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。

• 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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=685046
• 关键词: Structure; function; RZZ; complex; essential

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稳定性至关重要。在早期预言中，染色体在核包膜分解后分散到细胞质中。为了使沿中期板（细胞中部）的所有染色体对齐，将染色体附着在微管（MTS）上。动力学是一种有丝分裂特异性的结构，在G2阶段晚期组装在染色体的丝粒上，并用作MT附着的位置。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尚未对齐，也会激活MC并防止过早后期。 MC监视两个标准：1）Kinetochore-MT附件和2）姐妹Kinetochore紧张。 Kinetochore是一个平台，MT结合蛋白和MC蛋白对接，因此是MC调节的位置。 RZZ复合物是必不可少的动力学和MC组件。 RZZ复合物由三个亚基组成，即杆，ZW10和Zwilch，带有辅助蛋白ZWINT-1和脊柱。 RZZ综合体在调节有丝分裂中的几个重要作用，通过募集以下：1）MC效应，MAD1/MAD2； 2）MT电动机，Dynein/Dynactin； 3）在有丝分裂结束时Dynein/dynactin募集和MC沉默也是脊柱上的。在前载酶期间，染色体比对需要动力学的动力学募集。作为检查点沉默机制的一部分，去除检查点蛋白（如MAD2）也需要Dynein/dynactin从动力学到中期对准后的纺锤极。 ZWINT-1是KNL1（结构动物学蛋白）和RZZ复合物之间的连接器，是ZW10的稳定动力学定位所必需的。我们建议剖析如何组装RZZ复合物并在有丝分裂过程中发挥其功能的机制。我们以前已经映射了ZW10，Zwilch，Spindly和Zwint-1的动力学定位域。我们建议检查RZZ复合物亚基如何相互作用，组装并与Bub1，spindly和Mad1/Mad2相关联。我们建议识别RZZ相互作用突变体并检查其对已知RZZ功能的影响。为了消除内源性蛋白在突变体上的干扰，我们将使用siRNA敲低杆，ZW10或Zwilch，并用野生型构建体或相互作用突变体营救。动力学募集对于MC蛋白功能至关重要。关于指定募集途径的蛋白质蛋白质相互作用的分子细节很少。我们建议通过对RZZ复合物的结构 - 功能分析来填补知识的空白。鉴定RZZ复合物组装机理以及RZZ复合物及其伴侣之间的蛋白质 - 蛋白质相互作用对于理解动力学MC途径很重要。**