Optimizing knocksideways to visualize interactions between protein subcomplexes

PROJECT SUMMARY Genomic instability is a hallmark for cancers. More than 90% of cancer cells exhibit aneuploidy. The spindle assembly checkpoint is a fundamental mechanism that prevents chromosomal instability by halting premature chromosome segregation. In this project, the “knocksideways” techniques will be adapted to analyze the factors that contribute to kinetochore localization and activity of the MAD1:MAD2 complex. Targeting the MAD1:MAD2 complex to unattached kinetochores is a crucial step to activate the spindle assembly checkpoint. Here we use “knocksideways” in its broader meaning, which includes devising different ways to target a protein or a protein subcomplex away from its endogenous location to a distinct subcellular niche. Such spatial isolation strategy makes it possible to address interactions between “pre-fabricated” protein subcomplexes. If another protein subcomplex follows suit and is diverted to the new location, this implicates positive physical interactions between the two subcomplexes. Biochemical reconstitution and structural analysis is still the gold standard to establish interactions between protein subcomplexes. However, the knowledge of subunits and posttranslational modifications could be prerequisite for such reconstitution. On the other hand, traditional methods check dependency relationship between protein subcomplexes using knockdown or knockout of one component and analyzing the impact on other components. The dependency relationship cannot establish direct interactions nor the sufficiency of one subcomplex in recruiting additional subcomplexes. We propose to modify and expand two “knocksideways” schemes to analyze how the MAD1:MAD2 complex interacts with other protein subcomplexes, either as its kinetochore receptors or as its activity regulators. In our opinion, innovative use of the “knocksideways” analyses will help answer some key questions surrounding the MAD1:MAD2 complex. Such techniques will also be useful in studying the building of other sophisticated molecular machineries.