Award Details:
Award Type: Catalyst
Award #: C-008
Proposal Title: Multiplexed Imaging of Transient Molecular Complex Dynamics in vivo
PI(s): Lawrence Miller (UIC) and Jerrold Turner (UChicago)
Award Amount: $200,000.00
Application Cycle: Round 4 (Fall 2007)
Award Start Date: January 1, 2008
Award End Date: December 31, 2009
Abstract:
Proteins interact transiently within multi-component complexes to control a wide variety of biological processes such as the cell cycle, motility or immune response. In order to understand and model the molecular mechanisms that underlie biological function, researchers needs experimental tools that allow them to elucidate how the spatial and temporal regulation of a specific protein complex is coupled to a specific activity and a particular cellular response. Our research seeks to provide a general method for microscopically visualizing the location and stoichiometry of multiple protein-protein interactions in living cells in real time. Our approach is based on technology that allows the selective labeling of genetically encoded fusion proteins in living mammalian cells with cell-permeable small molecules. We will synthesize organic complexes of lanthanide ions such as terbium or europium that luminesce brightly with very long lifetimes and multiple emission maxima. The lanthanide probes will enable a facile form of lifetime imaging microscopy that will allow us to visualize and quantify resonance energy transfer between lanthanide-labeled targets and one or more fluorophore-labeled proteins. As an initial proof-of-concept, we will use the proposed lanthanide protein labeling and microscopy technologies to study the biochemical mechanism of cytoskeletal-mediated remodeling of the tight junction protein complex and its effect on the epithelial permeability, a problem not easily resolvable with existing experimental techniques. Given its generality, we anticipate that the proposed imaging technology will be easily adopted by other investigators for studying protein dynamics in a wide variety of cell types.