Investigating the role of Polo-like kinase in regulating synaptonemal complex dynamics

在有性繁殖生物体中，遗传信息从父母到后代的流动依赖于 减数分裂，一种由二倍体细胞产生单倍体配子的特殊细胞分裂。成功 减数分裂中的染色体分离需要同源染色体之间的配对、突触和交叉形成 第一阶段的染色体突触涉及一种拉链状的蛋白质结构的组装，称为 在两对同系物之间形成的联会复合体(SC)，用作交叉的支架 重组。最近的证据表明，干细胞具有液晶特性，允许染色体- 广泛的信号转导，以调节交叉的数量和分布。在线虫中，SC物质形式 在没有染色体轴的情况下的球形聚集体，称为多复合体，并需要招募因子 作为单一焦点的交叉形成，概括了其在正常减数分裂中的稳健交叉控制。尽管 SC的保守结构和功能，目前尚不清楚是什么导致了SC和 在减数分裂过程中其类液体性质是如何被调节的。类Polo蛋白(PLK)是一类 保守的细胞周期蛋白激酶，通过磷酸化波协调减数分裂前期事件。这 我的建议是基于我的初步数据，暗示PLK提供了SC和其 遗传易驯化模式生物秀丽线虫对交叉因子的亲和力。在此，我建议进一步 通过结合线虫遗传学、活体成像、生化纯化和 定量磷酸蛋白质组学。在目标1中，我将使用一种缺乏染色体轴的菌株作为实验 并对SC多复合体进行时间推移显微镜观察以确定PLK如何调节它们的融合， 球形度和周转率。液-液相分离通常通过磷酸化在空间和时间上进行调节。 在目标2中，我将测试PLK通过使其磷酸化来调节SC的动态特性的假设 减数分裂进程中的成分。我将从线虫中提纯生化量的SC物质 通过比较含有或不含有PLK-2和MAP的PLK介导的磷酸化位点的裂解产物的水平 利用质谱学和化学标记技术对SC中的磷酸肽进行分析。这一努力将得到补充。 通过我正在进行的使用磷酸特异性抗体的工作，我已经提出了几个PLK共识 两个平行SC组分SYP-5和SYP-6的无序C-末端中的基序。SYP-5和 SYP-6在进入减数分裂时以依赖于PLK的方式强烈磷酸化，这对 在减数分裂早期启动SC组装。我将继续对SC中的PLK磷酸盐进行表征 通过提高磷酸特异性抗体。我将确定保守的PLK的生物学意义 靶向突变的磷酸化位点。总体而言，这项工作将通过以下方式提供对机制的见解 其中保守的细胞周期激酶调节SC的动态，并将广泛适用于不同物种， 包括人类。

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 the mechanisms by which conserved cell cycle kinases regulate SC dynamics and will be broadly applicable across species, including humans.