Tie-Dyes for Detection of Protein Interactions. A Tool Towards Fluorescence Based Detection of Synapses

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To understand brain function one must have a knowledge of the organization and connectivity of neurons. Current techniques to map neuronal connectivity are either well suited to imaging synapses or well suited for large scale analysis of the brain. Fluorescence microscopy, especially when paired with CLARITY can image an entire brain, but cannot resolve synaptic junctions (~20nm). This work was undertaken to develop a tool that can circumvent this limitation and allow for detection of synapses for high throughput volumetric mapping of synapses.The tool developed has three components; an anchor a linker and a fluorogen. The anchor is composed of the HaloTag system which covalently binds the HaloTag Ligand. The fluorogen is Malachite Green which binds to the Fluorogen Activating Protein (FAP) to become highly fluorescent. The linker is a polymeric spacer that allows the fluorogen to probe the environment around the anchor for the FAP. The HaloTag Ligand, polymer, Fluorogen unit is called a tie-dye.The tie-dyes were synthesized with two different linker lengths; MG-halo and MG-80p-halo. Additionally, the system was tested for specificity of HaloTag-targeted FAP-signal using FAPs of two different binding affinities. The two FAPs and two tie-dyes were used to assess the effect of linker length and KD on the ability of the tie-dye to specifically bind to interacting proteins.Specific binding of the tie-dye to dimerized fusions of the HaloTag and FAP was demonstrated for three systems. The tie-dye system demonstrated a 100% increase in signal above baseline FAP signal upon of rapamycin-induced dimerization of FKBP/FRB. EGF/EGFR proximity was detected with the tie-dye at 300% activation above baseline FAP binding. Finally, the tie-dye system was applied to the detection of membrane apposition in a model HEK293 cell system to show 450% increase in specific FAP signal at contact sites.In all three cases, the proximity induced FAP signal is activated over background FAP fluorescence. Differences in signals above baseline FAP fluorescence depend on the relationship between tie-dye length and inter-receptor spacing, the affinity of the probe for the fluorogen activating protein, and to some extent the order with which the probe interacts with the protein tags. This validation of the tie-dyes will enable future work in application to high throughput detection of synapses. Additionally, the distance dependencies may allow this tool to act as a molecular ruler for intercellular geometries, forces, and overall dynamics.