Kinetochore Function in Vertebrate Cells

脊椎动物细胞的动粒功能

基本信息

批准号：
9274805
负责人：
Jennifer G DeLuca
金额：
$ 13.36万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9274805
关键词：
Address Affect Affinity Anaphase Binding Biochemical Biological Biological Assay Cell division Cells Centromere Chromatin Chromosome Segregation Chromosomes Collaborations Complex Computer Simulation Congenital Abnormality Congresses Data Defect Development Ensure Funding Generations Goals Grant Health Human Image In Vitro Kinetochores Knowledge Link Malignant Neoplasms Maps Mediating Microscopy Microtubules Mitosis Mitotic Mitotic Chromosome Modeling Molecular Monitor Nucleosomes Oncogenic Phosphorylation Phosphotransferases Play Population Positioning Attribute Process Protein Dephosphorylation Proteins Recruitment Activity Regulation Resolution Role Scaffolding Protein Signal Transduction Stress Testing Therapeutic aurora B kinase base cancer cell daughter cell design forging human disease in vivo insight mathematical model novel novel therapeutics prevent protein structure reconstitution research study response segregation single molecule therapeutic target tool tumor progression

项目摘要

DESCRIPTION (provided by applicant): Kinetochores are large protein structures assembled on centromeric chromatin that power and regulate chromosome segregation during mitosis. Properly functioning kinetochores are essential for mitotic cell division and for maintaining human health, as defective chromosome segregation is implicated as a causative factor in the formation of birth defects and in the initiation and progression of cancer. Kinetochores are considered the "orchestrators" of mitosis for several reasons. Kinetochores physically connect mitotic chromosomes to spindle microtubules (MTs) and transduce forces through this connection to congress chromosomes; they regulate the binding strength between chromosomes and MTs so that improper attachments are corrected; and, finally, they ensure that cells do not exit mitosis if chromosomes fail to attach or are incorrectly attached to MTs by regulating the spindle assembly checkpoint (SAC), which monitors kinetochore-MT attachment and prevents anaphase in such unfavorable conditions. The NDC80 complex plays a central role in the generation and regulation of attachments: it is the primary linkage between kinetochores and MTs, and phosphorylation of its Hec1 subunit by the essential Aurora B kinase (ABK) regulates kinetochore-MT attachment strength. The goals of this study are to understand how kinetochore-MT attachments are precisely regulated during mitotic progression and to determine how kinetochore-MT stability is linked to SAC signaling. These issues will be addressed in three Aims. The first Aim will investigate how NDC80-mediated kinetochore-MT attachment is regulated by phosphorylation and dephosphorylation and how the SAC detects stable NDC80-MT connections. Using a combination of in vivo, in vitro, and in silico approaches, the mechanisms governing Hec1 dephosphorylation during mitosis will be determined, the contributions of ABK and non-ABK mitotic kinases to Hec1 phosphorylation will be mapped, and how Hec1 regulates the recruitment and eviction of upstream SAC activators will be determined. In the second Aim, the mechanism for ABK-mediated regulation of kinetochore-MT attachment stability will be addressed by building on exciting preliminary data suggesting that kinetochore substrates, including Hec1, are phosphorylated by a population of ABK recruited specifically to kinetochores, rather than by a population of ABK at inner centromeres. To determine the molecular basis for this activity, cell biological approaches and in vitro reconstitution assays using purified proteins and nucleosome arrays will be employed. In the third Aim, unexplored roles of the kinetochore scaffold protein KNL1 will be investigated. How KNL1 recruits both SAC silencers and activators will be determined, and the functions of recently identified KNL1-interacting proteins will be characterized. In summary, this proposal will address how kinetochore-MT attachments are regulated and monitored during mitosis to ensure accurate chromosome segregation using multiple approaches, including quantitative cell-based high-resolution and super-resolution microscopy, biochemical reconstitution, single- molecule biophysical assays, and mathematical modeling.

描述（由申请人提供）：着丝粒是组装在着丝粒染色质上的大型蛋白质结构，在有丝分裂过程中为染色体分离提供动力和调节。着丝粒的正常功能对于有丝分裂细胞分裂和维持人类健康至关重要，因为有缺陷的染色体分离是一个致病因素。由于多种原因，动粒被认为是有丝分裂的“指挥者”。动粒将有丝分裂染色体与纺锤体微管 (MT) 物理连接，并通过这种连接将力转导至国会染色体；它们调节染色体和 MT 之间的结合强度，从而纠正不正确的附着，最后，它们确保细胞不会退出有丝分裂；通过调节纺锤体装配检查点 (SAC)，染色体无法附着或错误地附着至 MT，该检查点监控动粒-MT 附着并防止后期发生NDC80 复合物在附着的产生和调节中发挥着核心作用：它是着丝粒和 MT 之间的主要连接，其 Hec1 亚基被必需的 Aurora B 激酶 (ABK) 磷酸化可调节着丝粒 - MT 附着强度。本研究的目的是了解在有丝分裂过程中如何精确调节着丝粒-MT 附着，并确定着丝粒-MT 稳定性如何与SAC 信号传导。第一个目标将研究 NDC80 介导的着丝粒-MT 附着如何通过磷酸化和去磷酸化进行调节，以及 SAC 如何结合体内检测稳定的 NDC80-MT 连接。体外和计算机方法将确定有丝分裂过程中控制 Hec1 去磷酸化的机制，ABK 和非 ABK 有丝分裂激酶对有丝分裂的贡献将绘制 Hec1 磷酸化，以及 Hec1 如何调节上游 SAC 激活剂的招募和驱逐。在第二个目标中，将通过建立令人兴奋的初步数据来解决已确定的 ABK 介导的着丝粒-MT 附着稳定性调节机制。动粒底物（包括 Hec1）是由专门招募到动粒的 ABK 群体磷酸化的，而不是由内部的 ABK 群体磷酸化的。为了确定这种活性的分子基础，将采用细胞生物学方法和使用纯化蛋白和核小体阵列的体外重建测定，在第三个目标中，将研究着丝粒支架蛋白 KNL1 如何招募两者。 SAC 沉默子和激活子将被确定，并且最近鉴定的 KNL1 相互作用蛋白的功能将被表征。总之，该提案将解决如何进行的问题。动粒-MT附着在有丝分裂过程中受到调节和监测，以确保使用多种方法准确分离染色体，包括基于细胞的定量高分辨率和超分辨率显微镜、生化重建、单分子生物物理测定和数学建模。