TOOLS FOR SINGLE-CELL ANALYSIS OF DROSOPHILA METABOLISM

GÁNDARA, L.; WAPPNER, P.

Ciudad de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencia; 2017

It is a well stablished fact that tumor cells suffer a metabolic reprograming that makes them unable to fully oxidize carbon chains through mitochondrial catabolism, turning glycolysis into their main ATP source. Recently, the idea that this metabolic switch (called Warburg effect) could be a feature of cell proliferation and differentiation during normal development has started to be worked out. We have developed several tools to test this idea in Drosophila melanogaster development. We generated three different FRET sensors, each of them reporting intracellular levels of a different key metabolite. Pyronic senses pyruvate, Laconic senses lactate, whilst OGSOR senses 2-oxoglutarate (2-OG). The 2-OG sensor OGSOR was originally developed in a prokaryotic system, so we are in the process of analyzing its behavior in Drosophila tissues throughout development. Its emission spectrum, as well as its dynamic response to increasing levels of extracellular 2-OG, were stablished after driving its expression in several larval organs. We observed an increase in intracellular levels of 2-OG elicited by both oxygen and nutrient deprivation, as well as in cells in which mitochondrial 2-OG transporters have been genetically downregulated. This implies that most of the cytosolic 2-OG arises from glutamate deamination rather than from the Krebs cycle. In addition, we engineered in a Drosophila fly line a transcriptional reporter of the Pyruvate Dehydrogenase Kinase (PDHK), one of the central regulators of the metabolic switch. The PDHK transcriptional reporter revealed very specific tissues and cell types in which this enzyme is highly expressed, suggesting that these zones might undergo strong glycolytic catabolism. Analysis of these particular expression territories with the remaining tools (FRET sensors) that we have developed will light on the metabolic profile of differentiating cells during Drosophila development.