Dynamic molecular architecture of the synaptonemal complex

Simone Köhler; Michal Wojcik; Ke Xu; Abby F Dernburg

doi:10.1126/sciadv.adq9374

. 2025 Jan 22;11(4):eadq9374. doi: 10.1126/sciadv.adq9374

Dynamic molecular architecture of the synaptonemal complex

Simone Köhler ^1,^2,^*,^†, Michal Wojcik ^3,⁴, Ke Xu ^3,^4,^5,^6,^*, Abby F Dernburg ^1,^2,^5,^*

PMCID: PMC11753403 PMID: 39841849

Abstract

During meiosis, pairing between homologous chromosomes is stabilized by the assembly of the synaptonemal complex (SC). The SC ensures the formation of crossovers between homologous chromosomes and regulates their distribution. However, how the SC regulates crossover formation remains elusive. We isolated an unusual mutation in Caenorhabditis elegans that disrupts crossover interference but not SC assembly. This mutation alters the unique C terminal domain of an essential SC protein, SYP-4, a likely ortholog of the vertebrate SC protein SIX6OS1. We use three-dimensional stochastic optical reconstruction microscopy (3D-STORM) to interrogate the molecular architecture of the SC from wild-type and mutant C. elegans animals. Using a probabilistic mapping approach to analyze super-resolution image data, we detect changes in the organization of the synaptonemal complex in wild-type animals that coincide with crossover designation. We also found that our syp-4 mutant perturbs SC architecture. Our findings add to growing evidence that the SC is an active material whose molecular organization contributes to chromosome-wide crossover regulation.

A separation-of-function allele reveals a role of the ultrastructure of the synaptonemal complex in crossover regulation.

INTRODUCTION

Diploid genomes are partitioned during meiosis to produce haploid daughter cells. To this end, homologous chromosomes must pair and undergo crossover (CO) recombination. This process creates recombinant chromatids that give rise to genetically diverse progeny and also generates physical linkages that direct proper chromosome segregation.

Programmed double-strand breaks (DSBs) are induced during early meiotic prophase, but only a small subset of DSBs eventually lead to COs (1); the others, sometimes the vast majority of breaks, are repaired to yield non-CO products. In most eukaryotes, COs are nonrandomly far apart, often occurring only once per chromosome pair per meiosis. It is still unclear how this regulation of CO number and distribution is achieved.

The synaptonemal complex (SC) plays essential roles in CO formation and patterning. This protein structure assembles between homologous chromosomes during meiotic prophase, mediating and stabilizing parallel alignment along their lengths (2). In transmission electron micrographs, the SC appears as a periodic, ladder-like structure when stained with heavy metals (3). Assembly of the SC between chromosomes depends on intact chromosome “axes”; in Caenorhabditis elegans this structure comprises cohesin proteins and 4 associated HORMA-domain proteins (HTP-1, HTP-2, HTP-3, and HIM-3) (4–7). Prior work in several organisms has shown that extended coiled-coil SC proteins, often referred to as “transverse filaments,” span the distance between paired chromosome axes in a head-to-head (N terminus to N terminus) configuration, while other proteins localize to the more electron-dark region in the center, known as the “central element” (8). In vivo imaging has revealed that this ordered material shows dynamic, liquid-like behavior despite its highly regular appearance (9–13).

SC proteins self-assemble through a regulated process to form a repeating array that holds paired chromosome axes at a regular distance. These proteins contain extensive regions of potential coiled coils, some of which presumably mediate specific interactions with each other (14). SC proteins must also interact with proteins associated with the chromosome axes, since mutations in some axis components disrupt the assembly of SCs between chromosomes and can lead to self-assembly of “polycomplexes,” dynamic three-dimensional (3D) bodies with internal striations that recapitulate the periodic organization of SCs (6, 10, 15). Some SC proteins also contain peptide motifs that recruit proteins that contribute to CO patterning and regulate SC assembly and disassembly, including enzymes that mediate posttranslational modifications such as SUMOylation and phosphorylation (13, 16, 17). Very few of these self-assembly and regulatory interfaces within the SC have been identified or characterized in detail. A molecular understanding of the roles and regulation of the SC has been limited in part by a lack of structural information about the physical disposition of functional elements within the SC.

Seven structural proteins essential for SC assembly in C. elegans have been identified and named SYP-1–6 and SKR-1/2. SYP-1–4 are each unique, while SYP-5 and SYP-6 are paralogs resulting from a recent gene duplication (14, 18–23). SKR-1 and its paralog SKR-2 are also components of the SCF ubiquitin E3 ligase complex. Reduced expression of some SC proteins or mutations that impair or abolish synapsis can perturb CO regulation in C. elegans, as well as in rice or Arabidopsis (22, 24–30). Some of the conserved proteins required for CO designation localize along the SC before their accumulation at eventual CO sites across diverse organisms (10, 27, 31–35). It has therefore been proposed that diffusion of CO factors along the SC may govern CO patterning along the length of the chromosomes (34, 36, 37). This idea has been supported by direct visualization of diffusion of meiotic RING finger proteins along the SC in C. elegans (36, 38).

The length and/or fluorescence intensity of the SC can change during meiotic prophase. In C. elegans, SC proteins appear to become more stably associated with this structure during late prophase, concomitant with the appearance of six bright foci of the crossover factor COSA-1 per nucleus, which is indicative of CO designation (9, 11, 25). However, it is unclear whether this accumulation is involved in CO patterning or occurs downstream of this process (9, 11–13, 25).

On the basis of serial-section electron microscopy in various organisms, the central region of the SC is approximately 100 nm in width (axis-to-axis distance) and less than 100 nm in thickness. These dimensions are below the resolution limit of conventional light microscopy. Protein organization within the SC has previously been interrogated through physical interaction assays and immuno–electron microscopy (14, 39), but these methods have limited ability to reveal ultrastructure. Super-resolution fluorescence microscopy has emerged as a powerful tool to investigate the organization of macromolecular assemblies, including the SC (22, 40, 41). In previous work, we established methods to map proteins associated with synapsed chromosome axes using single-molecule localization microscopy in intact germline tissue from C. elegans (42). Here, we use a similar imaging method to define the organization of individual proteins within the SC central region. In some cases, we mapped two epitopes on the same protein separately and used a probabilistic method to define the orientation of these proteins based on the distributions of the epitopes. Our findings reveal that the organization of proteins within the SC changes during pachytene. In the course of this work, we also identified a unique separation-of-function mutation in an unusual SC protein, SYP-4, which disrupts CO interference and also alters SC ultrastructure. This work expands our understanding of the dynamic organization of the SC during meiosis and its role in CO patterning.

RESULTS

Perturbation of CO interference by a separation-of-function mutation in the C terminus of SYP-4

As part of ongoing efforts to map the 3D organization of the chromosome axis (42) and SC (22), we inserted epitopes into endogenous genes encoding C. elegans SC proteins SYP-1, SYP-2, SYP-3, and SYP-4 using CRISPR-Cas9 genome editing. The functionality of tagged alleles was assessed through brood analysis (table S1). Production of inviable embryos and male self-progeny are indicative of chromosome mis-segregation during meiosis (43). Wild-type hermaphrodites produce close to 100% viable embryos and approximately 0.2% male self-progeny.

Fortuitously, we obtained three different C-terminally tagged alleles of SYP-4 that had distinct effects on meiosis (Fig. 1, A and B). Insertion of a hemagglutinin (HA) tag at the C terminus (syp-4^ha) did not disrupt its function, as indicated by brood analysis (Fig. 1C). An independent insertion of HA at the C terminus also resulted in a noncoding insertion within the 3′ untranslated region (3′UTR; syp-4^ha3′UTR), which reduced protein expression to 22.9 ± 9% (mean ± SD) of that seen in syp-4^ha (Fig. 1B and fig. S1, A and B). A similar decrease to 25 to 30% of normal expression of other SYP proteins in response to partial RNAi was previously shown to perturb CO patterning (25). A third allele, designated as syp-4^CmutFlag, added a 3×FLAG tag at the C terminus and also altered the C terminal sequence due to a frame shift. Together with the 3×Flag tag, these modifications transformed the last 19 amino acids of SYP-4 from mostly polar and hydrophobic amino acids to a highly charged sequence (18 charged residues of 34) (Fig. 1A). Homozygous syp-4^ha3′UTR and syp-4^CmutFlag hermaphrodites produced some inviable embryos and male self-progeny (Fig. 1C and table S1), indicating that these mutations perturb the function of SYP-4. Both alleles were fully recessive in trans with a wild-type syp-4 allele but only partially rescued a syp-4 deletion allele (fig. S1C and table S1).

Immunofluorescence revealed continuous staining along the entire lengths of the chromosome axes in HA-tagged syp-4^ha animals (Fig. 2A). Some partial and delayed SC assembly was seen in syp-4^ha3′UTR animals, consistent with evidence that reduced expression of SYP-1, SYP-2, or SYP-3 can perturb synapsis (Fig. 2B) (24, 25). Unexpectedly, however, SC assembly appeared to be continuous and timely in syp-4^CmutFlag homozygotes (Fig. 2C), despite the high nondisjunction they displayed. In wild-type animals, SYP proteins accumulate within the SC during meiotic progression (11). To test whether SC loading was affected in syp-4^ha3′UTR or syp-4^CmutFlag animals, we analyzed the accumulation of two SC proteins, SYP-2 and SYP-5 (Fig. 2D and fig. S2A). In syp-4^ha3′UTR animals, accumulation of SYP-2 and SYP-5 was delayed, consistent with the evident perturbation of synapsis. By contrast, SC proteins initially increased in syp-4^CmutFlag animals but did not continue to accumulate as prophase progressed, despite apparently normal expression of other SC proteins (fig. S2B).

A reporter for CHK-2 kinase activity (44) also indicated a delay in meiotic progression in both syp-4^CmutFlag and syp-4^ha3′UTR animals (Fig. 2E and fig. S3A). CHK-2 is normally activated at meiotic entry and is then inactivated at mid-pachytene, concomitant with CO designation. Under conditions that impair synapsis and/or CO designation, CHK-2 activity is prolonged by a “CO assurance checkpoint” that promotes the formation of at least one CO per chromosome (44, 45). Consistent with prolonged CHK-2 activity, we also observed an extended region of nuclei positive for RAD-51 foci marking DSBs, and more foci per nucleus, in both syp-4^CmutFlag and syp-4^ha′3′UTR compared to wild-type animals (fig. S3, B and C). This may reflect either a delay in establishment of CO intermediates in these mutants or a defect in cells’ ability to detect their presence or to inactivate the CO assurance checkpoint, resulting in persistent DSBs. Six bivalents were consistently observed at diakinesis in all three tagged syp-4 strains, indicating that COs eventually occurred on all chromosomes (Fig. 2F). However, the structure of bivalents at diakinesis was altered in syp-4^ha3′UTR and syp-4^CmutFlag oocytes (fig. S4), indicating that CO number might be perturbed (46–48).

To analyze CO formation in our tagged syp-4 strains, we visualized COSA-1 tagged with green fluorescent protein (GFP), which marks designated CO sites at late pachytene (49). Wild-type nuclei display six bright GFP::COSA-1 foci in late pachytene, corresponding to a single CO site per chromosome (46, 49). Partial knockdown of SC proteins can elevate the number of GFP::COSA-1 foci to 7 to 9 per nucleus (25). Consistent with these previous findings, we observed 6 ± 0.2 (SD) GFP::COSA-1 foci in wild-type oocytes and 7.3 ± 1.1 foci in syp-4^ha3′UTR. We observed 10.9 ± 1.7 GFP::COSA-1 foci at late pachytene in syp-4^CmutFlag hermaphrodites (Fig. 3, A and B, and fig. S5A). The observed increase in RAD-51 foci in syp-4^CmutFlag animals (fig. S3, B and C) cannot explain the increase in GFP::COSA-1 foci, since CO homeostasis ensures that excessive double strand breaks, e.g., resulting from a high dose of ionizing radiation, do not result in a corresponding increase in GFP::COSA-1 foci in wild-type animals (25).

Fig. 3. — (A) Maximum-intensity projections of deconvolved 3D wide-field images of late pachytene nuclei stained for SYP-4 (SC) and GFP::COSA-1, which marks designated CO sites. Merged images are shown at the bottom. (B) Wild-type oocyte nuclei contain six GFP::COSA-1 foci, while both *syp-4^ha3′UTR* and *syp-4^CmutFlag* hermaphrodites show elevated numbers of foci. (C) Genetic mapping by whole-genome sequencing reveals a twofold increase in COs in *syp-4^CmutFlag* oocytes. The number of COs in wild-type and *syp-4^CmutFlag* oocytes is consistent with the number of GFP::COSA-1 foci observed cytologically (P values of 0.306 and 0.327, respectively). Boxes in (B) and (C) indicate mean ± SD. (D) To quantify CO interference strength in *syp-4^CmutFlag* oocytes, we quantified the CoC, which denotes the ratio of the observed number of double COs (GFP::COSA-1 foci) to the number expected based on a random (noninterfering) distribution. In wild-type animals, the CoC of GFP::COSA-1 foci is 0, corresponding to complete interference (purple dashed line; n = 88 chromosomes), while it is approximately 1 in *syp-4^CmutFlag* mutants (orange, n = 41), indicating a severe reduction in or loss of interference (gray line).

To test whether the marked increase in GFP::COSA-1 foci in syp-4^CmutFlag animals reflects an increase in COs, we also measured crossing-over genetically in syp-4^CmutFlag animals using whole-genome sequencing (see Materials and Methods). This analysis confirmed that the increase in GFP::COSA-1 foci in syp-4^CmutFlag animals indeed reflects an increase in COs (Fig. 3C and fig. S5B). We quantified CO interference in syp-4^CmutFlag by calculating the coefficient of coincidence (CoC; Fig. 3D and fig. S5C) (50). By this measure, CO interference was severely reduced in syp-4^CmutFlag oocytes. The increase in crossing-over may contribute to the high nondisjunction we observed, since excess COs impede proper chromosome segregation (48).

CO interference is a distance-dependent effect, and in many organisms longer chromosomes undergo more COs than shorter ones (51–54). However, we found that chromosome axes in syp-4^CmutFlag were shorter than in wild-type animals in mid-pachytene, before the appearance of bright GFP::COSA-1 foci (fig. S5D, left). Thus, changes in chromosome length cannot account for the observed increase in COs in syp-4^CmutFlag animals. In C. elegans, an increase in axis length occurs concomitantly with CO designation (25), suggesting that the increase in the number of COs should result in greater elongation of the chromosome axes lengths in syp-4^CmutFlag animals. The axes of syp-4^CmutFlag animals are similar to wild-type axes at late pachytene (fig. S5D, right).

Loss of “interference” in polycomplexes with C-terminally mutated SYP-4

We also examined the effect of syp-4^CmutFlag in the context of polycomplexes, the ordered assemblies of SC proteins that form in the nucleoplasm when SC cannot assemble between chromosomes. In wild-type C. elegans oocytes, polycomplexes are often observed transiently before synapsis, and larger polycomplexes are observed throughout prophase in mutants lacking the axis protein HTP-3 (6, 10, 15) (Fig. 4A). Despite an absence of DSBs, polycomplexes in htp-3 mutants recruit several pro-CO proteins, which undergo dynamic redistribution during meiotic progression (10). In particular, the RING finger proteins ZHP-3 and ZHP-4 are initially distributed throughout these bodies but later form smaller foci that remain associated with polycomplexes. These ZHP foci are also positive for COSA-1 and thus resemble the “recombination nodules” that normally mark designated CO sites along SCs in late pachytene (34). In htp-3 mutant animals that express wild-type SC proteins, most polycomplexes show a single focus of these pro-CO proteins at mid-prophase (1.1 ± 0.2; Fig. 4, A and D). Intriguingly, in htp-3 syp-4^CmutFlag double mutants, we frequently observed multiple GFP::COSA-1 foci (2.1 ± 0.8) associated with individual polycomplexes (Fig. 4, C and D), mirroring the ~2-fold increase in GFP::COSA-1 foci observed along bona fide SCs in syp-4^CmutFlag mutants. By contrast, when we combined htp-3 and syp-4^ha3′UTR, we observed small polycomplexes associated with a single (1.1 ± 0.2) COSA-1 focus (Fig. 4, B and D).

Fig. 4. — (A to C) Fluorescence micrographs show that polycomplexes (SYP-2, blue) in ∆*htp-3* hermaphrodites expressing wild-type SC proteins (A) or SYP-4^HA3′UTR (B) are usually associated with a single GFP::COSA-1 focus (yellow), but polycomplexes in ∆*htp-3 syp-4^CmutFlag* double mutants (C) often have more GFP::COSA-1 foci. DAPI is shown in gray. (D) Quantification of GFP::COSA-1 foci. Boxplots show mean ± SD and whiskers are extreme values. Solid lines highlight P values <0.1 (Mann-Whitney test), dashed lines are shown for P values >0.1. (E and F) Representative electron micrographs show that the internal structure of polycomplexes, which normally display parallel electron-dark bands at ~97-nm intervals, is disturbed in ∆*htp-3 syp-4^CmutFlag* (F), in which electron-dark features are disorganized and 46 nm apart. This may reflect a destabilization of the midline region, or “central element,” of the SC within polycomplexes.

We next investigated the organization of polycomplexes using transmission electron microscopy. As in other organisms, polycomplexes in C. elegans recapitulate the periodic organization of SCs and are thus presumed to represent stacks of SC layers that form a nonisotropic 3D liquid crystalline lattice. We observed these characteristic striated complexes in high-pressure frozen tissue from htp-3 mutant animals (Fig. 4E). By contrast, in htp-3 syp-4^CmutFlag double mutants, we observed nuclear aggregates that resembled polycomplexes, with electron-dense lateral regions interspersed with more electron-lucent central regions, but lacking the regular, striated pattern characteristic of polycomplexes in htp-3 single mutants (Fig. 4, E and F). The polycomplexes in htp-3 syp-4^CmutFlag double mutants appeared to be internally fragmented, rather than maintaining regular orientation over hundreds of nanometers (Fig. 4F) (10), and the distance between parallel electron-dark bands was much narrower (46.3 ± 1.2 nm versus 97.6 ± 1.5 nm in htp-3 polycomplexes; Fig. 4, E and F). Thus, syp-4^CmutFlag perturbs the organization of proteins within polycomplexes and also disrupts the distribution of pro-CO factors associated with these nuclear bodies.

Dynamic architecture of the SC in C. elegans

Our data suggested that syp-4^CmutFlag is a separation-of-function allele that perturbs CO regulation without impairing the ability of the SC to spread along chromosomes. To characterize its effects further, we next examined the ultrastructure of the SC in wild-type and syp-4^CmutFlag animals before and after CO formation. While transmission electron microscopy first revealed the ordered, striated structure of the SC and polycomplexes, its ability to define the molecular organization of these assemblies is limited. We therefore took advantage of 3D-STORM super-resolution microscopy (55, 56) to map the positions and orientation of epitopes within the SC, an approach that we have recently used to define the molecular architecture of meiotic chromosome axes and the orientation of two recently discovered components of the SC (22, 42) (fig. S6). To map the relative localization of protein epitopes, we imaged a chromosome axis protein as a spatial reference [HIM-3 tagged with mEos2 or mMaple3; (7)] in all samples using PALM (photoactivated localization microscopy; fig. S6, B and C). We selected regions in which paired axes were in the same focal plane so that all images were collected parallel to the SC and could then be co-oriented within the plane by rotation about the optical axis. Because paired axes are held at a constant distance when the SC assembles between them, we could average data from multiple images to define the distributions and average positions of protein epitopes. We used a 3D coordinate system in which the orientation of the chromosome axes defines the y axis, the x axis is thus orthogonal to both chromosome axes, and z is the optical axis. The distributions of each protein epitope were then mapped in x and z.

To investigate whether the organization of the SC along homologous chromosomes is affected by syp-4^CmutFlag, we analyzed the organization of one of the transverse filament proteins, SYP-1 (18, 39). To this end, we localized an antibody raised against the C terminal peptide sequence of SYP-1 (57). In frontal view, this SYP-1 epitope was resolved as two parallel strands, located at 42.3 ± 0.8 nm and 42.0 ± 1.2 nm off-center in early and late pachytene of wild-type animals, respectively (Fig. 5A). The width of the SC central region in C. elegans is ~96 nm (10); this epitope thus lies near the outer edges of the central region and within a few nanometers of HIM-3, the most proximal known component of the chromosome axis (42), as expected for a transverse filament protein (39).

Two discrete distributions could also be resolved for the SYP-1 C terminus in syp-4^CmutFlag mutants. However, the distance of the peaks from the center was reduced to 38.0 ± 2.7 nm and 30.5 ± 1.4 nm in early and late pachytene, respectively. This finding is consistent with the closer spacing between electron-dark regions in htp-3 syp-4^CmutFlag compared to htp-3 polycomplexes (Fig. 4, E and F).

We also measured the localization of the SYP-1 C terminus along the z axis (Fig. 5B). In wild-type animals, the distribution of localization events in z was narrower in early than in late pachytene (xz view; Fig. 5B and table S2). This is consistent with evidence that SYP-1 may accumulate along the SC later in pachytene (9, 11), which may result in a thicker complex. In contrast, in syp-4^CmutFlag mutant animals, the z distribution of SYP-1 C termini remained narrower than in wild-type animals in late pachytene (Fig. 5B), suggesting that this mutation may reduce the tendency of the SC to grow thicker concomitant with CO designation. This may be a consequence of the dysregulation of meiotic progression in this mutant, as indicated by prolonged CHK-2 activity described above.

Next, we measured whether the localization of the N terminus of SYP-1 is similarly altered during meiotic progression or in syp-4^CmutFlag mutants. SYP-1 undergoes N terminal acylation, and N terminal epitope tags on this protein disrupt SC assembly (58), but we identified a poorly conserved region close to the N terminus, which accommodated insertion of an HA epitope without impairing SC function (fig. S7 and table S1). Consistent with previous evidence of a transverse orientation for SYP-1 (34), the N terminus was confined to a single peak at the center of the SC along the x axis in both early and late pachytene in both wild-type and syp-4^CmutFlag mutant animals. However, the N terminal distribution did not change detectably during pachytene nor was it altered in the syp-4^CmutFlag strain (Fig. 5, C and D). This finding suggested that the increase in thickness we observed for the C terminus of SYP-1 is not caused by simple stacking of SYP-1 molecules along the z axis but may involve a reorganization of SYP-1 within the SC.

To better define the orientation of SYP-1 within the SC, we modeled the region spanning the two epitopes as a rigid rod and mapped the N terminal localization events to the C terminal distributions (fig. S8; see Materials and Methods). This approach indicated that SYP-1 lies nearly parallel to the plane of the SC in early pachytene and becomes more splayed in late pachytene (Fig. 5, E and F). This reorganization could result from either a change in orientation or conformation of the protein and may be correlated with more extensive condensation of SC proteins, resulting in reduced fluorescence recovery after photobleaching (FRAP), during late prophase (11).

In syp-4^CmutFlag mutant animals, SYP-1 is more diagonally oriented in early pachytene and is even more splayed in late pachytene. Overall, SYP-1 appears disorganized in syp-4^CmutFlag animals (Fig. 5, E and F). Thus, the syp-4^CmutFlag allele affects the organization of proteins within both polycomplexes and SCs assembled between homologous chromosome axes.

We tested whether the conformation of other SC components also changes during meiotic progression or in syp-4^CmutFlag homozygotes. We mapped the C termini of SYP-2 using an antibody raised against a C terminal peptide (19), the C terminal HA epitopes on SYP-3::HA and SYP-4::HA, the N terminus of GFP::SYP-3 (59), and a Flag epitope inserted into the middle of SYP-4. We also mapped the C termini of SYP-2 and SYP-4 in syp-4^CmutFlag mutant animals in late pachytene.

The results are summarized in table S2. All SYP-2, SYP-3, and SYP-4 epitopes localized near the midline of the SC in x, similar to “central element” proteins identified in other species (Fig. 6). We also observed consistent differences for most epitopes between early and late pachytene in wild-type worms and between wild-type and syp-4^CmutFlag animals. The C terminus of SYP-2 localized to the center of the SC in x in early pachytene and syp-4^CmutFlag animals, but we were able to resolve a bimodal distribution with peaks at 18.7 ± 2.4 nm from the center in wild-type animals in late pachytene (Fig. 6A). Similarly, the SYP-3 C terminus was slightly more central in early pachytene (12.3 ± 1.7 nm off center) than late pachytene (16.3 ± 4.5 nm, Fig. 6B). By contrast, a GFP tag at the N terminus of SYP-3 localized to the SC midline in both early and late pachytene, suggesting a head-to-head arrangement of SYP-3 molecules (Fig. 6B). The z distributions of SYP-2 and SYP-3 epitopes expand from early to late pachytene in wild-type but not in syp-4^CmutFlag animals, similar to our findings for SYP-1 (Fig. 6, A and B) further confirming that this mutant prevents or reduces the normal accumulation of SC proteins between homologs in late pachytene, as observed by confocal microscopy (Fig. 2D).

Fig. 6. — (A) 3D-STORM images reveal opposing reorganizations of the C terminus of SYP-2 (antibody targeting the last 20 amino acids of SYP-2) during pachytene in wild-type and *syp-4^CmutFlag* oocytes in both frontal (top) and cross-sectional (bottom) view. (B) Localizations of N (anti-GFP antibody targeting GFP::SYP-3; Table S1) and C termini of SYP-3 (anti-HA antibody targeting SYP-3::HA; table S1) in wild-type animals in early and late pachytene are shown. (C) Localizations of epitope tags inserted internally (anti-Flag antibody targeting SYP-4::intFlag; table S1) or at the C terminus of SYP-4 (anti-HA antibody targeting SYP-4^HA; table S1) in wild-type animals in early and late pachytene are shown. For comparison, the localization of the 3×Flag-tag in C-terminally mutated *syp-4^CmutFlag* animals is also shown. Scale bars in (A) apply to all images.

A change in the axial distribution in early vs. late pachytene was even more pronounced for the C terminus of SYP-4: While this epitope was confined to the central plane in early pachytene and in syp-4^CmutFlag animals, we resolved a bimodal distribution along the z axis in wild-type late pachytene (Fig. 6C). Together, our data reveal changes within the ultrastructure of the SC during pachytene and in syp-4^CmutFlag mutant animals.

DISCUSSION

Through super-resolution imaging, we have found that the ultrastructure of SCs changes during pachytene. While some of our data can be explained by the accumulation of proteins within the SC, we also see evidence of reorientation of components relative to each other (Fig. 7). Moreover, we observed differences in the ultrastructure of both polycomplexes and SCs in syp-4^CmutFlag mutants. In syp-4^CmutFlag homozygotes, SCs and polycomplexes appear disorganized, and the transverse filament protein SYP-1 assumes a more splayed orientation compared to wild-type SCs (Fig. 7). This aberrant SC ultrastructure is accompanied by an increase in the number of “designated COs” marked with bright GFP::COSA-1 foci, and a corresponding increase in genetically detectable COs. While previous studies have revealed that the integrity of the SC is important for CO interference (14, 18–22, 25, 26, 29, 30), our findings suggest that the SC ultrastructure may play a role in regulating CO distribution. However, how the SC mediates CO interference is still contentious (60): Recent findings revealing that regulatory factors can diffuse along or within the SC (38, 61) suggested that interference between early recombination intermediates is mediated by a coarsening process driven by diffusion of pro-CO factors along or within the SC (34, 36, 37, 62).

Fig. 7. — The models of SC organization generated by mapping of N and C terminal distributions of SYP proteins are shown for early (left, blue) and late (right, purple) pachytene in wild-type animals and late pachytene *syp-4^CmutFlag* mutant animals (center, orange; the wild-type late pachytene SC model is shown in the background for comparison). C termini are represented as cubes and N termini (central domain for SYP-4) as spheres. Upper panels show the mean ± 25% of the full width at half-maximum corresponding to the top 42% most likely localization events. The localization of HIM-3 in x is used to define axis localization. Gray loops represent sister chromatids of homologous chromosomes. Models are rendered using POV-ray (version 3.7.0).

Our fortuitous isolation of a separation-of-function allele of syp-4 reveals that changes in the ultrastructure of the SC are associated with severe defects in CO interference: Changes in the SC ultrastructure may modulate the binding or diffusion of CO regulatory factors within the SC. Perturbations applied to nematic liquid crystalline materials such as the SC (10) can change the orientation of molecules (63). Therefore, the designation of a CO event within the SC, which has been shown to distort the SC (35), may even trigger the reorganization of the SC we observed during pachytene in wild-type animals resulting in changes of the physical properties that may modulate the binding or diffusion of CO factors to establish interference along chromosomes (Fig. 7).

The C terminal domain of SYP-4 is unique among C. elegans SC proteins: It lacks sequences predictive of coiled-coil structure, is likely to be intrinsically disordered, and has a very unusual amino acid composition. It is acidic and rich in glycine, which may make it very flexible. Most distinctively, it contains repeated instances in which phenylalanine alternates with other residues, most often asparagine, to form (F-x-F-x-F) motifs, and a terminal FxFF motif. These features are conserved within nematodes, which facilitated our identification of a SYP-4 ortholog in Pristionchus pacificus, a distant relative of C. elegans (64). Moreover, the C terminus of SIX6OS1, an essential component of the central element in mice (65), contains very similar motifs, along with a similar amino acid composition and predicted disorder. An amino acid polymorphism in this domain was linked to elevated recombination rates in women in an Icelandic population (66). Thus, it seems likely that C terminal domain of SYP-4 is a unique interface involved in CO regulation, and that this function may be conserved between nematodes and mammals.

MATERIALS AND METHODS

Biological resources

A complete list of C. elegans strains used in this study can be found in table S1. All strains were cultured at 20°C using standard methods (67). GFP::SYP-3 is a single-copy transgene inserted by MosSCI (59). Other tags were inserted using CRISPR-Cas9 genome editing as described in (42). In brief, Cas9 and gRNA were delivered by microinjection, either encoded on a plasmid (68) or as Cas9-ribonucleoprotein complexes preassembled in vitro. Repair templates for small epitope tags were codon optimized for C. elegans (69) and synthesized as “Ultramers” by Integrated DNA Technologies (table S3).

Brood counts

To assess the impact of meiosis on progeny production in C. elegans mutants, individual L4 animals were transferred to fresh OP50 plates every 24 hours and the laid eggs and hatched L1s were counted after each transfer until no eggs were found. The surviving progeny were counted 2 to 4 days after to determine viability and the incidence of males. Note that some eggs and L1s are typically missed because of their small size, resulting in reported egg viabilities higher than 100%.

Immunofluorescence

Gonads were dissected from young adults (24 hours post-L4) and immunofluorescence was performed as described (70) with modifications described in (42). The following primary antibodies, all of which have been previously described, were used: goat anti–SYP-1 (1:500, affinity purified) (57), mouse anti-HA (1:500, monoclonal 2-2.2.14, Thermo Fisher Scientific catalog no. 26183-A647, RRID:AB_2610626), rabbit anti–SYP-2 (1:500, affinity purified) (71), rabbit anti–SYP-5 (1:500) (22), mouse anti-GFP (1:500, monoclonal, Roche catalog no. 11814460001, RRID:AB_390913), mouse anti-Flag (1:500, monoclonal M2, Sigma-Aldrich catalog no. F1804, RRID:AB_262044), chicken anti–HTP-3 (1:500) (71), rabbit anti–HIM-3 (1:500, SDQ4713 modENCODE project) (72), rabbit anti–HIM-8pT64 (1:2000) (44), and rabbit anti–RAD-51 (1:500) (57). Commercial secondary antibodies were raised in donkeys and fluorescently labeled with Alexa Fluor 488, Cy3, or Alexa Fluor 647 (1:500, Jackson ImmunoResearch and Invitrogen). Gonads were mounted in ProLong Gold antifade mountant (Thermo Fisher Scientific), and epifluorescence images were acquired on a DeltaVision Elite microscope (Applied Precision) using a 100× numerical aperture (NA) 1.4 oil-immersion objective. Images of nuclei at diakinesis (fig. S4) were acquired on a Zeiss LSM880 AiryFast system using a 63× NA 1.4 oil-immersion objective.

To quantify the length of the transition zone (TZ), we measured the region of the gonad from the first appearance of nuclei with condensed, crescent-shape chromosome morphology to the first row consisting of only pachytene nuclei (lacking crescent-shaped DNA) using the poly-line feature in Fiji (73). This was normalized by dividing by the length of the region from TZ entry to the end of pachytene, just before cells form a single file. Similarly, the “CHK-2 active zone” was defined as the length of the region containing nuclei with at least one bright focus of immunofluorescence using an antibody that recognizes phosphorylated HIM-8 and ZIM proteins (PZims), also normalized to the length of the region from TZ to end of pachytene.

RAD-51 foci were quantified from maximum intensity projections of the bottom (coverslip-adjacent) half of whole gonads in Fiji by (i) subtracting the background in 4′,6-diamidino-2-phenylindole (DAPI) and RAD-51 channels (50 pixel radius) and (ii) segmenting the nuclei by smoothing, blurring, and thresholding the DAPI channel. Holes in segmented nuclei were filled in manually, and a watershed transformation was applied to define nuclear masks. (iii) RAD-51 foci within each area were counted using the “Find Maxima” algorithm in Fiji.

To measure the lengths of SCs, SC or chromosome axis staining was traced manually in Fiji using the Simple Neurite Tracer plugin (74). Only chromosomes with well-defined end points and contiguous staining were traced. To measure the distribution of GFP::COSA-1 foci along individual chromosomes, maxima of the intensity in the GFP::COSA-1 channel along the 3D traces were identified using the “findpeaks” function in R followed by a manual assessment of the data for quality control. To obtain the number of foci per nucleus, GFP::COSA-1 foci were counted manually in 3D stacks in Fiji.

To quantify protein levels within the SC by immunofluorescence, imaging was performed on an Olympus IXplore SpinSR spinning disk confocal microscope with a 60× 1.42-NA oil-immersion objective using the SoRa disc. 3D stacks of wild-type (N2) and syp-4^ha3′UTR(ie27); meIs8 or syp-4^CmutFlag(ie25); meIs8 animals were acquired from the same slide. For each gonad, 3D image stacks were tiled using the BigStitcher plugin in Fiji. SCs were then segmented by thresholding DAPI and HTP-3 (chromosome axis) using Python (version 3.8). Specifically, foreground was set to intensity values larger than the Otsu threshold of the 25 or 10% brightest pixels of the DAPI and HTP-3 channels, respectively. The DAPI and HTP-3 masks were then multiplied to remove cytoplasmic background in the HTP-3 masks and obtain masks for SCs. Only SC masks larger than 100 voxels corresponding to 0.017 μm³ were used for further analysis. For each SC mask, the median SYP intensity was normalized to the median intensity of the corresponding HTP-3 signal. The regions of the gonads containing TZ and pachytene nuclei were divided into 10 zones of equal length, and the mean normalized SYP intensity, weighted by the volume of the SC mask, was calculated for each gonad. Independent experiments were normalized to corresponding wild-type controls. Figure S2A shows representative maximal intensity projections with equally scaled intensity values.

Analysis of CO interference

CO interference strength was analyzed by calculating the CoC by dividing the observed probability of finding two GFP::COSA-1 foci separated at a given distance by the expected likelihood of finding two foci at the given distance. No distinction between individual chromosomes was made, and all chromosome lengths were normalized. The expected likelihood was calculated for each genotype from the observed distributions of the GFP::COSA-1 foci.

STORM and PALM imaging

Super-resolution imaging of immunostained, intact germline tissue was carried out as described (42). For STORM, secondary antibodies raised in donkeys or goats labeled with Alexa Fluor 647 were used (1:500, Jackson ImmunoResearch and Invitrogen). Following acquisition of the immunofluorescence signal, a fluorescently tagged positional reference protein, mEos2::HIM-3 or mMaple3::HIM-3 (42) was imaged using PALM (75, 76). For early pachytene images, mEos2::HIM-3 was costained with a rabbit anti–HIM-3 antibody and a donkey anti-rabbit secondary antibody (Jackson ImmunoResearch) labeled with CF568-NHS ester to achieve a 2:1 dye-to-antibody ratio. All images were rotated about the optical axis such that the direction of the chromosome axes corresponds to the y axis. Aligned and averaged images (fig. S6) were then used to generate histograms of localization events in x and z (42). To systematically distinguish between mono- and bimodal distributions, we evaluated fits with one and two Gaussians using an analysis of variance (ANOVA) test in R. Distributions were considered to be bimodal when the ANOVA test indicated that the data were significantly different from a monomodal distribution (P < 0.05). The P values are listed in table S2. SDs of fit parameters were estimated by a subsampling approach using subsets of half the number of individual SC stretches (42). The results are summarized in table S2.

Statistical analysis

Sample sizes were not predetermined and experiments were not randomized. All imaging experiments were conducted at least twice; all other experiments, such as recombination mapping by whole-genome sequencing, were performed on multiple biological replicates, as indicated. The investigators were not blinded to allocation during experiments and outcome assessment. For STORM experiments, the total lengths and number of stretches analyzed are summarized in table S2. P values were calculated by two-sided Mann-Whitney-Wilcoxon tests in R (version 4.1.2).

Analysis of SC organization

To derive a model of SC organization from our data, we treated each protein as a rigid rod and determined which orientation was most consistent with the distributions of localization events mapped by STORM. We first removed the most extreme 2.5% localization events using squared Mahalanobis distances in R (version 3.4.2). We then mapped each localization event corresponding to the nth-percentile in x and the mth-percentile in z of the N terminal (or C terminal) distribution to a randomly selected localization event within the nth ± 7.5% in x and mth ± 7.5% in z of the corresponding C terminal (or N terminal) distribution. This analysis yields a distance between each pair of N and C terminal localization events and an orientation of the protein within the SC. Each localization event may be as far as 20 nm from the epitope, since we used unlabeled primary and dye-labeled secondary antibodies; thus, we expect that these distances may vary by ±40 nm relative to the true distance. Some pairs of localization events between the N- and C terminal distributions yield improbably short or long distances between N and C terminal antibodies. We restricted our analysis to mapping events that yield distances between the fifth and 95th percentile of all distances mapped. Using this approach, the distances for all conditions were between 11.6 and 120 nm (fig. S8B).

Recombination mapping

To map meiotic COs in syp-4^CmutFlag(ie25) homozygotes, this allele (which was generated in the N2 Bristol background) was introgressed into the divergent Hawaiian strain CB4856 by eight sequential crosses. Hawaiian and N2 strains homozygous for either wild-type syp-4 or syp-4^CmutFlag(ie25) were crossed to each other, and the hybrid F1 progeny were backcrossed to Hawaiian syp-4⁺ males. The resulting F2 progeny inherit one haploid set of CB4856 chromosomes and a set of potentially recombinant N2/CB4856 maternal chromosomes. To map recombination events, F2 hermaphrodites were plated individually, allowed to reproduce for one generation, and the genomic DNA of their pooled progeny was isolated by phenol-chloroform extraction. Illumina sequencing libraries were prepared as described in (77) and sequenced as 50–base pair single reads on a HiSeq2500 Illumina sequencer at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, with a coverage of 2 to 4× genomes per sample. Reads were mapped to reference genomes of the N2 (Wormbase release WS230) and CB4856 (78) strains. Genotypes were called using the multiplexed shotgun genotyping toolbox with default parameters (79). The aligned sequences are available through the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database under accession number SRP126693. COs that occurred in oogenesis were detected as transitions from Hawaiian/Bristol heterozygous stretches to homozygous Hawaiian stretches. The observed number of COs was doubled, since only half of the total that occur in each meiotic cell are inherited by individual progeny.

Electron microscopy

High-pressure freezing, freeze substitution, sample preparation, and microscopy were performed as described previously (10, 80, 81). Images were acquired on a Tecnai 12 transmission electron microscope (120 kV, FEI, Hillsboro, OR) equipped with a Gatan Ultrascan 1000 CCD camera (Pleasanton, CA).

Immunoblotting

To compare expression levels of SYP proteins, 120 adult worms were lysed by boiling in 40 μl of Laemmli sample buffer with β-mercaptoethanol for about 5 min, until particulate matter was not detected using a dissection stereomicroscope. Whole-worm lysates were electrophoresed using NuPAGE 4 to 12% polyacrylamide gradient gels and transferred to polyvinylidene difluoride membranes. Primary antibodies were rabbit (Thermo Fisher Scientific catalog no. PA1-985, RRID:AB_559366, used for HA::SYP-1) or mouse anti-HA (SYP-4) and mouse anti–α-tubulin (DM1A, Millipore catalog no. 05-829, RRID:AB_310035), each diluted 1:5,000. Horseradish peroxidase (HRP)–conjugated secondary antibodies (Jackson Immuno-Research Labs catalog no. 715-035-151, RRID:AB_2340771 and no. 711-035-152, RRID:AB_10015282) were detected using ECL reagents (Amersham). SYP-4::HA and tubulin were detected using the same HRP-conjugated anti-mouse 2° antibody. HA::SYP-1 was detected using HRP anti-rabbit/ECL and tubulin was detected by Cy3-conjugated anti-mouse secondary antibody (1:5,000). Images were recorded using a Chemidoc system (Bio-Rad) and quantified using Fiji.

Acknowledgments

Some C. elegans strains used in this work were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the NIH – Office of Research Infrastructure Programs (P40 OD010440). We thank the Advanced Light Microscopy Facility (ALMF) at the European Molecular Biology Laboratory (EMBL) for support. We thank Y. Kim for sharing the SYP-5 antibody.

Funding: This work was supported by a postdoctoral fellowship of the Human Frontier Science Program to S.K. (LT000903/2013-C), an NSF Graduate Research Fellowship (DGE 1106400) to M.W., NIH/NIGMS R35GM149349 and the Pew Charitable Trusts to K.X., and support to A.F.D. from the National Institutes of Health (R01 GM065591) and the Howard Hughes Medical Institute.

Author contributions: Conceptualization: S.K., M.W., K.X., and A.F.D. Methodology: S.K. and M.W. Investigation: S.K. and M.W. Formal analysis: S.K. and M.W. Visualization: S.K. and M.W. Supervision: K.X. and A.F.D. Writing–original draft: S.K. and A.F.D. Writing–review and editing: S.K., K.X., and A.F.D. Funding acquisition: S.K., M.W., K.X., and A.F.D.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. NGS datasets generated in this study are available on the NCBI SRA database under accession number SRP126693 (https://ncbi.nlm.nih.gov/sra/SRP126693). Code generated for this study is available on Zenodo (https://doi.org/10.5281/zenodo.14518632). Materials such as C. elegans strains generated for this study are available upon request.

Supplementary Materials

The PDF file includes:

Figs. S1 to S8

Tables S1 to S3

Legend for data S1

sciadv.adq9374_sm.pdf^{(5.7MB, pdf)}

Other Supplementary Material for this manuscript includes the following:

Data S1

sciadv.adq9374_data_s1.zip^{(4.7MB, zip)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S8

Tables S1 to S3

Legend for data S1

sciadv.adq9374_sm.pdf^{(5.7MB, pdf)}

Data S1

sciadv.adq9374_data_s1.zip^{(4.7MB, zip)}

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PERMALINK

Dynamic molecular architecture of the synaptonemal complex

Simone Köhler

Michal Wojcik

Ke Xu

Abby F Dernburg

Roles

Abstract

INTRODUCTION

RESULTS

Perturbation of CO interference by a separation-of-function mutation in the C terminus of SYP-4

Fig. 1. Identification of two partially functional syp-4 alleles.

Fig. 2. Synapsis is delayed by reduced expression of SYP-4 but not by the syp-4CmutFlag allele.

Fig. 3. CO interference is reduced in syp-4CmutFlag mutants.

Loss of “interference” in polycomplexes with C-terminally mutated SYP-4

Fig. 4. Irregular organization and an increase in GFP::COSA-1 foci in syp-4CmutFlag polycomplexes.

Dynamic architecture of the SC in C. elegans

Fig. 5. Super-resolution microscopy reveals distinct 3D organizations of SYP-1 during pachytene and in syp-4CmutFlag mutant animals.

Fig. 6. Individual components of the SC are distinctly reorganized during pachytene.

DISCUSSION

Fig. 7. A probabilistic model reveals distinct ultrastructural states of the SC.

MATERIALS AND METHODS

Biological resources

Brood counts

Immunofluorescence

Analysis of CO interference

STORM and PALM imaging

Statistical analysis

Analysis of SC organization

Recombination mapping

Electron microscopy

Immunoblotting

Acknowledgments

Supplementary Materials

The PDF file includes:

Other Supplementary Material for this manuscript includes the following:

REFERENCES AND NOTES

Associated Data

Supplementary Materials

ACTIONS

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Links to NCBI Databases

Fig. 2. Synapsis is delayed by reduced expression of SYP-4 but not by the syp-4^CmutFlag allele.

Fig. 3. CO interference is reduced in syp-4^CmutFlag mutants.

Fig. 4. Irregular organization and an increase in GFP::COSA-1 foci in syp-4^CmutFlag polycomplexes.

Fig. 5. Super-resolution microscopy reveals distinct 3D organizations of SYP-1 during pachytene and in syp-4^CmutFlag mutant animals.