Investigating the structure and function of the human centromere and kinetochore

项目摘要 在细胞分裂过程中染色体的精确分离是细胞分裂的最基本要求之一。 生物学如果没有适当的染色体分离，遗传信息就不能忠实地传递到不同的染色体上。 细胞和生物体的世代，导致严重的后果，包括细胞死亡，发育缺陷， 或癌症的进展。此外，癌细胞中染色体分离不当， 导致抗肿瘤炎症反应。染色体分离过程的中心是着丝粒， 纺锤体微管结合的染色体位点。着丝粒在表观遗传学上被定义为 存在含有组蛋白变体CENP-A的核小体。着丝粒染色质作为 动粒的基础，一种在CENP-A核小体上组装并介导 微管结合对着丝粒的研究对于更好地理解 染色体分离在健康和疾病，但我们对人类着丝粒的理解仍然存在 基本上不完整。该提案旨在回答有关结构和功能的基本问题， 着丝粒及其相关蛋白。大着丝粒蛋白重组的研究进展 复合物增加了我们对人类动粒结构的理解，但重建了 复合物只能近似体内结构，目前有多种竞争模型 着丝粒和动粒的结构和组织。新兴的低温电子技术 断层扫描（cryo-ET）提供了询问着丝粒和动粒结构的机会， 它们在玻璃体水合细胞内的原生环境。为此，在目标1中，将使用冷冻ET来获得第一个 在细胞周期的间期和有丝分裂阶段，人类着丝粒和动粒的原位结构。 该建议的第二个重点是阐明所需的着丝粒蛋白质之间的相互作用 对于着丝粒的基本功能，包括微管附着的形成和维持 着丝粒的身份。两种动粒蛋白，Ndc80和CENP-Q，都被证明有助于 微管结合在体外和体内，但其各自的作用，在微管结合在体内还没有 完全特征化。组成型着丝粒相关网络（CCAN）中的多种蛋白质具有 类似地被证明有助于CENP-A在着丝粒处的维持和沉积，但CCAN 包含多个相互关联的子复合体，其贡献从未被系统地测试过。在 目的2各内源基因座的诱变（和各野生型基因的快速消耗 产品）将用于阐明在体内和时间的准确性的相互作用，基础纺锤体 附着和维持着丝粒的同一性。这些实验将提供重要的见解， 人类着丝粒和动粒的结构和功能 分离和基因组的世代保真度。

Project Summary Accurate segregation of chromosomes during cell division is one of the most fundamental requirements in biology. Without proper chromosomal segregation, genetic information cannot be faithfully transmitted across cell and organismal generations, leading to severe consequences including cell death, developmental defects, or progression of cancer. Furthermore, improper chromosome segregation in cancer cells has been shown to lead to anti-tumor inflammatory responses. Central to the process of chromosome segregation is the centromere, the chromosomal locus at which spindle microtubules bind. The centromere is defined epigenetically by the presence of nucleosomes containing the histone variant CENP-A. Centromeric chromatin serves as the foundation of the kinetochore, a large protein complex which assembles on CENP-A nucleosomes and mediates microtubule binding. Research into the centromere is necessary to better understand the processes that underlie chromosome segregation in both health and disease, but our understanding of the human centromere remains largely incomplete. This proposal aims to answer fundamental questions about the structure and function of the centromere and its associated proteins. Recent advances in reconstitution of large centromeric protein complexes have increased our understanding of the structure of the human kinetochore, but reconstituted complexes can only approximate in vivo structures, and currently there are multiple competing models for the structure and organization of the centromere and kinetochore. The emerging technology of cryo-electron tomography (cryo-ET) provides the opportunity to interrogate the structure of the centromere and kinetochore in their native context within vitreous hydrated cells. To this end, in Aim 1 cryo-ET will be used to obtain the first in situ structures of the human centromere and kinetochore in the interphase and mitosis stages of the cell cycle. The second focus of this proposal is to elucidate the interactions among centromeric proteins that are required for the essential functions of the centromere, including formation of microtubule attachments and maintenance of centromeric identity. Two kinetochore proteins, Ndc80 and CENP-Q, have both been shown to contribute to microtubule binding in vitro and in vivo, but their respective roles in microtubule binding in vivo have not been fully characterized. Multiple proteins within the constitutive centromere-associated network (CCAN) have similarly been shown to contribute to maintenance and deposition of CENP-A at the centromere, but the CCAN contains multiple interconnected subcomplexes whose contributions have never been systematically tested. In Aim 2 mutagenesis of the respective endogenous gene loci (and rapid depletion of the respective wild type gene products) will be used to elucidate in vivo and with temporal accuracy the interactions that underlie spindle attachment and maintenance of centromeric identity. These experiments will provide important insights into the structure and function of the human centromere and kinetochore which are essential for proper chromosome segregation and genomic fidelity across generations.