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

项目摘要 当细胞分裂时，它必须准确地将其复制的染色体在两个子代之间分离 细胞。染色体分离的功能或调节上的错误会导致细胞存活率降低 无关的染色体。有趣的是，在癌细胞中经常发现额外的和不稳定的染色体，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在癌症和其他疾病进展中的影响，至关重要 为了确定在正常条件下染色体分离是如何发生的，以及实现这一分离的步骤 这一过程可能会出现故障，从而产生受损的基因组。动粒是一种~100组分的分子 一种将微管末端连接到染色体并利用解聚能力的机器 分离染色体的微管。重要的是，Kintochore必须保持它们与 在高张力下，染色体和微管末端。弱化动粒微管 连接可能会阻止细胞分裂或促进错误的染色体分离。而动毛虫 结合微管的成分大多是确定的，目前还不清楚这些成分是如何结合的 协调以承受调节和执行染色体分离所需的力量。对于这项建议，我 将使用纯化的成分重建最小的人类动粒微管界面 依恋强度。我将通过测试不同组合的承载强度来实现这一点 在微管末端纯化的动粒成分。此外，我将测试绑定的多个副本是否 组件通过组装成特定的几何形状或增加微管连接来加强微管连接 绑定交互的数量。附着强度将使用光学陷阱进行精确测量 操纵和测量绑定到微管末端的动粒组件所经历的力。 我对动粒微管附着界面的重建将建立一个最小系统，通过这个系统 测试承重强度在细胞分裂的功能和调节中的作用。重要的是，这 重组系统提供了一种检查天然动粒微管附着的方法 细胞外环境高度可控。通过引入以下内容来增加此系统的复杂性 调节信号分子将测试微管动粒连接如何控制细胞的进程 组织。识别染色体分离调节通路中容易出错的相互作用 将推动新的实验发展成为细胞分裂错误在癌症等方面的作用 疾病。了解显著的异常基因组是如何形成的，对于提高我们的鉴定很重要， 预防和治疗受损的基因组疾病，如癌症。

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.