Optogenetic Tuning Of Protein-Protein Binding In Bilayers Using Lovtrap
BIO-PROTOCOL(2020)
摘要
Modern microscopy methods are powerful tools for studying live cell signaling and biochemical reactions, enabling us to observe when and where these reactions take place from the level of a cell down to single molecules. With microscopy, each cell or molecule can be observed both before and after a given perturbation, facilitating better inference of cause and effect than is possible with destructive modes of signaling quantitation. As many inputs to cell signaling and biochemical systems originate as protein-protein interactions near the cell membrane, an outstanding challenge lies in controlling the timing, location and the magnitude of protein-protein interactions in these unique environments. Here, we detail our procedure for manipulating such spatial and temporal protein-protein interactions in a closed microscopy system using a LOVTRAP-based light-responsive protein-protein interaction system on a supported lipid bilayer. The system responds in seconds and can pattern details down to the one micron level. We used this technique to unlock fundamental aspects of T cell signaling, and this approach is generalizable to many other cell signaling and biochemical contexts.
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关键词
Optogenetics, In-vitro reconstitution, Cell signaling, LOV2, Microscopy, Supported lipid bilayers
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