Spatial Mutation of Membrane Protein Assembly Dynamics Using Nano-Actuators

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DESCRIPTION (provided by applicant): Angiopoietins stimulate proangiogenic signals of endothelial cells (ECs) via ligand-induced assembly and translocation of Tie2 receptors and modulate several key processes in ECs including proliferation, migration, and assembly. However, it is still unclear how these biomolecular regulators coordinate highly complex morphogenesis such as vascular sprouting, migration, branching, and network formation. To understand molecular mechanisms of angiogenesis with high spatio-temporal complexity, the spatiotemporal dynamics of Tie2 must be monitored in real-time with high resolution. Here, I propose an innovative approach to investigate dynamic assembly and spatial distribution changes of Tie2 using a new magneto-plasmonic nano-actuation system. Unlike conventional microscopy that only provides a static assessment of cell status, the proposed system allows us to visualize biomolecular events of the signaling molecules continuously over the entire angiogenesis period as well as throughout morphogenetic development. Additionally, by using magnetic tweezing capability of the nano-actuation system, control of Tie2 spatial distributions is possible, enabling the study of how spatially polarized Tie2 distribution influences the cell's behavior and fate. With this new technique, specifically, I will challenge the following questions: 1. How do the assembly state and distribution of Tie2 change after stimulation with various ligands such as angiopoietins? 2. Are the assembly states of Tie2 related to the different subcellular locations such as cell periphery, cell-to-cell contacts, and cell-to-substratum contacts? 3. How do spatially polarized distributions of Tie2 influence a cell's behavior?