Releasing the brake: how kinetochore localized phosphatases allow silencing of the spindle-assembly checkpoint and consequently mitotic exit upon chromosome bi-orientation.

释放刹车：动粒定位磷酸酶如何使纺锤体装配检查点沉默，从而在染色体双向时退出有丝分裂。

• 批准号: 268630125
• 负责人: Dr. Debora Bade
• 金额: --
• 依托单位: Universitair Medisch Centrum Utrecht
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/268630125?language=en
• 关键词: Releasing; brake; how; kinetochore; localized

Genomic instability is a major contributor to solid tumor formation. Chromosomal instability, one of the modes of genomic instability, is the main cause for aneuploidy, a deviation from the diploid chromosome set. Aneuploidy occurs in 70% of all solid tumors. It is the consequence of chromosome segregation errors. As the spindle assembly checkpoint (SAC) monitors chromosome-spindle interactions and prevents mitotic progression before all chromosomes are attached, it protects against aneuploidy. Accordingly, several studies emphasized the importance of SAC proteins in tumor suppression. Importantly, two strategies that involve targeting the SAC have shown promise for anti-cancer therapy. The first is chronic activation by microtubule poisons, a strategy already in clinical use. The second is preventing mitotic exit, which was proposed to be more efficient in cancer cell killing than the microtubule poison. It will therefore be valuable to uncover mechanisms of SAC silencing required for timely/efficient mitotic exit.I will investigate how SAC silencing is achieved on a molecular level. I will specifically concentrate on how regulated dephosphorylation triggers SAC silencing and mitotic progression. Three aims will be pursued. First, I will identify the targets of the two phosphatases required for anaphase onset, namely PP1 and PP2A-B56. For this, I will use BioID and APEX purification approaches. In parallel, I will perform quantitative phospho-proteomics from cell lines in which localization of the phosphatases to kinetochores is specifically disrupted. In the second aim, I will address how dephosphorylation of the uncovered targets promotes SAC silencing. I will therefore use RNAi techniques and also work with the corresponding phospho-mimetic mutants. Finally, I will study how the phosphatases themselves are regulated in space and time. Together, these insights will contribute to fundamental understanding of the spatiotemporal control of SAC silencing that ensures timely and error-free chromosome segregation.

基因组不稳定性是实体瘤形成的主要因素。染色体不稳定性是基因组不稳定性的一种形式，是非整倍体的主要原因，非整倍体是一种偏离二倍体染色体组的现象。非整倍体发生在70%的实体瘤中。这是染色体分离错误的结果。由于纺锤体组装检查点（SAC）监测染色体-纺锤体相互作用，并在所有染色体附着之前阻止有丝分裂进程，因此它可以防止非整倍性。因此，一些研究强调了SAC蛋白在肿瘤抑制中的重要性。重要的是，涉及靶向SAC的两种策略已显示出抗癌治疗的前景。第一种是微管毒物的慢性激活，这是一种已经在临床上使用的策略。第二个是阻止有丝分裂退出，这被认为比微管毒素更有效地杀死癌细胞。因此，这将是有价值的，以揭示及时/有效的有丝分裂退出所需的SAC沉默机制。我将研究SAC沉默是如何在分子水平上实现的。我将特别关注调节去磷酸化如何触发SAC沉默和有丝分裂进程。将追求三个目标。首先，我将确定后期启动所需的两种磷酸酶的靶标，即PP 1和PP 2A-B56。为此，我将使用BioID和APEX纯化方法。与此同时，我将从磷酸酶定位到着丝粒被特异性破坏的细胞系中进行定量磷酸蛋白质组学研究。在第二个目标中，我将讨论未被覆盖的靶点的去磷酸化如何促进SAC沉默。因此，我将使用RNAi技术，并与相应的磷酸模拟突变体。最后，我将研究磷酸酶本身是如何在空间和时间上进行调节的。总之，这些见解将有助于从根本上理解SAC沉默的时空控制，确保及时和无错误的染色体分离。