The synaptonemal complex aligns meiotic chromosomes by wetting

Abstract

During meiosis, the parental chromosomes are drawn together to enable exchange of genetic information. Chromosomes are aligned through the assembly of a conserved interface, the synaptonemal complex, composed of a central region that forms between two parallel chromosomal backbones called axes. Here, we identify the axis-central region interface in C. elegans, containing a conserved positive patch on the axis component HIM-3 and the negative C terminus of the central region protein SYP-5. Crucially, the canonical ultrastructure of the synaptonemal complex is altered upon weakening this interface using charge-reversal mutations. We developed a thermodynamic model that recapitulates our experimental observations, indicating that the liquid-like central region can assemble by wetting the axes without active energy consumption. More broadly, our data show that condensation drives tightly regulated nuclear reorganization during sexual reproduction.

Fig. 1.. The HORMA domain of HIM-3 is required for axis interactions with the SC-CR.

(A) Top left: Assembled synaptonemal complex with the darkly stained parental chromosomes to its side (left) and of the polycomplexes that form in cohesin(−) worms (right) as seen in negative negative stain electron micrographs [adapted from (17)]. Bottom left: Interpretive diagrams colored magenta for the SC-CR, green for the axes (also called lateral or axial elements), and blue for chromatin. (B) Models depicting the worm components of the axis (top) and the SC-CR (bottom). The position of each of these components within the synaptonemal complex is based on (12, 25, 29, 30, 51). The pairs HTP-1/2, SYP-5/6, and SKR-1/2 are each partially redundant with each other. (C) Pachytene nuclei from worms of the indicated genotypes stained for the SC-CR component SYP-5 (red) and the axis components HIM-3 (green) and HTP-3 (magenta). The merged images on the right also show DNA [4′,6-diamidino-2-phenylindole (DAPI), blue]. The HTP-3 antibody weakly cross-reacts with the nucleolus. Scale bar, 1 μm. Gene models of HIM-3, with the HORMA domain and the closure motif highlighted, are shown to the right. Regions deleted are denoted by red brackets. See fig. S1A for images of the gonads. (D) Quantification of the images in (C). The enrichment at polycomplexes relative to the nucleoplasm was done using line scans. Normalized HIM-3 enrichment was calculated by dividing HIM-3 enrichment by SYP-5 enrichment. N = 14 and 40 for him-3⁺ and him-3^ΔC-term. Brown-Forsythe and Welch analysis of variance (ANOVA) P = 0.09, 0.005, and 0.64 for SYP-5, HIM-3, and HIM-3 (normalized). ns, not significant.

Fig. 2.. Axis interactions with the SC-CR are mediated by a positive patch on the HORMA domain of HIM-3.

(A) Structural models of the meiotic HORMA proteins, with surface charge plotted in a red-blue scale. The structures of HTP-1 and HIM-3 are from (26). The models of HTP-3 and the three HIM-3 mutants were generated in AlphaFold (45). Bottom: Secondary structural models, with amino acids constituting the positive patch on HIM-3 (positions 170, 171, 174, 177, and 178), and the analogous positions in HTP-1 and HTP-3 are shown as surfaces colored according to charge. Note that the positive patch on HIM-3 is located away from the closure motif binding pocket; see fig. S2A. (B) Pachytene nuclei from worms of the indicated genotypes stained for the SC-CR component SYP-5 (red) and the axis components HIM-3 (green) and HTP-3 (magenta). The merged images on the right also show DNA (DAPI, blue). The HTP-3 antibody weakly cross-reacts with the nucleolus. Scale bar, 1 μm. See fig. S2D for images of the gonads and fig. S3 for similar analysis in live gonads. (C to E) Quantification of the images in (B). The enrichment at polycomplexes relative to the nucleoplasm was done using line scans. Normalized enrichment (E) was calculated by dividing HIM-3 enrichment by SYP-5 enrichment. N = 14, 15, 12, and 12 for him-3(+), him-3^KK170-171EE, him-3^R174E, and him-3^KK177-178EE. (C) Brown-Forsythe and Welch ANOVA P[him-3(+) versus him-3^KK170-171EE] = 0.99; P[him-3(+) versus him-3^R174E] = 0.99; P[him-3(+) versus him-3^KK177-178EE] > 0.99. (D) Brown-Forsythe and Welch ANOVA P[him-3(+) versus him-3^KK170-171EE] = 0.0002; P[him-3(+) versus him-3^R174E] = 0.0015; P[him-3(+) versus him-3^KK177-178EE] = 0.0002. (E) Brown-Forsythe and Welch ANOVA P[him-3(+) versus him-3^KK170-171EE] = 0.0053; P[him-3(+) versus him-3^R174E] = 0.036; P[him-3(+) versus him-3^KK177-178EE] = 0.0078.

Fig. 3.. Lowering SC-CR affinity for the axes perturbs synapsis.

(A) Total self-progeny from hermaphrodites of the indicated genotypes. (B) Percentage of males among self-progeny of hermaphrodites of the indicated genotypes, indicative of meiotic X chromosome non-disjunction. (C) Pachytene nuclei stained for the SC-CR component SYP-5 (red) and the axis component HIM-3 (green), with merged images shown on the right. Note the extensive asynapsis in the him-3 mutants (i.e., axes lacking SC-CR staining) despite loading of the mutated HIM-3 proteins onto the axis. Scale bar, 10 μm. See fig. S4A for images of the gonads. (D) Quantification of the images in (C), indicating a smaller number of synapsed chromosomes in him-3 mutants. (E) Chiasmata number deduced from the number of DAPI bodies at diakinesis. Wild-type animals undergo one chiasma per chromosome, for a total of six chiasmata per nucleus. (F) STED microscopy images of pachytene nuclei stained for the SC-CR component SYP-2 (red in the merged image) and the axis component HTP-3 (green in the merged image). An example of a line scan through a synapsed chromosome is shown above the HTP-3 staining in wild-type animals. Scale bar, 1 μm. (G) Quantification of different synapsis phenotypes in STED images, as shown in (F). “Wide synapsis” indicates parallel axes separated by more than 150 nm, as shown in the top nucleus from him-3^KK170-171EE animals. “Loose axis associations” indicate axes wrapped around SC-CR structures, as shown in the bottom nucleus from him-3^KK170-171EE animals. (H) Inter-axes distance in the indicated genotypes, measured from nuclei stained as in (F). Distance was measured only between parallel axes that had unilamellar SYP-2 staining. See Materials and Methods for full statistical information. ****P ≤ 0.0001.

Fig. 4.. The C terminus of SYP-5 contributes to SC-CR interactions with the axis.

(A) The C terminus of SYP-5 (amino acids 515 to 547), with positively and negatively charged residues colored in blue and red, respectively, and charge reversing mutations below. (B) Pachytene nuclei of the indicated genotypes stained for the SC-CR component SYP-2 (red) and the axis component HIM-3 (green). The merged images on the right also show DNA (DAPI, blue). The syp-5^6K mutant fails to form polycomplexes, likely due to perturbed self-interactions of the SC-CR. Scale bar, 2 μm. See fig. S6A for images of the gonads. (C to E) Quantification of the enrichment of SYP-2 and HIM-3 to polycomplexes. While the SC-CR is less enriched at polycomplexes in syp-5^5K animals, these polycomplexes recruit more HIM-3. In (E), HIM-3 enrichment is normalized to SYP-2 enrichment. (F) STED microscopy images of pachytene nuclei stained for SYP-2 (red in the merged image) and the axis component HTP-3 (green). Scale bar, 1 μm. (G) The number of SC-CR structures per pachytene nuclei in the indicated genotypes. (H) Inter-axes distance in the indicated genotypes, measured from nuclei stained as in (F). Distance was measured only for him-3^KK170-171EE syp-5^6K mutants, where the parallel axes exhibited unilamellar SC-CR staining, and is compared to the data from Fig. 3H. (I) Quantification of synapsis phenotypes in STED images, as shown in (F). See Fig. 3G for more details. (J) Anti–HIM-3 Western blot showing the interaction of purified HTP-3–HIM-3 complex containing His₆-tagged HTP-3 and wild-type (WT) HIM-3, which served as “prey,” with “bait” biotinylated peptides spanning residues 528 to 547 of SYP-5, either unmodified or phosphorylated at S541 (SYP-5 Phos). The interaction was qualitatively weaker with charge reversal mutations in HIM-3. See fig. S8 for complete blots and additional controls. See Materials and Methods for full statistical information.

Fig. 5.. Parameters for the thermodynamic model of synaptonemal complex assembly.

(A) Parameters used to model synaptonemal complex assembly. Sources: (12, 25, 52, 53). (B) Distance between the “ladder rungs” in negative stain electron microscopy images from (17). Each point represents an individual measurement between adjacent “rungs.” N = 28 and 27 for polycomplexes and assembled SC-CR. Welch’s t test P = 0.12. (C) Total nuclear fluorescence of GFP–HIM-3 and GFP–SYP-3 in pachytene nuclei, yielding a ratio of 1:1.2 GFP–SYP-3 to GFP–HIM-3. N = 100 and 90 for GFP–SYP-3 and GFP–HIM-3. Welch’s t test P < 0.0001. (D) The fraction of GFP–SYP-3 on chromosomes in animals of the indicated genotypes is significantly lower in him-3^R174E and him-3^KK170-171EE mutants. N = 15, 15, and 21 for him-3(+), him-3^R174E, and him-3^KK170-171EE. Brown-Forsythe and Welch ANOVA P[him-3(+) versus him-3^KK107-171EE] < 0.0001; P[him-3(+) versus him-3 ^KK107-171EE] < 0.0001; P(him-3(+) versus him-3 ^KK107-171EE] = 0.0011. (E) Polycomplex volume calculated based on the dimensions of polycomplexes in negative stain electron microscopy images from (17). Given the mostly spherical appearance of polycomplexes, the z dimension is assumed to be the average of the widths and height. Each point indicates a single polycomplex. N = 10. A.U., arbitrary units.

Fig. 6.. Results of the thermodynamic model of synaptonemal complex assembly.

(A) Predicted number of synapsed chromosomes as a function of $\mathrm{\alpha }=\frac{e_{\text{SH}}}{e_{\text{SS}}}$. The condensate volume is held constant V_c = 0.1 μm³. Dashed arrows indicate how the number of synapsed chromosomes in wild type and him-3^R174E allows to deduce the values of α. (For simplicity, we ignore here the slight reduction (8%) in V_c in him-3^R174E worms.) (B) Contour plot of the predicted number of synapsed chromosomes (black lines) as a function of V_c and α. The orange and yellow lines indicate threshold SC-CR thickness of 90 and 100 nm, respectively, derived based on the total condensate volume, the dimensions of the SC-CR, and the number of synapsed chromosomes. The green, blue, and red asterisks denote the position of wild-type, him-3^R174E, and him-3^KK170-171EE worms, respectively. The black arrow and asterisk indicate the effect of combining the syp-5 mutations with him-3^KK170-171EE. Top right: Example images of the mutations shown on the contour plot, with the axis stained in green and the SC-CR in red. See Supplementary Text for more details.