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; 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.

在性生殖的生物中，遗传信息从父母到后代的流动依赖于 减数分裂是一种专门的细胞分裂，在该细胞分裂中，单倍体配子是由二倍体细胞产生的。成功的 减数分裂中的染色体分离需要配对，突触和跨界形成 预言期间的染色体。突触涉及拉链样蛋白结构的组装，称为 突发型复合物（SC）在两个配对同源物和功能之间形成跨界的脚手架 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，以调节交叉的数量和分布。在秀丽隐杆线虫中，SC材料形式 在没有染色体轴的情况下，球形骨料（称为多复合物），需要募集因子 对于单一焦点的跨界形成，请概括其在正常减数分裂中的稳健跨界控制。尽管 SC的保守结构和功能，尚不清楚是什么驱动SC和SC的相位分离 在减数分裂进程期间，如何调节其液体样性能。类似马球的激酶（PLK）是一个家庭 保守的细胞周期激酶，通过磷酸化的波来编排减数分裂预言事件。这 提案是基于我的初步数据暗示，PLK提供了SC及其ITS的类似液体样特性 在遗传上可牵引的模型有机体C.秀丽隐杆线虫中，对跨因子的亲和力。在这里我建议进一步 通过结合秀丽隐杆线虫遗传学，现场成像，生化纯化和 定量磷酸蛋白质组学。在AIM 1中，我将使用缺乏染色体轴的菌株作为实验 平台并执行SC多复合物的延时显微镜，以确定PLK如何调节其融合， 球形和营业额。通常通过磷酸化在空间和时间上调节液态液相。 在AIM 2中，我将测试以下假设，即PLK通过磷酸化来调节SC的动态特性 减数分裂进展过程中的成分。我将从秀丽隐杆线虫中净化生化量的SC材料 有或没有PLK-2和MAP PLK介导的磷酸化位点的裂解物通过比较水平 使用质谱和化学标记在SC中的磷酸肽。这项努力将得到补充 通过我正在进行的使用磷酸化抗体的工作，我已经针对多个PLK共识提出了这些抗体 两个副SC分量SYP-5和SYP-6的无序的C末端尾部中的基序。 Syp-5和 SYP-6以PLK依赖性的方式对减数分裂的入口进行了稳健的磷酸化，这对于 在早期减数分裂预言中启动SC组装。我将继续表征SC内的PLK磷酸材料 通过升高磷酸特异性抗体。我将确定保守PLK的生物学意义 通过靶向诱变，磷酸化位点。总体而言，这项工作将提供有关机制的见解 哪些保守细胞周期激酶可以调节SC动力学，并将广泛适用于物种， 包括人类。