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

点击翻译按钮获取中文摘要

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.