A major objective of high resolution imaging of cells is to visualize the molecular organization of cellular components and to determine if specific components participate in the same macromolecular complex. Efforts to address these issues have tackled the problem from two opposite perspectives: 1. Improvements in microscopy have enabled resolution of spectrally distinct signals at a molecular scale under ideal conditions; and 2. Energy transfer approaches have allowed detection of complexes where two proteins are in very close contact. This proposal introduces a third approach that fills the gap in resolution between the two former methods and avoids many of the compromises inherent in them. This approach is based on formation of a fluorescent complex when the association between two non-fluorescent protein fragments is facilitated by tethering the fragments in a macromolecular complex. Earlier adaptations of this approach are known as bimolecular fluorescence complementation (BiFC) and ubiquitin-mediated fluorescence complementation (UbFC). These earlier approaches have significant limitations that are due to the lack of optimization of the fluorescent protein fragments (BiFC probes) for purposes of the assay. The proposed research seeks to eliminate these limitations through systematic in vitro evolution and protein design strategies. The research team will develop novel BiFC probes that will enable imaging of cellular processes that have not been previously imaged due to limitations inherent in existing methods. To validate the universal utility of the BiFC probes, they will be used to image molecular events involving bacteria, plants and animal cells. Public Health Relevance: Many cellular functions require that multiple components come together to form a complex. We propose to develop new methods that will enable us to see the complexes formed by specific combinations of components. These methods are analogous to linking a light bulb to one component and a battery to the other. This work will improve our understanding of interactions among cellular components that are important for the health of the cell and the organism. ? ? ?
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