Abstract
Proteostasis is critical for cellular function and longevity, especially in long-lived cells including neurons. A major component of proteostasis is the regulated degradation and replacement of proteins to ensure their quality and appropriate abundance. The regulation of synaptic vesicle protein turnover in neurons is important for understanding synaptic communication, yet it is incompletely understood, partly due to limited tools for assessing protein turnover in vivo. Here, we present ARGO (Analysis of Red-Green Offset), a fully genetically encoded, ratiometric fluorescence imaging method that visualizes and quantifies protein turnover with subcellular resolution in vivo. ARGO involves cell-specific labeling of the protein-of-interest with both RFP and GFP, followed by Cre/Lox-mediated removal of GFP (pulse) and periodic ratiometric imaging to track protein turnover (chase). This approach is inexpensive, modular, and scalable for use in genetically tractable experimental organisms. Using ARGO, we examined the turnover of Synaptogyrin/SNG-1, an evolutionarily conserved, integral SV protein, in adult Caenorhabditis elegans neurons. Our findings support the model that SV proteins are sorted for degradation at the synapse, then trafficked to the neuron cell body to complete degradation. We show that the rate of presynaptic SNG-1 turnover is consistent across synapses within a single neuron, indicating a cell-wide super-pool for SV protein degradation. Our results further suggest that, contrary to prevailing models, neither the surveillance nor the sorting of SV proteins for degradation is a rate-limiting step for SNG-1 turnover; rather, the rate-limiting step is the clearance of sorted-for-degradation SNG-1 from the presynapse.
Article Summary How proteins are turned over within subcellular compartments is not well understood, in part because the phenomenon is difficult to quantify. The authors developed a simple, genetically encoded method to quantify the turnover of a protein-of-interest using fluorescence microscopy. They used this method to begin to assess synaptic vesicle protein turnover in vivo, as this is important for synaptic function. They found that synaptic vesicle protein Synaptogyrin/SNG-1 is sorted for degradation at the synapse but degraded in the neuron cell body, and the turnover rate depends on animal age but is constant across presynapses within a neuron.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The biorxiv doi has been added to the reference for the Zhong et al., 2024 manuscript.
