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Abstract

Homologous recombination plays an important role in meiosis as it allows for the exchange of genetic information between homologous chromosomes that secures the diversity of the progeny. An excess of double-strand breaks is introduced and processed into recombination intermediates to achieve homologous recombination. However, only a limited subset of these recombination intermediates is designated to become crossovers. Any imbalance in the distribution of crossovers may lead to genomic instability and errors in chromosome segregation with devastating consequences on the health of the progeny. However, what is the main regulatory mechanism and when exactly crossovers are designated remain highly disputed questions. In this study, I harnessed recent advances in image analysis and 3D dual-colour single-molecule localisation microscopy (SMLM) to investigate the timing and mechanism of crossover designation in Caenorhabditis elegans This study combines in vivo confocal microscopy with a custom analysis pipeline for segmentation, foci detection, and tracking to identify the timing of crossover designation. Recombination intermediates are initially detected as dim, diffraction-limited foci of a pro-crossover factor, while designated crossovers at later stages are marked by bright foci. Interestingly, two populations of dim recombination intermediates that have differing association dynamics of the pro-crossover factors could be identified. Similarly, ultrastructures that are consistent with break repair intermediates that are destined to become crossovers were identified at earlier stages than previous data suggested. Together, these findings indicated that crossover designation happens earlier than previously thought. To obtain these data, the experimental procedures had to be optimised and adapted. These optimisations now set a basis for future investigation of crossover regulation, quantitative analysis of time-lapse movies of the C. elegans germline, and the re-purposing of the SMLM data analysis tools for describing the 3D-SMLM localisations of the synaptonemal complex components and associated proteins. Indeed, the optimised immunohistochemistry protocol compatible with 3D-SMLM imaging was invaluable in investigating the organisation and function of novel synaptonemal complex components. 3D-SMLM imaging showed that SYP-5/6 proteins are transversal filaments that span the width of the synaptonemal complex. Furthermore, 3D-SMLM imaging proved that Skp1-related proteins (SKR-1/2 in C. elegans) localise to the central region of the synaptonemal complex. This result was contrary to the chromosome axis localisation observed in mice and provided further insight into the possible roles of these proteins within the synaptonemal complex C. elegans. 3D-SMLM imaging combined with the appropriate sample preparation broadened the understanding of the structure of the synaptonemal complex in C. elegans.