Glycolysis regulates Hedgehog signalling via the plasma membrane potential

Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator—the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady‐state levels of ATP and stabilizes Smoothened (Smo), the 7‐pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full‐length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N‐acylethanolamides—lipoprotein‐borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo—a key step in pathway activation—in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.