Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy

Abstract

During prophase I of meiosis, chromosomes become organized as loop arrays around the proteinaceous chromosome axis. As homologous chromosomes physically pair and recombine, the chromosome axis is integrated into the tripartite synaptonemal complex (SC) as this structure's lateral elements (LEs). While the components of the mammalian chromosome axis/LE-including meiosis-specific cohesin complexes, the axial element proteins SYCP3 and SYCP2, and the HORMA domain proteins HORMAD1 and HORMAD2-are known, the molecular organization of these components within the axis is poorly understood. Here, using expansion microscopy coupled with 2-color stochastic optical reconstruction microscopy (STORM) imaging (ExSTORM), we address these issues in mouse spermatocytes at a resolution of 10 to 20 nm. Our data show that SYCP3 and the SYCP2 C terminus, which are known to form filaments in vitro, form a compact core around which cohesin complexes, HORMADs, and the N terminus of SYCP2 are arrayed. Overall, our study provides a detailed structural view of the meiotic chromosome axis, a key organizational and regulatory component of meiotic chromosomes.

Keywords: STORM; chromosome axis; expansion microscopy; meiosis; synaptonemal complex.

Fig. 1.

The ExSTORM procedure produced homogenously expanded samples for meiotic chromosome axis proteins, with improved resolution. (A) A STORM image (magenta) of a hypotonically spread mouse pachytene spermatocyte stained for SYCP3. (B) The magnified view of 1 meiotic chromosome within the boxed region in A. (C) ExSTORM image (green) of the same chromosome after expansion. (D) Overlay of (B) preexpansion image (magenta) and (C) postexpansion image (green). Overlapped signals are shown in white. (E) The magnified view of the blue boxed region in D. (F) Line profiles of SYCP3 intensity taken along the yellow line in E, with ExSTORM data showing sharper peak distributions than STORM data. (G) Expansion ratio values (diamonds) calculated by comparing preexpansion and postexpansion STORM images from 5 independent expansion samples, with mean value (red line) and SD (black bar) of 4.09 ± 0.14. (H) FRC measurement of resolution shows a 2D resolution of 52.2 nm for the STORM image (B) and 14.8 nm for the ExSTORM image (C). Similar FRC analysis on 5 independent expansion samples yields resolutions of 48.5 ± 7.3 nm (SD) for STORM and 17.9 ± 4.4 nm (SD) for ExSTORM. (I) Root-mean-square (RMS) length measurement error [mean (squares) + 1 SD (bars) for 5 independent expansion samples] in between preexpansion and postexpansion STORM images following the method in ref. . The measurement error is 10 nm at length scale of 1 µm, suggesting a distortion of 1%. (J) Zoom-in view of (I) at 0- to 100-nm length range. The measurement error is 0.96 nm at length scale of 50 nm, suggesting a distortion of 1.9%. (Scale bars: 2 μm in A; 500 nm in B–D; 200 nm in E.)

Fig. 2.

Two-color ExSTORM images of spread chromosomes immunostained for SYCP2 and SYCP3. (A) Schematic of the M. musculus SYCP2 protein, with its N-terminal domain (NTD; residues 1 to 394), closure motif (blue), and C-terminal coiled-coil (CC) domain indicated. SYCP2 likely interacts with HORMADs and SYCP3 through its closure motif and CC domain, respectively. The immunogenic regions for the SYCP2-N and SYCP2-C antibodies are marked. (B) Two-color ExSTORM image on spread chromosomes labeled with SYCP3 and SYCP2 C terminus. (C–E) Zoom-in views of the boxed region shown in B, with SYCP2 C terminus showing a similar width to SYCP3, as analyzed by distance distributions (F) (from 3 cells, 4 chromosomes), with Gaussian fits in SI Appendix, Fig. S16A. (G) Two-color ExSTORM image on spread chromosomes labeled with SYCP3 and SYCP2 N terminus. (H–J) Zoom-in views of the boxed region shown in G, with SYCP2 N terminus showing broader width than that of SYCP3, as analyzed by distance distributions (K) (from 2 cells, 4 chromosomes), with Gaussian fits in SI Appendix, Fig. S16B. (Scale bars: 1 μm in B and G; 200 nm in C–E and H–J.)

Fig. 3.

Two-color ExSTORM images of spread chromosomes immunostained for meiotic HORMADs. (A–C) ExSTORM image on a pachytene chromosome labeled with SYCP3 and HORMAD1. (D) Distance distributions (from 4 cells, 6 chromosomes) showing that HORMAD1 has broader width than SYCP3, with Gaussian fits in SI Appendix, Fig. S16D. (E–G) An axial ExSTORM image on a pachytene chromosome labeled with SYCP3 and HORMAD1, showing most HORMAD1 signals are outside the SYCP3 core. (H–J) ExSTORM image on a pachytene chromosome labeled with SYCP3 and HORMAD2. (K) Distance distributions (from 3 cells, 5 chromosomes) showing that HORMAD2 spans wider than SYCP3, with Gaussian fits in SI Appendix, Fig. S16E. (Scale bars: 200 nm.)

Fig. 4.

Two-color ExSTORM images of spread chromosomes immunostained for cohesin subunits. (A) Schematic of a meiotic cohesin complex containing 4 subunits: 2 SMCs (purple and cyan), 1 kleisin (green), and 1 stromal antigen protein (orange oval). (B–D) ExSTORM image of a meiotic chromosome labeled with SMC3 C terminus and SYCP3, with SMC3 showing broader width along LE than that of SYCP3, as analyzed by distance distributions (from 4 cells, 5 chromosomes) in E, with Gaussian fits in SI Appendix, Fig. S16H. (F) ExSTORM image of a meiotic chromosome labeled with RAD21L and SYCP3, with distance distributions (from 4 cells, 4 chromosomes) shown in G, and Gaussian fits in SI Appendix, Fig. S16I. (H) ExSTORM image of a meiotic chromosome labeled with STAG3 and SYCP3, with distance distributions (from 3 cells, 6 chromosomes) shown in I, and Gaussian fits in SI Appendix, Fig. S16J. (Scale bars: 200 nm.)

Fig. 5.

Model of meiotic chromosome axis organizations in mouse pachytene chromosomes. (A) Overlay of the localization distributions of the major LE/axis components, determined from their 2-color experiments with respect to SYCP3. The ±1 SD boundaries for the SYCP3 distribution curves over these experiments are shown as dashed lines. The distance coordinate is plotted with respect to the N terminus of SYCP1, i.e., the middle of SC central region. (B) Model of the protein organizations in synapsed meiotic chromosome axes in the axial view.