Phosphofructokinase relocalizes into subcellular compartments with liquid-like properties in vivo.

Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.