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Structure and function of the RZZ complex, an essential mitotic checkpoint complex.

RZZ 复合体（一种重要的有丝分裂检查点复合体）的结构和功能。

基本信息

批准号：
RGPIN-2016-06466
负责人：
Chan, Gordon
金额：
$ 2.4万
依托单位：
University of Alberta
依托单位国家：
加拿大
项目类别：
Discovery Grants Program - Individual
财政年份：
2020
资助国家：
加拿大
起止时间：
2020-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710301
关键词：
Structure function RZZ complex essential

项目摘要

Accurate chromosome segregation is essential for genomic stability. In early prophase, chromosomes disperse into the cytoplasm after nuclear envelope breakdown. To align all the chromosomes along the metaphase plate (middle of the cell), chromosomes are attached to microtubule (MTs). The kinetochore is a mitosis-specific structure that is assembled on the centromere of the chromosome during the late G2 phase, and serves as the site for MT attachment. The mitotic checkpoint (MC) is a failsafe mechanism that prevents premature segregation of chromosomes. Even if a single chromosome has not been aligned at the metaphase plate, the MC is activated and prevents premature anaphase. The MC monitors two criteria: 1) kinetochore-MT attachment and 2) sister kinetochore tension. The kinetochore serves as a platform where MT-binding proteins and MC proteins dock and therefore, is the site of MC regulation. The RZZ complex is an essential kinetochore and MC component. The RZZ complex is composed of three subunits, Rod, Zw10 and Zwilch, with accessory proteins Zwint-1 and Spindly. The RZZ complex plays several important roles in regulating mitosis by kinetochore recruitment of the following: 1) the MC effectors, Mad1/Mad2; 2) the MT motor, dynein/dynactin; and 3) Spindly, which is also required for dynein/dynactin recruitment and MC silencing at the end of mitosis. The kinetochore recruitment of dynein/dynactin is required for chromosome alignment during prometaphase. As part of the checkpoint silencing mechanism, dynein/dynactin is also required for the removal of checkpoint proteins, such as Mad2, from the kinetochore to the spindle pole after metaphase alignment. Zwint-1, a linker between Knl1 (a structural kinetochore protein) and the RZZ complex, is required for stable kinetochore localization of Zw10. We propose to dissect the mechanism of how the RZZ complex is assembled and exerts its function during mitosis. We have previously mapped the kinetochore localization domains of Zw10, Zwilch, Spindly and Zwint-1. We propose to examine how the RZZ complex subunits interact, are assembled and associate with Bub1, Spindly, and Mad1/Mad2. We propose to identify RZZ interaction mutants and examine their effects on known RZZ functions. To eliminate interference of the endogenous proteins on the mutants, we will use siRNA to knockdown Rod, Zw10 or Zwilch and rescue with either the wildtype construct or interaction mutants. Kinetochore recruitment is critical for MC protein function. Few molecular details are known regarding the protein-protein interaction that specifies the recruitment pathway. We propose to fill the gaps of knowledge by structure-function analysis of the RZZ complex. Identification of the mechanism of assembly of the RZZ complex and the protein-protein interactions between the RZZ complex and its partners is important for the understanding of the kinetochore MC pathway.

准确的染色体分离对于基因组的稳定性至关重要。在前期早期，染色体在核膜破裂后分散到细胞质中。为了使所有染色体沿着中期板（细胞的中间）排列，染色体附着在微管（MT）上。动粒是一种有丝分裂特异性结构，在G2晚期组装在染色体的着丝粒上，并作为MT附着的位点。有丝分裂检查点（MC）是防止染色体过早分离的故障安全机制。即使单个染色体在中期板上没有对齐，MC也被激活并防止过早的后期。MC监测两个标准：1）动粒-MT附着和2）姐妹动粒张力。动粒作为MT结合蛋白和MC蛋白对接的平台，因此是MC调节的位点。RZZ复合物是一种重要的动粒和MC组分。RZZ复合物由Rod、Zw 10和Zwilch三个亚基以及辅助蛋白Zwint-1和Spindly组成。RZZ复合物通过以下动粒募集在调节有丝分裂中起几个重要作用：1）MC效应子，Mad 1/Mad 2; 2）MT马达，动力蛋白/动力肌动蛋白;和3）纺锤体，其也是有丝分裂结束时动力蛋白/动力肌动蛋白募集和MC沉默所需的。动力蛋白/动力肌动蛋白的动粒募集是前中期染色体排列所必需的。作为检查点沉默机制的一部分，动力蛋白/动力肌动蛋白也是中期对齐后从动粒到纺锤体极去除检查点蛋白（如Mad 2）所必需的。Zwint-1是Knl 1（一种结构动粒蛋白）和RZZ复合物之间的连接体，是Zw 10稳定的动粒定位所必需的。我们建议剖析RZZ复合物如何组装并在有丝分裂过程中发挥其功能的机制。我们以前映射的动粒定位域Zw 10，Zwilch，Spindly和Zwint-1。我们建议研究RZZ复合物亚基如何相互作用，组装和关联Bub 1，纺锤形，和Mad 1/Mad 2。我们建议确定RZZ相互作用的突变体，并研究其对已知的RZZ功能的影响。为了消除内源蛋白对突变体的干扰，我们将使用siRNA敲低Rod、Zw 10或Zwilch，并用野生型构建体或相互作用突变体进行拯救。动粒募集对MC蛋白功能至关重要。很少有分子的细节是已知的蛋白质-蛋白质相互作用，指定的招聘途径。我们建议通过RZZ复合物的结构-功能分析来填补知识的空白。确定RZZ复合物的组装机制以及RZZ复合物与其伙伴之间的蛋白质-蛋白质相互作用对于理解动粒MC途径是重要的。