An optimized reporter of the transcription factor hypoxia- inducible factor 1α reveals complex HIF-1α activation dynamics in single cells

Immune cells adopt a variety of metabolic states to support their many biological functions, which include fighting path-ogens, removing tissue debris, and tissue remodeling. One of the key mediators of these metabolic changes is the tran-scription factor hypoxia-inducible factor 1 alpha (HIF-1 alpha). Single-cell dynamics have been shown to be an important determi-nant of cell behavior; however, despite the importance of HIF-1 alpha, little is known about its single-cell dynamics or their effect on metabolism. To address this knowledge gap, here we opti-mized a HIF-1 alpha fluorescent reporter and applied it to study single-cell dynamics. First, we showed that single cells are likely able to differentiate multiple levels of prolyl hydroxylase inhi-bition, a marker of metabolic change, via HIF-1 alpha activity. We then applied a physiological stimulus known to trigger meta-bolic change, interferon-gamma, and observed heterogeneous, oscil-latory HIF-1 alpha responses in single cells. Finally, we input these dynamics into a mathematical model of HIF-1 alpha-regulated metabolism and discovered a profound difference between cells exhibiting high versus low HIF-1 alpha activation. Specifically, we found cells with high HIF-1 alpha activation are able to meaning-fully reduce flux through the tricarboxylic acid cycle and show a notable increase in the NAD+/NADH ratio compared with cells displaying low HIF-1 alpha activation. Altogether, this work demonstrates an optimized reporter for studying HIF-1 alpha in single cells and reveals previously unknown principles of HIF-1 alpha activation.