Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy.

The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20-30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.

Quantitative basis of meiotic chromosome synapsis analyzed by electron tomography.

The synaptonemal complex is a multiprotein complex, which mediates the synapsis and recombination between homologous chromosomes during meiosis. The complex is comprised of two lateral elements and a central element connected by perpendicular transverse filaments (TFs). A 3D model based on actual morphological data of the SC is missing. Here, we applied electron tomography (ET) and manual feature extraction to generate a quantitative 3D model of the murine SC. We quantified the length (90 nm) and width (2 nm) of the TFs. Interestingly, the 80 TFs/µm are distributed asymmetrically in the central region of the SC challenging available models of SC organization. Furthermore, our detailed 3D topological analysis does not support a bilayered organization of the central region as proposed earlier. Overall, our quantitative analysis is relevant to understand the functions and dynamics of the SC and provides the basis for analyzing multiprotein complexes in their morphological context using ET.

Electron tomography of mouse LINC complexes at meiotic telomere attachment sites with and without microtubules.

Telomere movements during meiotic prophase I facilitate synapsis and recombination of homologous chromosomes. Hereby, chromosome movements depend on the dynamic attachment of meiotic telomeres to the nuclear envelope and generation of forces that actively move the telomeres. In most eukaryotes, forces that move telomeres are generated in the cytoplasm by microtubule-associated motor proteins and transduced into the nucleus through the LINC complexes of the nuclear envelope. Meiotic LINC complexes, in mouse comprised of SUN1/2 and KASH5, selectively localize to the attachment sites of meiotic telomeres. For a better understanding of meiotic telomere dynamics, here we provide quantitative information of telomere attachment sites that we have generated with the aid of electron microscope tomography (EM tomography). Our data on the number, length, width, distribution and relation with microtubules of the reconstructed structures indicate that an average number of 76 LINC complexes would be required to move a telomere attachment site.

EM Tomography of Meiotic LINC Complexes.

Electron microscope (EM) tomography is a powerful technique that enables the three-dimensional analysis of subcellular structures at high resolution. We have applied this method to the quantitative analysis of LINC complex distribution and interaction with the cytoskeleton in meiotic cells from male mice. In this chapter, we describe methods to generate and analyze the tomograms.