Hypoxia-inducible factor 1 (HIF-1) is a master regulator of oxygen homeostasis and cellular adaptation to hypoxia. Its α-subunit (HIF-1α) is continuously synthesized and, under normoxia, degraded via oxygen-dependent posttranslational modifications mediated by prolyl hydroxylases and the von Hippel–Lindau complex. Despite extensive biochemical characterization, the real-time intracellular dynamics of HIF-1α remain poorly understood.
In this work, the photoconvertible fluorescent protein Eos was employed to visualize and analyze HIF-1α behavior in living cells. Eos fluoresces green irreversibly switches to red upon 365–405 nm illumination, allowing time-resolved tracking of protein populations. HIF-1α was genetically fused to monomeric EosFP, and several mutants targeting key hydroxylation (P402A, P564G) and phosphorylation (S576A, S657A) sites were generated and verified by Sanger sequencing. Tandem Eos (tdEos) constructs yielded higher signal intensity but displayed unphysiological cytoplasmic aggregation, prompting the exclusive use of the monomeric EosFP fusion for imaging experiments.
Under both wide-field and confocal microscopy, HIF-1α-Eos fusion proteins were tracked even under normoxic conditions. The study provided valuable insights into the dynamics of HIF-1α. The speed of the processes was unexpectedly fast. Additionally, the observed increase in the green signal following photoconversion is a novel finding, offering further insights into the underlying mechanisms of photoconversion. The experiments demonstrated that EosFP photoconversion worked efficiently with 365 nm excitation, producing minimal photobleaching at moderate illumination. Notably, the first exposure to the UV LED caused a 20–30% increase in green fluorescence intensity—a behavior not previously described for EosFP.
Confocal laser scanning microscopy enabled precise regional photoconversion using a 405 nm laser, revealing that red-converted HIF-1α-EosFP redistributed rapidly throughout the nucleus within seconds, achieving a homogeneous signal within about one minute. This fast mobility highlights the transient nature of HIF-1α interactions and demonstrates that Eos-based imaging can capture even short-lived nuclear dynamics.
Stable HEK293 knock-in cell lines were created using the TALEN system targeting the AAVS1 locus, and correct integration was verified by junction PCR. However, fluorescence signals from knock-in constructs remained below the detection threshold, despite confirmed mRNA and protein expression by qPCR and Western blot. These results suggest that EosFP tagging or promoter strength may limit detectable expression levels for low-abundance transcription factors.
This approach demonstrated both the potential and the limits of Eos-based imaging to capture fast transcription factor dynamics. It established a methodological framework combining molecular cloning, gene editing, and advanced microscopy for investigating oxygen-regulated proteins. The study also highlights how modulating HIF-1α via agents such as IOX2 (a prolyl-hydroxylase inhibitor) and MG132 (a proteasome inhibitor) could guide therapies by promoting or inhibiting its degradation, providing new perspectives for cancer and cardiovascular disease treatment strategies.
