|Institution:||University of Pennsylvania|
|Keywords:||Chemical Biology; Chemical Dimerization; Mad1; Mitotic Checkpoint; Rapamycin; Spindle Assembly Checkpoint; Biology; Chemistry; Molecular Biology|
|Full text PDF:||http://repository.upenn.edu/edissertations/1015
Cellular processes such as growth, migration, signaling and cell division require choreographed interactions between dozens or hundreds of proteins carefully organized in time and space. In order to test hypotheses about complex cellular functions, it is desirable to experimentally perturb the interactions of individual proteins that perform these functions with a level of spatial and temporal control commensurate with the time and space scales over which the system is naturally organized. Inducible protein dimerization offers the ability to experimentally control protein-protein interactions. Inducible dimerization can be used to test the immediate effects of dimerizing two proteins, or it can be engineered to create or destroy a protein or change a protein's localization. Several different techniques for inducible dimerization using small molecules or light have been developed, each with its own strengths and weaknesses. Ultimately, only light-inducible dimerization offers the potential for both temporal and spatial experimental control. In this thesis, I describe the application of inducible dimerization to further our understanding of a complex signaling network, the Mitotic Checkpoint, which monitors chromosome segregation and is regulated by the localization of its constituent checkpoint proteins. I discovered that relocalizing a single key checkpoint protein, Mad1, to kinetochores at metaphase is sufficient to reactivate the checkpoint. I also describe the development of a novel photochemical technique which has allowed us to achieve light-induced dimerization at centromeres, a cellular compartment which has not been successfully targeted by previously reported light-inducible dimerization systems. This technology enables us to perform experimental biology on living cells with a new level of spatial and temporal control.