In order to understand the frequency of exchange of sarcomeric proteins in the muscle, two major state-of-the-art techniques were employed in the REBUILD project:
1) Mass-spectrometry combined with labelled aminoacids. Animals are fed a diet where the sole source of Lysine is a heavier isotope (Lys8) that causes a mass shift of 8Da on a mass spectra, upon digestion of all proteins with an enzyme called lysyl endopeptidase (LysC). In this manner, we can identify newly synthesized proteins after defined time points via incorporation of Lys8 and comparing the ratio of light (before the shift in food source) vs heavy (after introducing the Lys8 food) peptides. This heavy:light ratio informs on the rate of replacement for any given protein in the muscle cell, since mass-spectrometry allows for unbiased detection of the entire proteome.
2) RNA-Sequencing of dissected muscle fibers. To better understand regulators of protein replacement, we performed performed massive next-gen sequencing of RNA from dissected flight muscle of Drosophila after different activity interventions and at several age points, detailed below.
3) A novel “exercise chamber” to stimulate and track flight of free-flying Drosophila. We developed and built a large “Flydome” to stimulate flight-on-demand of populations of hundreds of flies, simultaneously, and combined it with camera-vision and dedicated software to track and quantify flight of all flies. This allowed to obtain muscle from “exercised” flies which we then compared to control flies and “non-flyers” – flies that are unable to contract their flight muscles, due to genetic ablation of their motor neurons. These 3 mechanical/activity interventions (control, exercise, no-flight) we employed to obtain muscle samples with different activation histories, and also, from flies at different stages along their lifespan. These samples were analysed via the techniques mentioned above.
4) Genetic tools for observing directly protein exchange. In order to confirm and study in depth the mechanisms involved in protein turnover, new genetic tools that take advantage of existing regulatory mechanisms were developed. We combined temporal and cell-specific enhancers (both via tissue-specific GAL4 enhancers and flipable fluorescent reporters) with, so-called nanobodies, to control incorporation of green fluorescent proteins attached to newly synthesized sarcomeric proteins. This allowed observation of introduction of new components in sarcomeres after a experimentally defined stimulus.
