Figure 1

Sensors for studying cellular bioenergetics. (a) Diagram of glucose catabolism. Glucose is broken down at glycolysis and then can be fully oxidized to CO₂ at the mitochondria (left), or alternatively, partially oxidized to lactate (right); pyruvate (red) stands as the branching point between the two alternative pathways. (b) Schematic representation of the FRET sensors^12,13,14. The donor and acceptor fluorophores, CFP and YFP, are represented in blue and yellow, respectively. Binding of the corresponding metabolite (green) to its binding domain elicits a conformational change that separates CFP from YFP, and FRET ceases. (c,d) FRET maps of the Laconic signal of wing imaginal discs in which different expression levels of the sensor were attained by introducing either 1 or 2 copies of the tub-Laconic construct. Note that the apparent FRET signal obtained after subtracting a constant background value is dependent on the expression levels of the sensor, while this dependency is largely suppressed after applying the linear unmixing algorithm (compare 1x vs 2x Tub-Laconic and the region pointed by arrows to the rest of the tissue). A high FRET signal in the color code shown on the right must be associated to low lactate levels. Scale bar: 100 μm. Dotted lines show the region in which the FRET signal was measured. (e) Quantification of Laconic expression and of the FRET signal obtained after constant background subtraction (CB) or applying the linear unmixing algorithm (LU) in wing discs with 1 or 2 copies of Tub-Laconic. Data represent the media +/− SD; p = 1.4292E-05 for expression levels; p = 0.0358 for FRET signal (LU); Student’s T-test; n ≥ 20 per group.