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Mechanisms of mitotic regulation

有丝分裂调节机制

基本信息

批准号：
10798363
负责人：
P. TODD STUKENBERG
金额：
$ 25万
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：有丝分裂调控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。斯图肯伯格实验室研究有丝分裂调控已有22年，最近发现，人类乳腺肿瘤失调了大量有丝分裂调节因子的转录，以产生高度非整倍体的肿瘤。许多最不受调控的转录本是极光B激酶，它的复杂的成员和一个信号网络，控制着丝粒体内的多个有丝分裂事件每条染色体。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，这两项突破已经使新的实验路线能够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激酶的客体复合体在体内外进行液-液相分离。对该激酶存在于凝集物中的洞察使许多体外反应得以重建。例如，在生理浓度和条件下，极光B激酶在冷凝物中被激活。它还阐明了Aurora如何磷酸化运动中枢并激活的惊人机制下游的蛋白激酶。凝析油还可以激活微管捆绑活动，这是可溶性的不存在的蛋白质。事实上，CPC快速生成凝聚体发出的微管平行束结构中心体的相反极性代表了一种新的微管活性。第二，我们已经确定了早期CPC的新功能，包括在着丝粒上产生R-环。这项新活动是通过首先从有丝分裂染色体中纯化复合体而发现的，我们在有丝分裂染色体上鉴定了32个R环的调节因子。 R-环既指导CPC的定位，又是姐妹染色单体凝聚所必需的。的研究。 Aurora B对R环的调节提出了Aurora B控制的另一条令人兴奋的研究路线有丝分裂转录。为了提高我们实验的严密性和威力，斯图肯伯格实验室开发了将体外重组、电子计算机建模和细胞分析中关键点突变的替换相结合的工具。这些研究的结果将为染色体乘客的失调提供重要的洞察力复杂的驱动癌细胞的非整倍体，以及如何更好地结合Aurora抑制剂来有效治疗癌症。