Development of single-molecule ubiquitination mediated fluorescence complementation to visualize protein
Marius F Ifrim1, Aleksandra Janusz-Kaminska2, Gary J Bassell2
1Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, Weiskotten Hall, 766 Irving Avenue, Syracuse, NY 13210, USA; Department of Cell Biology, Emory University School of Medicine, 615 Michael St., Atlanta, GA 30322, USA.
Abstract:
The ubiquitination/proteasome system is important for the spatiotemporal control of protein synthesis and degradation at synapses, while dysregulation may underlie autism spectrum disorders (ASDs). However, methods allowing direct visualization of the subcellular localization and temporal dynamics of protein ubiquitination are lacking. Here we report the development of Single-Molecule Ubiquitin Mediated Fluorescence Complementation (SM-UbFC) as a method to visualize and quantify the dynamics of protein ubiquitination in dendrites of live neurons in culture. Using SM-UbFC, we demonstrate that the rate of PSD-95 ubiquitination is elevated in dendrites of FMR1 KO neurons compared with wild-type controls. We further demonstrate the rapid ubiquitination of the fragile X messenger ribonucleoprotein, FMRP, and the AMPA receptor subunit, GluA1, which are known to be key events in the regulation of synaptic protein synthesis and plasticity. SM-UbFC will be useful for future studies on the regulation of synaptic protein homeostasis.
