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Reconstitution of the load-bearing attachments between the human kinetochore and microtubule ends

人体着丝粒和微管末端之间承载附件的重建

基本信息

批准号：
9330686
负责人：
LUKE ANDREW HELGESON
金额：
$ 5.71万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2019-07-31
项目状态：
已结题

项目摘要

Project Summary When a cell divides it must accurately segregate its duplicated chromosomes between the two daughter cells. Errors in the function or regulation of chromosome segregation can produce less viable cells with extraneous chromosomes. Interestingly, extra and unstable chromosomes are often found in cancer cells but it is unknown if genome damage created by these segregation errors can promote cancer formation or growth. To understand the impact of these abnormal genomes in cancer and other disease progression it is essential to determine how chromosome segregation occurs under normal conditions and the steps at which this process can malfunction to produce damaged genomes. The kinetochore is a ~100 component molecular machine that connects microtubule ends to chromosomes and harnesses the power of depolymerizing microtubules to segregate chromosomes. Importantly, kinetochores must maintain their connection to chromosomes and microtubule ends while under high tension. Weakening kinetochore microtubule connections can halt cell division or promote incorrect chromosome segregation. While the kinetochore components that bind microtubules are mostly determined, it is still unknown how these components coordinate to bear the forces necessary to regulate and perform chromosome segregation. For this proposal, I will use purified components to reconstruct the minimal human kinetochore microtubule interface at its native attachment strength. I will accomplish this by testing the load-bearing strength of different combinations of purified kinetochore components at microtubule ends. Additionally, I will test if multiple copies of the binding components strengthen microtubule attachment by assembling into a specific geometry or increasing the number of binding interactions. Attachment strength will be measured using an optical trap to precisely manipulate and measure the forces experienced by the kinetochore components bound to microtubule ends. My reconstitution of the kinetochore microtubule attachment interface will establish a minimal system by which to test the role of load-bearing strength in the function and regulation of cell division. Importantly, this reconstituted system provides a means by which to examine native kinetochore microtubule attachments in a highly controllable environment outside of cells. Increasing the complexity of this system by introducing regulatory signaling molecules will test how microtubule kinetochore attachments control the progression of cell division. Identification of error-prone interactions within the regulatory pathways of chromosome segregation will promote the development of novel experiments into the role of cell division errors in cancer and other diseases. Understanding how significant aberrant genomes form is important to improving our identification, prevention and treatment of damaged genome diseases, such as cancer.

项目概要当细胞分裂时，它必须准确地将其复制的染色体分离到两个子细胞之间细胞。染色体分离的功能或调节错误会产生存活率较低的细胞外来染色体。有趣的是，癌细胞中经常发现额外且不稳定的染色体，但它目前尚不清楚这些分离错误造成的基因组损伤是否会促进癌症的形成或生长。要了解这些异常基因组对癌症和其他疾病进展的影响，至关重要确定正常条件下染色体分离是如何发生的以及发生这种分离的步骤过程可能会发生故障，产生受损的基因组。着丝粒是由约 100 个成分组成的分子将微管末端连接到染色体并利用解聚力量的机器微管分离染色体。重要的是，动粒必须保持与染色体和微管在高张力下终止。着丝粒微管减弱连接可以阻止细胞分裂或促进不正确的染色体分离。虽然动粒结合微管的成分大部分是确定的，但仍不清楚这些成分是如何形成的协调以承担调节和执行染色体分离所需的力量。对于这个提议，我将使用纯化的成分在其天然状态下重建最小的人类动粒微管界面附着力。我将通过测试不同组合的承载强度来实现这一点微管末端的纯化着丝粒成分。此外，我将测试绑定的多个副本组件通过组装成特定的几何形状或增加微管附着来加强微管附着结合相互作用的数量。将使用光阱精确测量附着强度操纵和测量与微管末端结合的动粒成分所经历的力。我对动粒微管附着界面的重建将建立一个最小的系统测试承重强度在细胞分裂功能和调节中的作用。重要的是，这重建系统提供了一种检查天然动粒微管附件的方法高度可控的细胞外环境。通过引入增加了该系统的复杂性调节信号分子将测试微管动粒附着如何控制细胞的进展分配。鉴定染色体分离调控途径中容易出错的相互作用将促进新实验的发展，研究细胞分裂错误在癌症和其他疾病中的作用疾病。了解异常基因组的形成对于改善我们的识别非常重要，预防和治疗受损基因组疾病，例如癌症。