Mechanisms of mitotic regulation

有丝分裂调节机制

• 批准号: 10551950
• 负责人: P. TODD STUKENBERG
• 金额: $ 67.01万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10551950
• 关键词: Aneuploidy; Cellular Assay; Centromere; Centrosome; Chromosome Segregation; Chromosomes; Complex; Defect; Ensure; Event; Generations; Genetic Transcription; Genomic Instability; Human; In Vitro; Investigation; Kinetochores; Liquid substance; Malignant Neoplasms; Mammary Neoplasms; Microtubule Bundle; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Mitotic spindle; Modeling; Phase; Phosphorylation; Phosphotransferases; Physical condensation; Physiological; Prophase; Proteins; Reaction; Regulation; Signal Transduction; Sister Chromatid; Structure; Transcript; aurora B kinase; cancer cell; cancer therapy; cohesion; in silico; in vivo; inhibitor; insight; member; mutant; novel; reconstitution; tool; tumor

Project Summary/Abstract: Mechanisms of mitotic regulation Defects in the segregation of chromosomes in mitosis underlie aneuploidy and other structural rearrangements found in human cancers. The Stukenberg lab has studied mitotic regulation for 22 years and recently shown human breast tumors dysregulate the transcription of a large numbers of mitotic regulators to generate highly aneuploid tumors. Many of the most dysregulated transcripts were the Aurora B kinase, its complex members and a signaling network that controls multiple mitotic events from the inner centromere of every chromosome. The next phase of the Stukenberg lab will build on two recent breakthroughs that have enabled new lines of experimentation to elucidate how these proteins ensure accurate chromosome segregation and how dysregulation by tumors lowers this fidelity. First, we recently demonstrated that the Chromosome Passenger Complex containing Aurora B kinase undergoes liquid-liquid phase separation in vitro and in vivo. The insight that the kinase exists in a condensate enabled the reconstitution of a number of reactions in vitro. For example, Aurora B kinase activates in condensates under physiological concentrations and conditions. It has also elucidated surprising mechanisms for how Aurora phosphorylates kinetochores and activates downstream kinases. Condensates also activate a microtubule bundling activity that is not seen with soluble proteins. In fact, CPC rapidly generates microtubule parallel bundled structures that emanate from coacervates with the opposite polarity of centrosomes representing a novel microtubule activity. Second, we have identified new functions of the CPC in prophase including the generation of R-loops in the centromere. This new activity was found by first purifying the complex from mitotic chromosomes where we identify 32 regulators of R-loops. R-loops both direct the localization of the CPC and are required for cohesion of sister chromatids. The study of Aurora B regulation of R-loops has suggested another exciting line of investigation of Aurora B in controlling mitotic transcription. To increase the rigor and power of our experimentation the Stukenberg lab has developed tools to combine in vitro reconstitution, in silico modeling and replacement of key point mutants in cell assays. The result of these studies will provide important insight into how dysregulation of the chromosome passenger complex drive aneuploidy in cancer cells and how to better combine Aurora inhibitors to effectively treat cancer.

项目概要/摘要： 有丝分裂调控机制 在有丝分裂中染色体分离的缺陷是非整倍性和其他结构性的基础。 在人类癌症中发现的基因重排。Stukenberg实验室研究有丝分裂调控已有22年， 最近显示，人类乳腺肿瘤使大量有丝分裂调节因子的转录失调， 产生高度非整倍体肿瘤。许多失调最严重的转录物是极光B激酶，其 复杂的成员和一个信号网络，控制多个有丝分裂事件从内部着丝粒的 每一个染色体Stukenberg实验室的下一阶段将建立在最近的两项突破上， 使新的实验线路，以阐明这些蛋白质如何确保准确的染色体分离 以及肿瘤的失调如何降低这种保真度。首先，我们最近证明， 含Aurora B激酶的乘客复合物在体外和体内经历液-液相分离。 激酶存在于冷凝物中的见解使得能够在体外重建许多反应。 例如，极光B激酶在生理浓度和条件下在缩合物中活化。它 还阐明了极光如何磷酸化动粒并激活 下游激酶。冷凝物还激活微管捆绑活性，这在可溶性 proteins.事实上，CPC迅速产生微管平行捆绑结构，从凝聚层发出 中心体的相反极性代表了一种新的微管活性。第二，我们确定了 CPC在前期的新功能包括在着丝粒中产生R环。这项新活动 通过首先从有丝分裂染色体中纯化复合物发现，其中我们鉴定了32个R环调节器。 R环既指导CPC的定位，又是姐妹染色单体凝聚所必需的。研究 极光B对R环的调节提出了极光B在控制中的另一个令人兴奋的研究方向 有丝分裂转录为了增加我们实验的严谨性和力量，Stukenberg实验室开发了 结合体外重建、计算机模拟和细胞测定中关键点突变体替换的联合收割机工具。 这些研究的结果将提供重要的见解如何失调的染色体乘客 复杂驱动癌细胞中的非整倍体以及如何更好地联合收割机Aurora抑制剂来有效治疗癌症。