In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons

Protein translation has been implicated in different forms of synaptic plasticity but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based upon incorporation of non-canonical amino acids into proteins followed by chemo–selective fluorescent tagging via click chemistry. Following brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse–chase–like application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain–promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single particle tracking and quantum dots. The newly synthesized proteins exhibited a broad range of diffusive behaviors as expected if the pool of labeled proteins was heterogeneous.