A cryo-fixation protocol to study the structure of the synaptonemal complex

Abstract

Genetic variability in sexually reproducing organisms results from an exchange of genetic material between homologous chromosomes. The genetic exchange mechanism is dependent on the synaptonemal complex (SC), a protein structure localized between the homologous chromosomes.Current structural models of the SC are based on electron microscopy, super resolution, and expansion microscopy studies using chemical fixatives and sample dehydration of gonads, which are methodologies known to produce structural artifacts. We have developed a novel electron microscopy samplepreparation approach where pachytene cells are isolated from mouse testis by FACS, followed by cryo-fixation and cryo-substitution to achieve visualization of a close-to-native structure of the SC. We found that the central region of the SC was wider than previously recognized, and the transverse filaments more densely packed in the central region. Furthermore, we identified a structure nucleating the central element of the SC.

Current Progress in Expansion Microscopy: Chemical Strategies and Applications

“…In addition, chemical fixation might also cause artifacts in structures of biological components (Table ). − To circumvent these limitations, more recently, Laporte et al applied cryofixation in ExM (termed as Cryo-ExM), enabling visualization of native cellular components in ExM . In Cryo-ExM, through rapidly plunging samples into liquid ethane-propane, samples are instantaneously fixed in a vitreous state, avoiding damage to the ultrastructure.…”