Infrared Laser Heating Reveals a Reversible Phase Transition of Phosphofructokinase 1 in Mammalian Cells

Metabolic enzymes can operate in concerted networks, so-called metabolons. The glucosome is a multi-enzyme complex that includes enzymes of glycolysis and gluconeogenesis and regulates glucose metabolism in mammalian cells. The mechanism of how glucosomes assemble remains largely unknown. Fluorescence recovery after photobleaching (FRAP) experiments suggest that glucosomes are liquid-like, but direct evidence of a phase transition mechanism is missing. Here, we developed a novel method to measure the phase transition of condensates in living cells by combining infrared laser heating with confocal microscopy of fluorescently labeled phosphofructokinase 1 liver-type (PFKL). We showed that glucosomes form and dissolve reversibly in HeLa cells upon temperature modulation, allowing us to determine the condensation temperature. We studied cells under osmotic stress to show that glucosomes retain their phase behavior despite a significant decrease in the mobility of PFKL. We propose that this method is a versatile tool to study the phase behavior of biomolecular condensates, yielding complementary information to well-established techniques such as FRAP.