Optical Analysis of Hypoxia Inducible Factor (HIF)-1 Complex Assembly: Imaging of Cellular Oxygen Sensing

Abstract

Hypoxia is a common phenomenon that occurs in a variety of diseases such as cardiovascular ischemia, anemia, and cancer. Cellular oxygen sensors measure changes in tissue oxygenation and induce responses aimed at restoring sufficient supply with oxygen. Genetic adaptation to hypoxia is under control of hypoxia-inducible factors (HIFs), of which two highly homologous subunits HIF-1α and HIF-2α are regulated by oxygen tension. Together with HIF-1β (=ARNT; aryl hydrocarbon receptor nuclear translocator) they form transcriptionally active complexes under hypoxia which drive the expression of hypoxia inducible genes. The meaning of different HIF complexes, i.e., HIF-1α/ARNT versus HIF-2α/ARNT with respect to target gene or tissue specificity has not been fully resolved. We applied modern microscopic methods like fluorescence resonance energy transfer (FRET) to elucidate protein-protein interactions and fluorescence recovery after photo-bleaching (FRAP) to study mobility of HIF proteins inside the nuclei of living cells. We found differences both in nuclear mobility and the assembly of HIF-1 versus HIF-2 which might help to better understand the assembly of HIF complexes.

Hypoxia‐inducible factor‐1α regulates the interleukin‐6 production by B cells in rheumatoid arthritis

“…The enrichment of B cell cytoplasmic HIF‐1α suggests elevated transcription and accumulation, or reduced degradation, which is related to the pro‐inflammatory microenvironment and hypoxic conditions in RA synovial tissue. Since only a small fraction of the HIF‐1α complex will transport into the nucleus when performing its function, and its mobility in the nucleus, 40 , 41 detecting intranuclear HIF‐1α, especially in formalin‐fixed tissue, is challenging.…”

Melatonin for prevention of fetal lung injury associated with intrauterine inflammation and for improvement of lung maturation

“…For immunohistochemistry (IHC), slides were incubated in PBS containing 0.05% Triton X-100 (Sigma-Aldrich) and 5% normal goat serum (Invitrogen) for 30 minutes. Lung tissues were incubated with an endothelial marker, rabbit anti-vimentin antibody (1:200; Abcam), 41 a chemokine receptor that has angiogenic effects on microvascular endothelial cells, C-X-C motif chemokine receptor 2 (CXCR2) antibody (1:200; Abcam), 29 a marker of oxidative stress, hypoxia-inducible factors (HIF)-1β (1:200; Abcam), 42 surfactant protein B (SP-B) (1:400; Abcam). 43 donkey anti-rat Alexa Fluor 488, and donkey anti-rabbit Alexa Fluor 568 fluorescent secondary antibodies were used (1:500; Life Technologies) along with DAPI counterstaining.…”

Quantitative Phase Imaging Detecting the Hypoxia-Induced Patterns in Healthy and Neoplastic Human Colonic Epithelial Cells

“…Among hypoxia imaging techniques such as positron emission tomography (PET), single photon emission computed tomography (SPECT), electron paramagnetic resonance (EPR), and magnetic resonance imaging (MRI), optical techniques (bioluminescence and fluorescence) still play a major role in the examination of hypoxia at the cell and tissue level [ 16 , 17 ]. The hypoxic environment may also be detected using hyperspectral imaging supported by fluorescent/phosphorescent boron nanoparticles [ 18 ], specific near-infrared optical imaging probes [ 19 ], two-photon fluorescence microscopy [ 20 , 21 , 22 ], and fluorescence resonance energy transfer (FRET) [ 23 , 24 ], which were implemented to monitor hypoxia in solid tumors or living cells. However, it should be noted that the main disadvantages of fluorescence imaging related to the use of immunofluorescence for staining are associated with permanent modification of the sample, photobleaching, and phototoxicity or interference caused by other target proteins.…”