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Investigating the structure and function of the human centromere and kinetochore

研究人类着丝粒和着丝粒的结构和功能

基本信息

批准号：
10643935
负责人：
Kathryn Kixmoeller
金额：
$ 5.27万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10643935
关键词：
Antineoplastic Agents Area Auxins Binding Biological Assay Biology Black race Cell Cycle Cell Cycle Stage Cell Death Cell division Cells Cellular biology Centromere Chromatin Chromosome Segregation Chromosome abnormality Chromosomes Complex Cryo-electron tomography Defect Deposition Development Disease Elements Emerging Technologies Epigenetic Process Foundations G1 Phase Generations Genomics Health Histone H3 Histones Human Hydration status In Situ In Vitro Inflammatory Response Infrastructure Interphase Interphase Cell Kinetochores Maintenance Malignant Neoplasms Maps Mediating Metaphase Methods Microtubules Mitosis Mitotic spindle Modeling Molecular Machines Mutagenesis N-terminal Nature Nucleosomes Pennsylvania Process Proteins Research Role Small Interfering RNA Structure Testing Uncertainty Universities Variant Weight-Bearing state Work cancer cell centromere protein A centromere protein C chromosome missegregation design experimental study gene product genetic information genomic locus in vivo insight protein complex reconstitution segregation stoichiometry transmission process tumor tumor progression

项目摘要

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.

项目摘要在细胞分裂过程中对染色体的准确分离是生物学。如果没有适当的染色体分离，遗传信息就不能忠实地传递细胞和生物体世代，导致严重后果，包括细胞死亡，发育缺陷，或癌症的进展。此外，癌细胞中不适当的染色体分离已被证明是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置。着丝粒是由表观遗传学定义的存在含组蛋白变异体CENP-A的核小体。着丝粒染色质作为动粒的建立，它是一个大的蛋白质复合体，组装在CENP-A核小体上并介导微管结合。对着丝粒的研究对于更好地理解其背后的过程是必要的健康和疾病中的染色体分离，但我们对人类着丝粒的理解仍然存在基本上是不完整的。这项提议旨在回答有关联合国儿童基金会结构和功能的基本问题着丝粒及其相关蛋白。大着丝粒蛋白重组研究进展复合体增加了我们对人类动粒结构的理解，但却被重组了络合物只能近似体内结构，目前有多种竞争模型着丝粒和着丝粒的结构和组织。新兴的低温电子技术体层摄影术(冷冻-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中的天然环境。为此，在AIM中，将使用1号冷凝器获得第一个细胞周期间期和有丝分裂期着丝粒和着丝粒的原位结构。这项建议的第二个重点是阐明着丝粒蛋白之间的相互作用着丝粒的基本功能，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都被证明对微管在体外和体内的结合，但它们在体内微管结合中的作用尚未得到研究完全有特点的。结构性着丝粒相关网络(CCAN)中的多个蛋白质同样被证明有助于CENP-A在着丝粒的维持和沉积，但Ccan 包含多个相互关联的亚络合物，其贡献从未经过系统测试。在……里面目的2诱变各自的内源基因座(和迅速耗尽各自的野生型基因产品)将被用来在活体内并以时间上的准确性阐明纺锤体背后的相互作用着丝粒特性的依附和维持。这些实验将为我们提供对人类着丝粒和着丝粒的结构和功能，它们是正常染色体所必需的种族隔离和世代间的基因组保真度。