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.

在有性生殖生物中，遗传信息从亲代到后代的流动依赖于 减数分裂，一种特殊的细胞分裂，其中由二倍体细胞产生单倍体配子。成功的 减数分裂中的染色体分离需要同源体之间的配对、突触和交叉形成 突触涉及拉链状蛋白质结构的组装，称为 联会复合体 (SC)，在两对同源物之间形成，并充当交叉支架 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。在秀丽隐杆线虫中，SC 材料形成 没有染色体轴的球形聚集体，称为多复合物，并招募所需的因子 将交叉形成作为单个焦点，概括其在正常减数分裂中强大的交叉控制。尽管 SC 的保守结构和功能，目前尚不清楚是什么驱动 SC 和 在减数分裂过程中如何调节其液体样特性。 Polo 样激酶 (PLK) 是一个家族 通过磷酸化波协调减数分裂前期事件的保守细胞周期激酶。这 该提案基于我的初步数据，暗示 PLK 提供了 SC 的液体特性及其 与遗传易处理模型生物线虫中交叉因子的亲和力。在此我建议进一步 通过结合线虫遗传学、实时成像、生化纯化和 定量磷酸蛋白质组学。在目标 1 中，我将使用缺乏染色体轴的菌株作为实验 平台并对 SC 多聚复合物进行延时显微镜检查，以确定 PLK 如何调节其融合， 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，我将检验以下假设：PLK 通过磷酸化 SC 来调节 SC 的动态特性。 减数分裂过程中的成分。我将从线虫中纯化生化量的 SC 材料 使用或不使用 PLK-2 裂解物，并通过比较 PLK-2 介导的磷酸化位点的水平来绘制 PLK 介导的磷酸化位点图谱 使用质谱和化学标记对 SC 内的磷酸肽进行分析。这项努力将得到补充 通过我正在进行的使用磷酸化特异性抗体的工作，我针对几个 PLK 共识提出了该抗体 两个旁系同源 SC 组件 SYP-5 和 SYP-6 的无序 C 末端尾部内的基序。 SYP-5和 SYP-6 在减数分裂进入时以 PLK 依赖性方式被强烈磷酸化，这对于 在早期减数分裂前期启动 SC 组装。我将继续表征 SC 内的 PLK 磷酸位点 通过产生磷酸化特异性抗体。我将确定保守 PLK 的生物学意义 通过定向诱变进行磷酸化位点。总的来说，这项工作将通过以下方式提供对机制的见解： 保守的细胞周期激酶调节 SC 动态并将广泛适用于跨物种， 包括人类。