In sexually reproducing organisms, the flow of genetic information from parent to offspring relies on meiosis, a specialized cell division where haploid gametes are produced from diploid cells. Successful chromosome segregation in meiosis requires pairing, synapsis, and crossover formation between homologous chromosomes during prophase I. Synapsis involves the assembly of a zipper-like protein structure called the synaptonemal complex (SC) that forms between two paired homologs and functions as a scaffold for crossover recombination. Recent evidence shows that the SC has liquid crystalline properties, allowing for chromosome- wide signal transduction to regulate the number and distribution of crossovers. In C. elegans, SC materials form spherical aggregates in the absence of chromosome axes, called polycomplexes, and recruit factors required for crossover formation as a single focus, recapitulating its robust crossover control in normal meiosis. Despite the conserved structure and function of the SC, it remains unknown what drives phase separation of the SC and how its liquid-like properties are regulated during meiotic progression. Polo-like kinases (PLKs) are a family of conserved cell-cycle kinases that orchestrate meiotic prophase events via waves of phosphorylation. This proposal is based on my preliminary data hinting that PLKs provide the liquid-like properties of the SC and its affinity to crossover factors in the genetically tractable model organism C. elegans. Here I propose to further elucidate the role of PLKs by combining C. elegans genetics, live imaging, biochemical purification, and quantitative phosphoproteomics. In Aim 1, I will use a strain lacking chromosome axes as an experimental platform and perform time-lapse microscopy of SC polycomplexes to determine how PLKs modulate their fusion, sphericity, and turnover. Liquid-liquid phase separation is often regulated in space and time by phosphorylation. In Aim 2, I will test the hypothesis that PLKs regulate dynamic properties of the SC by phosphorylating its components during meiotic progression. I will purify biochemical quantities of SC materials from C. elegans lysates with or without PLK-2 and map PLK-mediated phosphorylation sites by comparing levels of phosphopeptides within the SC using mass spectrometry and chemical labeling. This effort will be complemented by my ongoing work using phospho-specific antibodies, which I have raised against several PLK consensus motifs within the disordered C-terminal tails of two paralogous SC components, SYP-5 and SYP-6. SYP-5 and SYP-6 are robustly phosphorylated upon meiotic entry in a PLK-dependent manner, and this is critical for initiating SC assembly in early meiotic prophase. I will continue to characterize PLK phosphosites within the SC by raising phospho-specific antibodies. I will determine the biological significance of conserved PLK phosphorylation sites by targeted mutagenesis. Overall, this work will provide insights into th...