Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3220.
Howard Hughes Medical Institute, Chevy Chase, MD 20815.
Proc Natl Acad Sci U S A. 2017 Jun 13;114(24):E4734-E4743. doi: 10.1073/pnas.1702312114. Epub 2017 May 30.
When cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC) and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the 3D organization of components within the axes, which include cohesin complexes and additional meiosis-specific proteins. Here, we investigate the molecular organization of meiotic chromosome axes in through STORM (stochastic optical reconstruction microscopy) and PALM (photo-activated localization microscopy) superresolution imaging of intact germ-line tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using 3D averaging, we determined the position of all known components within synapsed chromosome axes to high spatial precision in three dimensions. We find that meiosis-specific HORMA domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Although cohesin complexes containing the kleisin REC-8 protrude above and below the plane defined by the SC, complexes containing COH-3 or -4 kleisins form a central core, which may physically separate sister chromatids. This organization may help to explain the role of the chromosome axes in promoting interhomolog repair of meiotic double-strand breaks by inhibiting intersister repair.
当细胞进入减数分裂时，它们的染色体重新组织为线性染色质环阵列，锚定在中央轴上。减数分裂染色体轴形成联会复合体 (SC) 组装的平台，并在其他减数分裂过程中发挥核心作用，包括同源配对、重组和染色体分离。然而，对于轴内成分的 3D 组织，包括黏合复合物和其他减数分裂特异性蛋白质，知之甚少。在这里，我们通过对完整生殖系组织进行 STORM（随机光学重建显微镜）和 PALM（光激活定位显微镜）超分辨率成像，研究了减数分裂染色体轴的分子组织。通过用光可转换荧光蛋白标记一个轴蛋白 (HIM-3)，我们建立了其他成分的空间参考，这些成分使用针对通过 CRISPR/Cas9 基因组编辑插入的表位标签的抗体进行定位。通过 3D 平均，我们以高精度确定了在三维空间中所有已知成分在联会染色体轴内的位置。我们发现，减数分裂特异性 HORMA 结构域蛋白在黏合复合物和 SC 中心区域之间跨越一个间隙，这与其在 SC 组装中的关键作用一致。我们的数据进一步表明，两种不同的减数分裂黏合复合物在轴内的排列明显不同：尽管含有 kleisin REC-8 的黏合复合物突出在 SC 定义的平面之上和之下，但含有 COH-3 或 -4 kleisin 的复合物形成一个中央核心，这可能在物理上分离姐妹染色单体。这种组织可能有助于解释染色体轴在促进减数分裂双链断裂的同源修复中的作用，通过抑制姐妹染色单体修复。
