SUMO localizes to the central element of synaptonemal complex and is required for the full synapsis of meiotic chromosomes in budding yeast

Abstract

The synaptonemal complex (SC) is a widely conserved structure that mediates the intimate alignment of homologous chromosomes during meiotic prophase and is required for proper homolog segregation at meiosis I. However, fundamental details of SC architecture and assembly remain poorly understood. The coiled-coil protein, Zip1, is the only component whose arrangement within the mature SC of budding yeast has been extensively characterized. It has been proposed that the Small Ubiquitin-like MOdifier, SUMO, plays a role in SC assembly by linking chromosome axes with Zip1's C termini. The role of SUMO in SC structure has not been directly tested, however, because cells lacking SUMO are inviable. Here, we provide direct evidence for SUMO's function in SC assembly. A meiotic smt3 reduction-of-function strain displays reduced sporulation, abnormal levels of crossover recombination, and diminished SC assembly. SC structures are nearly absent when induced at later meiotic time points in the smt3 reduction-of-function background. Using Structured Illumination Microscopy we furthermore determine the position of SUMO within budding yeast SC structure. In contrast to previous models that positioned SUMO near Zip1's C termini, we demonstrate that SUMO lies at the midline of SC central region proximal to Zip1's N termini, within a subdomain called the "central element". The recently identified SUMOylated SC component, Ecm11, also localizes to the SC central element. Finally, we show that SUMO, Ecm11, and even unSUMOylatable Ecm11 exhibit Zip1-like ongoing incorporation into previously established SCs during meiotic prophase and that the relative abundance of SUMO and Ecm11 correlates with Zip1's abundance within SCs of varying Zip1 content. We discuss a model in which central element proteins are core building blocks that stabilize the architecture of SC near Zip1's N termini, and where SUMOylation may occur subsequent to the incorporation of components like Ecm11 into an SC precursor structure.

Figure 1. SUMO-diminished strains exhibit reduced sporulation and elevated crossover levels.

(A) Western blot detecting SUMO in lysates from control (smt3Δ/+, LFT36) and SUMO-diminished (P_SCC1[SMT3]/smt3Δ, LFT46) cells at 0, 12, 15 18 and 24 hours of sporulation. Bar graph in (B) gives the ratio of conjugated SUMO protein levels in LFT36 over conjugated SUMO protein levels in LFT46 (solid bars), as well as the ratio of unconjugated (free) SUMO levels in LFT36 over LFT46 (open bars) at each time point. (C) smt3Δ/+ (LFT36, closed circles) and P_SCC1[SMT3]/smt3Δ (LFT46, open squares) cells were sporulated and then assessed for the formation of multinucleate products over a time course (time points indicated on x axis). (For each strain at any given time point, the average between two independent experiments is indicated (in total, >800 nuclei were scored for each timepoint.) In (D), control and P_SCC1[SMT3]/smt3Δ cells were sporulated and surface spread on glass slides at the time points indicated in order to assess whether SUMO-diminished strains enter into and progress through meiotic prophase efficiently. The fraction of nuclei that had entered meiosis at each time point was recorded based on Red1 staining (not shown), while the fraction of nuclei that had reached the pachytene stage of meiotic prophase (or post-MI) were recorded based on the morphology of the DAPI-stained nucleus. Meiotic (Red1-positive) nuclei without pachytene morphology were grouped as the “Leptotene/Zygotene” (pre-pachytene) or “Diplotene” (post-pachytene) category. (E) Cartoon above graph indicates the genetic intervals used to calculate map distances on chromosome III. CEN3 is marked with an adjacent hygromycin resistance cassette and the RAD18 locus is marked with ADE2. Bar graph displays map distances (cM) for the three intervals (x axis) measured in control and SUMO-diminished strains. Error bars represent the standard error of map distance. See Table 2 for precise values and for map distances additionally calculated using tetrad analysis.

Figure 2. SC appears discontinuous in SUMO-diminished pachytene nuclei.

(A) Shown are surface-spread meiotic nuclei from smt3Δ/+ (LFT37) and P_SCC1[SMT3]/smt3Δ (LFT47) ndt80/ndt80 strains that had been sporulated for 24 hours. DAPI (white, first column) and antibodies to Red1 (red), Zip1 (green), SUMO (white, 3rd column; red, 4th column) were used to stain nuclei and assess SC formation. Scale, 1 µm. (B) displays the percentage of nuclei (n>50 per column) from control or SUMO-diminished strains that exhibit either continuous (black), discontinuous (black/white hatched), or dotty pattern of Zip1, Red1, or SUMO staining.

Figure 3. Induced Zip1 assembles full SC stretches in a SUMO-dependent manner.

Cartoon in (A) depicts the Zip1 induction experiment conducted, as described (, this work) using LFT51 (control) and LFT65 (sumo-diminished) strains. Bar graph indicates the extent of SC assembly (Zip1 staining) on chromosomes at each time point following induction (n>50 for each time point). Number on each column indicates the percentage of each set of nuclei that exhibit a polycomplex (Zip1 aggregate) structure. Western blots in (B) show SUMO levels in cellular lysates from the sporulated cells in this experiment (left blot; SUMO signal is 2–8 fold reduced in SUMO-diminished as compared to SMT3+ cells) and Zip1 levels in uninduced and induced strains (right blot; Zip1 runs at ∼100 kD, whereas the lower band is nonspecific). (C) Examples of meiotic surface-spread chromosomes from each strain after induction of ZIP1 expression. Chromosome spreads have been labeled with DAPI (DNA, white), antibodies to Zip1 (red) and SUMO (green). Arrows indicate polycomplex structures. Scale, 1 µm.

Figure 4. SUMO is positioned at the central element of the budding yeast SC.

(A) Surface-spread meiotic nuclei from an ndt80 homozygous strain (AM447). Top row: nuclear spreads are stained with antibodies that target the Zip1 N terminus (green) and SUMO (red), in addition to DAPI to visualize DNA (white, blue in merged image). Bottom row: nuclei are stained with antibodies targeting a C terminal fragment of Zip1 (green) in addition to SUMO (red) and DAPI (white, blue in merged image). Inset zooms in on a portion of indicated SC. Scale, 1 µm. Images were acquired using a structured illumination microscope (Applied Precision's OMX V4, access kindly provided by Stanford Neuroscience Microscopy Service, Stanford University) that can resolve the SC central element (Zip1-N staining) from the remainder of the SC (where Zip1-C is located). The presence of a slight offset between Zip1-N and SUMO staining patterns can be attributed to a chromatic aberration during imaging, as shown in (B). (B) TetraSpeck Beads (Invitrogen), stained uniformly with both red and green fluorescent dyes, were imaged using the same mount and oil conditions as were used to image meiotic nuclei. The resultant red and green bead patterns failed to completely overlap, indicating that a slight chromatic aberration exists for the imaging conditions used. The cartoon in (C) illustrates a model for budding yeast SC structure, with SUMO and Ecm11 (see Figure 5) positioned at the N termini of Zip1 within the SC central region.

Figure 5. Ecm11-MYC is positioned at the central element of the budding yeast SC.

Surface-spread meiotic nuclei from an ECM11-MYC ndt80 homozygous strain (AM2712). Top row: nuclear spreads are stained with antibodies that target the Zip1 N terminus (green) and MYC (red), in addition to DAPI to visualize DNA (white, blue in merged image). Bottom row: nuclei are stained with antibodies targeting a C terminal fragment of Zip1 (green) in addition to MYC (red) and DAPI (white, blue in merge). Boxed insets show portions of each image with increased zoom. The images were acquired using Applied Precision's V4 OMX structured illumination microscope at Stanford Neuroscience Services (Stanford University) as described in Figure 4. Scale, 1 µm.

Figure 6. V5-SUMO continuously incorporates into previously established, full-length SC during meiotic prophase.

The strain in (A), AM2905, is homozygous for an ndt80 mutation, carries two copies of the GAL4-ER transgene, and is heterozygous for both ZIP1-GFP and V5-SMT3 under the transcriptional control of the P_GAL1 promoter. Most ndt80 mutant meiotic nuclei in this strain background exhibit full-length SC by 24 hours of sporulation. Each strain was induced to express ZIP1-GFP and V5-SMT3 at 26 hours of sporulation, and then assessed at 1, 2 and 3 hours following induction. Uninduced cells were also assessed at 3 hours post-induction. Representative images of surface-spread nuclei from induced cells are shown, with top rows showing nuclei with lower levels of induced SC component incorporation and bottom rows showing SC component incorporation that is almost completely coincident with Zip1. Staining is as follows: DAPI (DNA), white; Zip1 (and/or Zip1-GFP), red; Zip1-GFP, green, V5-SUMO, blue. Scale, 1 µm. The stacked column graph in (B) indicates the fraction of nuclei with full-length Zip1 that exhibited None/Foci (open), Dotty (boxed), Dotty-linear (dotted lines), or Linear (solid) patterns of induced Zip1-GFP or V5-SUMO patterns on previously-established SC (n = 30). In (C), membranes with immobilized proteins from lysates of samples taken at the induction (START, 26 hours of sporulation), 1 hour, 2 hour and 3 hour post-induction, and a 3 hour uninduced sample were stained with anti-V5, then stripped and re-probed with anti-SUMO. Lane one contains sample from a strain containing no V5 tag, lane 2 contains AM2905 that has been induced for 12 hours, lane 3 contains MagicMarker protein standards (kDa) (Invitrogen), and lanes 4–8 contain AM2905 lysates at various time points. Unconjugated V5-SUMO and SUMO are indicated in the image. Graph at right plots the relative level of unconjugated V5-SUMO to total V5-SUMO (open boxes) and of unconjugated [V5-SUMO+SUMO] to total [V5-SUMO+SUMO] (closed circles) in each of lanes 4–8.

Figure 7. Ecm11-MYC continuously incorporates into previously established, full-length SC during meiotic prophase.

The strain in (A), AM2865, is homozygous for an ndt80 mutation, carries two copies of the GAL4-ER transgene, and is heterozygous for both ZIP1-GFP and ECM11-MYC under the transcriptional control of the P_GAL1 promoter. Each strain was induced and assessed as described in Figure 6. Representative images of surface-spread nuclei from uninduced or induced cells are shown; staining is as follows: DAPI (DNA), white; Zip1 (and/or Zip1-GFP), red; Zip1-GFP, green, Ecm11-MYC, blue. Scale, 1 µm. The stacked column graph in (B) indicates the fraction of nuclei with full-length Zip1 that exhibited None/Foci (open), Dotty (boxed), Dotty-linear (dotted lines), or Linear (solid) patterns of induced Zip1-GFP or Ecm11-MYC patterns on previously-deposited SC (n = 30). In (C), membranes with immobilized proteins from lysates of ECM11-MYC/ecm11Δ ndt80/ndt80 (AM2892) samples (left) and AM2865 (right) taken at the induction START (26 hours of sporulation), 1 hour, 2 hour and 3 hour post-induction, and a 3 hour uninduced sample were stained with anti-MYC, stripped and re-probed with anti-α-tubulin. Hemizygous ECM11-MYC lysates were included as a reference for baseline Ecm11 levels at each timepoint. Numbers at left give molecular weight positions (kDa). Graphs in (D) at right plot the relative levels of unSUMOylated Ecm11-MYC (closed circles), SUMOylated Ecm11-MYC (open squares) and multi-SUMOylated Ecm11-MYC (closed triangles) in each lane (at each timepoint of the time course experiments); the level of unSUMOylated Ecm11-MYC at 26 hours in Ecm11-MYC/ecm11Δ is set at 1. Relative levels between lanes were normalized using the tubulin staining.

Figure 8. Ecm11(K5R,K101R)-MYC continuously incorporates into previously established, full-length SC during meiotic prophase.

The strain in (A), AM2910, is homozygous for an ndt80 mutation, carries two copies of the GAL4-ER transgene, and is heterozygous for both ZIP1-GFP and ECM11(K5R,K101R)-MYC under the transcriptional control of the P_GAL1 promoter. Each strain was induced and assessed as described in Figure 6. Representative images of surface-spread nuclei from uninduced or induced cells are shown; staining is as follows: DAPI (DNA), white; Zip1 (and/or Zip1-GFP), red; Zip1-GFP, green, Ecm11(K5R,K101R)-MYC, blue. Scale, 1 µm. The stacked column graph in (B) indicates the fraction of nuclei with full-length Zip1 that exhibited None/Foci (open), Dotty (boxed), Dotty-linear (dotted lines), or Linear (solid) patterns of induced Zip1-GFP or Ecm11(K5R,K101R)-MYC patterns on previously-established SC (n = 30). In (C), membranes with immobilized proteins from lysates of AM2910 cells taken at the induction (START, 26 hours of sporulation), 1 hour, 2 hour and 3 hour post-induction, and a 3 hour uninduced sample were stained with anti-MYC, stripped and re-probed with anti-α-tubulin. Numbers at left give molecular weight positions. Graph in (D) plots the level of unSUMOylated Ecm11(K5R,K101R)-MYC (closed circles), SUMOylated Ecm11(K5R,K101R)-MYC (open squares) and multi-SUMOylated Ecm11(K5R,K101R)-MYC (closed triangles) at each timepoint during the induction, relative to the level of unSUMOylated Ecm11-MYC at 26 hours in Ecm11-MYC/ecm11Δ samples (from western in Figure 7D). Ecm11 levels were made comparable with blot in Figure 7C by setting the level of Ecm11(K5R,K101R) signal in lane 2 (27 hour timepoint, 1 hour of induction) to the level of Ecm11-MYC signal timepoint in lane 7 (27 hour timepoint, 1 hour of induction) of blot in Figure 7C. Relative levels between lanes were normalized using the tubulin staining.

Figure 9. A model describing the multimeric assembly of Zip1, Ecm11 and SUMO within SC.

Cartoon images shows possible intermediate steps in a dynamic SC assembly process. Zip1 (blue, darker blue at Zip1-N termini) units are stacked with Ecm11 complex proteins (purple) arranged near Zip1 N termini. Ecm11 components are SUMOylated (yellow) to varying extents. Multiple layers of this basic arrangement of Zip1, Ecm11 and SUMO comprise more mature SC structures.