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

研究 Polo 样激酶在调节联会复合体动力学中的作用

• 批准号: 10679711
• 负责人: Ariel Larissa Gold
• 金额: $ 4.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679711
• 关键词: Affinity; Aneuploidy; Biochemical; Biological; C-terminal; Caenorhabditis elegans; Cell Cycle; Cell division; Chemicals; Chromosome Pairing; Chromosome Segregation; Chromosomes; Communication; Complement; Consensus; Data; Diploid Cells; Disease; Down Syndrome; Ensure; Event; Failure; Family; Fluorescence Recovery After Photobleaching; Generations; Genetic; Genetic Crossing Over; Genome; Germ Cells; Haploidy; Homologous Gene; Human; Image; Immunofluorescence Immunologic; Infertility; Label; Length; Liquid substance; Maps; Mass Spectrum Analysis; Mediating; Meiosis; Meiotic Prophase I; Microscopy; Mitosis; Mitotic; Mutagenesis; Mutation; Nature; Organism; PLK1 gene; Parents; Pattern; Phase; Phospho-Specific Antibodies; Phosphopeptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Process; Property; Prophase; Regulation; Reproduction; Research; Role; Signal Transduction; Sister Chromatid; Site; Spontaneous abortion; Structure; Synaptonemal Complex; Tail; Testing; Therapeutic Intervention; Time; Work; age related; design; developmental disease; genetic information; in vivo; insight; model organism; offspring; phosphoproteomics; polo-like kinase kinase 1; protein complex; protein structure; recruit; response; scaffold; tool; transmission process

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.

在有性繁殖的生物体中，遗传信息从亲本到后代的流动依赖于 减数分裂，一种特殊的细胞分裂，其中单倍体配子由二倍体细胞产生。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、联会和交换形成。 染色体在前期I。突触涉及拉链状蛋白质结构的组装，称为 联会复合体：在两对同源物之间形成的联会复合体（SC），作为交换的支架 重组最近的证据表明，SC具有液晶特性，允许染色体- 广泛的信号转导来调节交叉的数量和分布。In C. elegans，SC材料表 在没有染色体轴的情况下的球形聚集体，称为多复合体，以及所需的募集因子 对于作为单焦点的交叉形成，概括了其在正常减数分裂中的鲁棒交叉控制。尽管 由于SC的保守结构和功能，目前尚不清楚是什么驱动了SC的相分离， 在减数分裂过程中其液体样特性是如何被调节的。Polo样激酶（PLK）是一个家族， 保守的细胞周期激酶，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的液体性质及其 对遗传上易处理的模式生物C中的交叉因子的亲和力。优美的在此，我建议进一步 结合C. elegans遗传学，活体成像，生化纯化， 定量磷酸蛋白质组学在目标1中，我将使用缺乏染色体轴的菌株作为实验菌株。 平台和进行SC多复合物的延时显微镜检查以确定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。 在目的2中，我将检验PLKs通过磷酸化SC的磷酸化来调节SC的动态特性的假设。 减数分裂进程中的组分。我将从C. elegans 有或没有PLK-2的裂解物，并通过比较PLK-2的水平来定位PLK介导的磷酸化位点。 使用质谱法和化学标记法测定SC内的磷酸肽。这一努力将得到补充 通过我正在进行的使用磷酸化特异性抗体的工作，我已经提出了反对几个PLK共识 在两个旁系同源SC组分SYP-5和SYP-6的无序C-末端尾部内的基序。SYP-5和 SYP-6在减数分裂进入后以PLK依赖的方式被强烈磷酸化，这对于 在减数分裂前期早期启动SC组装。我将继续在SC内表征PLK磷酸盐 通过产生磷酸化抗体我将确定保守的PLK的生物学意义 通过靶向诱变获得磷酸化位点。总的来说，这项工作将通过以下方式提供对机制的深入了解： 其中保守的细胞周期激酶调节SC动力学并将广泛应用于物种， 包括人类