allele is indicated on the x axis). All strains carry an ndt80 null allele, which causes a meiotic arrest that ensures maximal enrichment of mid-meiotic prophase stage cells at 24 hours after placement into sporulation medium [44]. Meiotic extracts were prepared at 24 hours after placement into sporulation media, as previously described [13, 38]. Strains included in this analysis are: AM2712 (ZIP1), MP39 (zip1[Δ2–9]), SYC96 (zip1[Δ10–14]), SYC97 (zip1[Δ15–20]), AM3719 (zip3), AM3662 (zip1[F4A,F5A]), AM3628 (zip1[N3A,R6A,D7A]), AM3656 (zip1[P14A,P16A]), AM2784 (zip1Δ), K986 (zip1[I18A,F19A]). B) The stacked bar graph plots the percentage of mono-SUMOylated (dark shaded bar), poly-SUMOylated (light bar), or hyper-SUMOylated (gray shaded bar) forms of Ecm11-MYC detected in each strain at 24 hours after placement into sporulation media. The absence of a light or gray bar in some strains indicates that this form of Ecm11 was detected in less than 1% of the total population. Error bars represent the range of values from three independent meiotic cultures, except in the case of the zip1[F4A, F5A] strain, where only one experiment was performed. Data plotted is listed in S6 Table. * Note zip1[I18A, F19A] is not present in the blot shown in (A).
Fig 9.
zip1[N3A, R6A, D7A] and zip1[F4A, F5A] and zip1[I18A, F19A] mutants resemble corresponding small deletion alleles with respect to synapsis.
Panels show representative surface-spread mid-meiotic prophase nuclei from zip1[N3A, R6A, D7A] (K969; top panel including 3 rows), zip1[F4A, F5A] (AM4067; middle panel including two rows) and zip1[I18A, F19A] (K985; bottom panel including three rows) mutants, with genotypes indicated at left. All strains carry an ndt80 null allele, which allows meiotic cultures to accumulate at mid-late prophase stages when full-length SCs are normally present. Mid-meiotic prophase chromosomes are stained with DAPI to label DNA (white), anti-Zip1 (green), and anti-MYC to label the epitope-tagged Ecm11 expressed in these strains (magenta). The merge between Zip1 and Ecm11-MYC channels is shown in the final column.
Fig 10.
Zip1 residues 2–14 promote the localization of Zip3 to Zip1 polycomplex structures.
A) Four groups of panels each show three representative images of Zip1 polycomplex structures in spo11 meiotic prophase nuclei expressing either wild-type ZIP1 (AM4174; far left group), zip1[Δ2–9] (AM4253; second group), zip1[Δ10–14] (AM4173; third group), or zip1[Δ15–20] (AM4256; far right group). Zip1 polycomplex is shown in green (first row) on surface-spread meiotic prophase nuclei. The localization of Zip3-MYC (second row, magenta) and Zip4-HA (fourth row, magenta) is also assessed. Merged images between either Zip3-MYC and Zip1 or Zip4-HA and Zip1 are shown in the third and fifth rows, respectively. The number of polycomplexes (n = 20) for which Zip3 is nearly fully coincident with Zip1 is displayed at the bottom of the corresponding strain’s images (n = 20). (B) Three groups of panels each show three different surface spread spo11 meiotic nuclei with a Zip1 polycomplex structure (labeled by anti-Zip1; green, top row). The localization of Zip3-MYC (second row, magenta) and Zip4-HA (fourth row, magenta) is also assessed on these surface-spread meiotic prophase nuclei. Merged images between either Zip3-MYC and Zip1 or Zip4-HA and Zip1 are shown in the third and fifth rows, respectively. Nuclei at the far left correspond to strains homozygous for zip1[N3A, R6A, D7A] (AM4350); the middle panel corresponds to strains homozygous for zip1[F4A, F5A] AM4340; and nuclei in the far right panel correspond to strains homozygous for zip1[Δ21–163] (AM4343). Scale bar, 1 μm.
Fig 11.
Residues 2–14 are required for Zip1’s capacity to recruit Zip3 to recombination initiation sites.
Chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR) to monitor the association of Zip3-FLAG with three DSB sites (GAT1, BUD23, ERG1, dark blue, green and light blue lines, respectively) as well as to centromere and axis sites (magenta and red lines, respectively) in SK1 strains carrying ZIP1 (ORD9670), zip1[Δ2–9] (AM3946/VBD1872), zip1[Δ10–14] (AM3951/VBD1873), or zip1Δ (ORD9689) alleles. x axes indicate number of hours after placement in sporulation medium. The relative abundance of indicated chromosomal sites detected by qPCR in Zip3-FLAG immunoprecipitates is expressed as a percentage of the abundance of each site detected in the input, prior to immunoprecipitation. Values are the average ± standard deviation from two independent experiments. A single experiment was performed in the case of the zip1 null strain. Data plotted is listed in S6 Table.
Fig 12.
Zip3-MYC foci localize to the SC central element sub-structure in wild-type meiotic nuclei and are diminished in number and intensity in zip1[Δ2–9] and zip1[Δ10–14] strains.
Images show representative surface-spread meiotic nuclei labeled with antibodies against the chromosomal axis protein Red1 (magenta) and antibodies against the MYC epitope to label Zip3-MYC (green). (A) In wild-type strains (AM4171), structured illumination microscopy reveals Zip3-MYC foci directly in between lengthwise-aligned homologous chromosome axes in mid-meiotic prophase nuclei, reflecting the embedded distribution of Zip3 complexes within the central region of SCs. (B) The position of Zip3-MYC foci in between lengthwise-aligned axes is unchanged but the number of robust Zip3-MYC foci is severely diminished in zip1[Δ2–9] strains (AM4277). Zip3-MYC’s location within the central element substructure of the SC in this strain is also indicated in this image, where Zip3-MYC (green) is co-labeled with antibodies that target Ecm11-Gmc2 (magenta). Scale bar, 1 μm.
Fig 13.
Msh4-MYC localizes to the SC central element sub-structure.
Images show representative surface-spread meiotic nuclei from a wild-type strain (K1268) carrying a MYC-tagged MSH4 gene. The surface-spread nuclei are labeled with antibodies against Ecm11-Gmc2 central element proteins (magenta, top row), or the chromosomal axis protein Red1 (magenta, bottom row), in conjunction with antibodies against the MYC epitope (green). The increased resolving power of structured illumination microscopy reveals that Msh4-MYC foci directly embed within the central element substructure of the SC (see zoomed inset in top row). Scale bar, 1 μm.
Fig 14.
Msh4-MYC foci are reduced in number and diminished in size in zip1[Δ2–9] and zip1[Δ10–14] strains.
Images show representative surface-spread meiotic nuclei labeled with antibodies that target Ecm11-Gmc2 (magenta, top rows) or antibodies against the chromosomal axis protein Red1 (magenta, bottom rows) in conjunction with antibodies that target the MYC epitope to label Msh4-MYC (green). (A) In wild-type strains (AM4278; top row), conventional fluorescence microscopy reveals Msh4-MYC foci directly embedded within the SC central element substructure, consistent with the position of Zip3 complexes (which colocalize with Msh4 [28]) within the central region of SCs as shown in Fig 12. The position of Msh4-MYC foci within the central region of the SC is unchanged but the number of robust Msh4-MYC foci is severely diminished in zip1[Δ2–9] strains (AM4274; middle row) and in zip1[Δ15–20] strains (AM4265; bottom panel including two rows). (B) The distribution of Msh4-MYC foci with respect to Red1-labeled chromosome axes is shown for wild type (K1268; top row), zip1[Δ10–14] strains (AM4270; middle row), as well as zip1 null strains (AM4263; bottom row). Scale bar, 1 μm.
Fig 15.
Adjacent conserved regions within Zip1’s N terminus coordinate meiotic crossing over with synapsis: A model.
A) Cartoon illustrates the likelihood of the indicated secondary structure (alpha helical = purple; beta sheet = green; unstructured = pink) across the length of the 875 residue Zip1 protein. Secondary structure prediction was performed using JNet (http://www.compbio.dundee.ac.uk/www-jpred/). The lower line plot indicates the relative conservation of individual amino acid residues across the entire Zip1 protein, using homologs from the following 18 fungal species: Saccharomyces cerevisiae, Candida glabrata, Lachancea lanzarotensis, Tetrapisispora blattae, Tetrapisispora phaffii, Kazachstania naganishii, Vanderwaltozyma polyspora, Torulaspora delbrueckii, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Kazachstania africana, Naumovozyma dairenensis, Naumovozyma castellii, Kluyveromyces marxianus, Kluyveromyces dobzhanskii. Eremothecium cymbalariae, Ashbya gossypii, Ashbya aceri. Conservation scores are calculated in JalView 2.10 [67] based on multiple sequence alignment (http://www.jalview.org/help/html/calculations/conservation.html). The conservation score is based on the physio-chemical properties of the amino acid residues and is given in arbitrary units from 0–11, where 11 is the most conserved (scores are listed in S6 Table). Stronger conservation is indicated by a longer line above and below the axis. The per-residue Jalview scores were plotted as an area graph in Excel and mirrored in Adobe Illustrator such that the amplitude of the plot signifies conservation: The maximum deviation from the center line indicates a maximum conservation (score = 11), whereas no deviation indicates no conservation (score = 0). Data plotted is listed in S6 Table. The cartoon in (B) illustrates the possibility that Zip1’s N terminal 20 residues represent adjacent functionalities: The region corresponding to residues 2–14 is required for MutSγ crossing over, perhaps through a direct interaction with the crossover regulator (and putative E3 SUMO ligase) Zip3, but dispensable for SC assembly per se. Our prior characterization of the zip1[Δ21–163] internal deletion allele indicates that residues between amino acids 21 and 163 are essential for Zip1’s SC assembly function but completely dispensable for MutSγ crossing over [24]. Data in the current study indicates that residues 15–20 are essential for Zip1’s SC assembly and Ecm11 SUMOylation activity (as demonstrated by the zip1 null phenocopy displayed by zip1[Δ15–20] and zip1[I18A, F19A] strains). We note that the 15–20 region also plays a role in Zip1’s pro-crossover activity (indicated by gray dotted arrow). We speculate that the adjacency between the functionally distinct regions of Zip1’s N terminus may mechanistically underlie the coordination between MutSγ crossing over and synapsis, by providing a scaffold for direct molecular communication (blue arrow) between crossover factors and synapsis proteins.