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

准确的染色体分离对基因组的稳定性至关重要。在早期，核膜破裂后，染色体分散到细胞质中。为了将所有染色体沿着中期板(细胞中部)排列，染色体被附着在微管(MTS)上。动粒是一种有丝分裂特有的结构，在G2期后期组装在染色体着丝粒上，是MT附着的场所。有丝分裂检查点(MC)是一种防止染色体过早分离的故障保护机制。即使没有一条染色体在中期平板上对齐，MC也会被激活，从而防止早熟后期。MC监测两个指标：1)动粒-MT连接和2)姐妹动粒张力。动粒是MT结合蛋白和MC蛋白对接的平台，因此是MC调节的部位。RZZ复合体是动粒和MC的重要组成部分。RZZ复合体由Rod、Zw10和ZWilch三个亚基组成，带有辅助蛋白Zwint-1和Spindly。RZZ复合体通过以下动粒募集在有丝分裂中发挥重要作用：1)MC效应器Mad1/MAD2；2)MT马达，dynein/dynactin；3)纺锤形，这也是有丝分裂结束时dynein/dynactin募集和MC沉默所必需的。在前中期，动力蛋白/动力蛋白的动粒募集是染色体配对所必需的。作为检查点沉默机制的一部分，dynein/dynactin还需要在中期对齐后将检查点蛋白，如MAD2，从着丝粒移至纺锤体极。ZWINT-1是Knl1(一种结构动粒蛋白)和RZZ复合体之间的连接物，是Zw10稳定的动粒定位所必需的。我们建议剖析RZZ复合体在有丝分裂过程中如何组装并发挥其功能的机制。我们先前已经定位了Zw10、ZWilch、Spinidly和Zwint-1的动粒定位结构域。我们建议研究RZZ复合亚单位是如何相互作用、组装并与Bub1、Spindly和Mad1/Mad2相关联的。我们建议识别RZZ相互作用突变体，并检查它们对已知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击倒Rod、Zw10或Zwilch，并用野生型构建或相互作用突变体拯救。动粒募集是MC蛋白功能的关键。关于指定招募途径的蛋白质-蛋白质相互作用，人们知之甚少。我们建议通过对RZZ复合体的结构-功能分析来填补知识空白。识别RZZ复合体的组装机制以及RZZ复合体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。

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.