The subcellular dynamics of ubiquitination has important roles in many biological processes from gene expression to synaptic function and impairments may directly result and/or contribute to neurological disease. While past studies using biochemical fractionation or microscopy on fixed cells suggest the importance of the ubiquitination-proteasome system (UPS) in synaptic protein homeostasis and neuronal function, the methods used do not allow for the direct visualization of subcellular localization and dynamics of ubiquitination. The transient nature of the ubiquitinated products hinders their detection and prevents the analysis of the rate and how the rate of protein ubiquitination may be affected by synaptic plasticity and/or altered in neurological diseases. We have recently developed a new single molecule method, Single Molecule Ubiquitination Mediated Fluorescence Complementation (SM-UbFC), using split-Venus-based fluorescent reporters to visualize and quantify the subcellular dynamics of ubiquitination in live neurons. This method will be applied to elucidate new mechanisms of synapse and RNA biology in health and disease using cultured mouse cortical neurons and human iPSC derived neurons. Aim-1 tests the hypothesis that plasticity-inducing stimuli regulate ubiquitination of synaptic scaffolding proteins and glutamate receptor subunits directly in dendritic spines, which is altered in fragile x syndrome. We will further develop, optimize and apply SM-UbFC to detect dynamic changes in the ubiquitination of synaptic proteins in mouse and human iPSC derived neurons in response to plasticity inducing stimuli. Aim- 2 tests the hypothesis that the Cdh1-APC E3 ligase complex regulates RNA granules at synapses by ubiquitination of multiple RNA binding proteins, focusing on candidates we have recently identified in a mass spectrometry analysis of the Cdh1 interactome. These include FMRP, FXR1P and Caprin-1. We will further develop, optimize and apply SM-UbFC to detect dynamic changes in the ubiquitination of these RNA binding proteins in RNA granules using mouse cortical neurons and human iPSC derived neurons. We will further test the hypothesis that RNA granule dynamics are altered in FMR1 KO neurons or by disease linked mutations in Caprin-1. The model systems selected in Aims1+2 will uncover new mechanisms of ubiquitination dynamics regulating the postsynaptic density and glutamate receptors (Aim-1) and RNA granules localized to synapses (Aim-2). The SM-UbFC method is anticipated to be useful for future studies to elucidate mechanisms regulating ubiquitination dynamics and understanding how they may go awry in neurological diseases leading to dysregulation of synaptic protein homeostasis. This sensitive method provides a potential tool to assess efficacy of therapeutic strategies to correct defects in ubiquitination dynamics in neurological disease.