Monitoring Synaptic Protein Dynamics with Higher Temporal, Spatial, and Molecular Resolutio

Memory acquisition and storage are fundamental for an organism's survival and depend on plasticity at chemical synapses to strengthen communication between neural circuits. However, studying the molecular basis of these events is technically challenging due to the wide range of temporal and spatial scales of these events as well as the molecular diversity of synapses. Because synaptic plasticity and strengthen depend on the availability of new proteins at the synapse, the TimeSTAMP (TS) reporter was developed to selectively visualize new protein copies. The TS reporter uses the activity of the cis-acting HCV protease, separating a protein of interest from a visual element, such that only in the presence of an inhibitor to this protease does the protein of interest become visible. A drug-dependent, continuous fluorescence readout was added to the original reporter based on split YFP (TS:YFP) to dynamically image both new and old protein copies in living cells, thereby extending the temporal resolution of the reporter. Furthermore, a genetically encoded EM tag (miniSOG) was incorporated into TS:YFP (TS:YSOG) to provide correlated ultrastructural snapshots of marked proteins, thereby extending the spatial resolution of the reporter. Furthermore, a novel technique to resolve multiple colors within EM samples was developed to image TS tags with respect to other synaptic markers by EM. To make these techniques applicable for in vivo experiments, several methods were investigated to enhance delivery of this impermeant protease inhibitor across the blood brain barrier, including pro-drug synthesis, mannitol-induced osmotic shock, and peptide-mediated transcytosis. These TS reporters would be useful for monitoring specific synaptic protein dynamics in the context of long-term memory, but key regulators that coordinate plasticity events to maintain synaptic stability over the lifetime of the organism have not yet been identified. Therefore, instead of systematically attaching this growing list of synaptic proteins to TS, a synapse-wide proteomic screen was designed to identify key regulators in maintaining stable synapses from which targets for TS tagging could be identified. This screen identified many novel synaptic proteins that have had little attention in mammalian systems and would be good candidates to study with the described TS reporters.