Mechanisms of mitotic regulation

有丝分裂调节机制

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要：有丝分裂调节机制有丝分裂中染色体分离的缺陷是非整倍体和其他结构的基础在人类癌症中发现的重排。 Stukenberg 实验室研究有丝分裂调控已有 22 年之久最近表明，人类乳腺肿瘤会失调大量有丝分裂调节因子的转录，从而产生高度非整倍体肿瘤。许多最失调的转录本是 Aurora B 激酶，它的复杂的成员和控制来自内部着丝粒的多个有丝分裂事件的信号网络每一条染色体。斯图肯伯格实验室的下一阶段将建立在最近的两项突破的基础上启用新的实验线来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，我们最近证明了染色体含有 Aurora B 激酶的 Passenger Complex 在体外和体内进行液-液相分离。激酶存在于冷凝物中的认识使得能够在体外重建许多反应。例如，Aurora B 激酶在生理浓度和条件下在冷凝物中激活。它还阐明了 Aurora 如何磷酸化动粒并激活的令人惊讶的机制下游激酶。冷凝物还可以激活微管成束活性，这是可溶性物质所没有的。蛋白质。事实上，CPC 快速生成源自凝聚层的微管平行束状结构具有相反极性的中心体代表一种新的微管活性。其次，我们已经确定 CPC 前期的新功能包括在着丝粒中生成 R 环。此次新活动是通过首先从有丝分裂染色体中纯化复合物而发现的，我们在其中鉴定了 32 个 R 环调节因子。 R 环既指导 CPC 的定位，又是姐妹染色单体凝聚所必需的。研究的 Aurora B 对 R 环的调节提出了 Aurora B 在控制中的另一条令人兴奋的研究路线有丝分裂转录。为了提高实验的严谨性和力量，Stukenberg 实验室开发了结合体外重建、计算机模拟和细胞分析中关键点突变体替换的工具。这些研究的结果将为了解染色体乘客的失调如何提供重要的见解癌细胞中复杂的驱动非整倍性以及如何更好地结合 Aurora 抑制剂来有效治疗癌症。