In vitro Reconstitution of the C. elegans Synaptonemal Complex

Sexually reproducing organisms depend on the precise segregation of chromosomes during meiosis to ensure
the inheritance of the genome through the formation of haploid gametes. Defects in this process can lead to
infertility and the production of gametes with an abnormal number of chromosomes, known as aneuploidy. To
successfully segregate chromosomes during meiosis, homologous chromosomes must pair, synapse, and
undergo crossover recombination during prophase I. Synapsis is defined by the formation of a highly
conserved, zipper-like proteinaceous structure called the synaptonemal complex (SC) that links two homologs
together and serves as a scaffold for crossover recombination. The SC is a tripartite structure comprised of two
parallel stretches of chromatin-associated axial elements and a central region that connects the two. Despite
its conserved role and appearance across most eukaryotes, little is known about its molecular architecture and
the mechanisms that underlie the assembly of higher-order SC structures. The nematode Caenorhabditis
elegans has been an excellent model organism for studying meiotic mechanisms. In C. elegans, the SC central
region has been known to be composed of six interdependent coiled-coil proteins SYP-1, SYP-2, SYP-3 SYP-
4, and SYP-5/6. Recently, we have identified that two paralogous Skp1-related (SKR) proteins, SKR-1/2, which
are adaptors of the Skp1-Cul1-F-box (SCF) ubiquitin ligase complex, play moonlighting functions as structural
components of the SC. Their identification has enabled me to reconstitute the SC central region in vitro using a
bacterial protein expression system. SKR-1 and all previously known SYP proteins are purified together
throughout multiple purification steps at stoichiometric ratios, suggesting that the complete set of SC
components has been identified in C. elegans. Here, I propose to use these purified components as a platform
to address long-standing questions regarding the molecular architecture and assembly mechanisms of the SC.
In Aim 1, I will determine the stoichiometry of the SC central region proteins using size-exclusion
chromatography coupled with multi-angle light scattering (SEC-MALS) and Mass Photometry (MP). I will then
construct a map of the SC by identifying areas of protein-protein interactions via cross-linking mass
spectrometry. In Aim 2, I will investigate the necessary requirements that potentiate and regulate SC assembly.
Recent evidence has suggested that the SC exhibits liquid-crystalline properties that allow for rapid diffusion
and condensation of pro-crossover factors along chromosome lengths, which in turn controls the number and
distribution of crossovers. I will induce the formation of phase-separated droplets using purified components
and assess their behavior under conditions that are known to affect SC assembly in vivo. Ultimately, this work
will provide crucial insights into the organization and assembly requirements of the SC and establish a
foundation for understanding the SC structure in other eukaryotic organisms.

Grant Number: 5F31GM153163-02
NIH Institute/Center: NIH
Principal Investigator: Brenda Cesar-Hernandez