Investigation of protein synthesis in Drosophila larvae using puromycin labelling

Abstract

Translational control of gene expression is an important regulator of growth, homeostasis and aging in Drosophila. The ability to measure changes in protein synthesis in response to genetic and environmental cues is therefore important in studying these processes. Here we describe a simple and cost effective approach to assay protein synthesis in Drosophila larval cells and tissues. The method is based on the incorporation of puromycin into nascent peptide chains. Using an ex vivo approach, we label newly synthesized peptides in larvae with puromycin and then measure levels of new protein synthesis using an anti-puromycin antibody. We show that this method can detect changes in protein synthesis in specific cells and tissues in the larvae, either by immunostaining or western blotting. We find that the assay reliably detects changes in protein synthesis induced by two known stimulators of mRNA translation - the nutrient/TORC1 kinase pathway and the transcription factor dMyc. We also use the assay to describe how protein synthesis changes through larval development and in response to two environmental stressors - hypoxia and heat-shock. We propose that this puromycin-labelling assay is a simple, but robust method to detect protein synthesis changes at the levels of cells, tissues or whole body in Drosophila.