# Single-Molecule Imaging of Ubiquitination Dynamics in Neurons

> **NIH NIH R21** · EMORY UNIVERSITY · 2023 · $430,375

## Abstract

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.

## Key facts

- **NIH application ID:** 10817362
- **Project number:** 1R21NS133933-01A1
- **Recipient organization:** EMORY UNIVERSITY
- **Principal Investigator:** GARY J BASSELL
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $430,375
- **Award type:** 1
- **Project period:** 2023-09-15 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10817362

## Citation

> US National Institutes of Health, RePORTER application 10817362, Single-Molecule Imaging of Ubiquitination Dynamics in Neurons (1R21NS133933-01A1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10817362. Licensed CC0.

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