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

项目摘要 当一个细胞分裂时，它必须准确地将它的复制染色体分离在两个子细胞之间。 细胞染色体分离的功能或调节的错误可以产生较少的活细胞， 外来染色体有趣的是，在癌细胞中经常发现额外的和不稳定的染色体， 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。 为了了解这些异常基因组在癌症和其他疾病进展中的影响， 为了确定在正常条件下染色体分离是如何发生的，以及这一过程的步骤， 这一过程可能会发生故障，产生受损的基因组。动粒是一个由约100个组分组成的分子 连接微管末端和染色体并利用解聚能力的机器 微管分离染色体。重要的是，动粒必须保持它们与 染色体和微管末端，而在高张力下。弱化动粒微管 连接可以阻止细胞分裂或促进不正确的染色体分离。而动粒 结合微管的成分主要是确定的，仍然不知道这些成分是如何 协调以承受调节和执行染色体分离所必需的力。对于这一提议，我 将使用纯化的成分在其天然的位置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， 纯化的微管末端的动粒组分。此外，我将测试是否有多个绑定副本 组分通过组装成特定的几何形状或增加微管的分子量来加强微管的附着。 绑定交互的数量。将使用光学陷阱测量附着强度，以精确 操纵和测量结合到微管末端的动粒组分所经受的力。 我对动粒微管附着界面的重建将建立一个最小的系统， 以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方法，通过它来检查天然动粒微管附件中， 高度可控的细胞外环境。增加了系统的复杂性， 调节信号分子将测试微管动粒附着如何控制细胞的进展， 师.染色体分离调控途径中易错相互作用的鉴定 将促进新的实验发展到细胞分裂错误在癌症和其他疾病中的作用， 疾病了解显著的异常基因组是如何形成的对提高我们的识别能力很重要， 预防和治疗受损基因组疾病，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.