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。在早期前期，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（细胞中间）排列，染色体附着在微管（mt）上。着丝点是一种有丝分裂特异性结构，在G2期后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）是一种防止染色体过早分离的失效保护机制。即使单个染色体没有在中期板上对齐，MC也被激活并防止过早的后期。MC监测两个标准：1)着丝点- mt附着和2)姐妹着丝点张力。着丝点作为mt结合蛋白和MC蛋白对接的平台，因此是MC调控的位点。RZZ复合体是一个重要的着丝点和MC成分。RZZ复合物由三个亚基Rod、Zw10和Zwilch组成，并有辅助蛋白Zwint-1和Spindly。RZZ复合体在调节有丝分裂中发挥着以下几个重要作用：1)MC效应体Mad1/Mad2；2) MT电机，dynein/dynactin；3)细长，这也是有丝分裂末期dynein/dynactin募集和MC沉默所必需的。动蛋白/动蛋白的着丝点募集是前期染色体排列所必需的。作为检查点沉默机制的一部分，dynein/dynactin也需要在中期对齐后将检查点蛋白（如Mad2）从着丝点移到纺锤极。Zwint-1是Knl1（一种结构着丝粒蛋白）和RZZ复合物之间的连接物，是Zw10稳定着丝粒定位所必需的。我们建议剖析RZZ复合物在有丝分裂过程中如何组装和发挥其功能的机制。我们之前已经绘制了Zw10、Zwilch、Spindly和Zwint-1的着丝点定位域。我们建议研究RZZ复合物亚基是如何相互作用、组装并与Bub1、Spindly和Mad1/Mad2结合的。我们建议鉴定RZZ相互作用突变体，并研究它们对已知RZZ功能的影响。为了消除内源性蛋白对突变体的干扰，我们将使用siRNA敲低Rod、Zw10或Zwilch，然后用野生型构建体或相互作用突变体进行修复。着丝粒的招募对MC蛋白的功能至关重要。关于蛋白质-蛋白质相互作用指定招募途径的分子细节知之甚少。我们建议通过RZZ复合物的结构-功能分析来填补这方面的知识空白。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-蛋白相互作用对于理解着丝点MC途径具有重要意义。

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.