Visualization of the three-dimensional organization of hypoxia-inducible factor-1α and interacting cofactors in subnuclear structures

Berchner‐Pfannschmidt, Utta; Wotzlaw, Christoph; Merten, E.; Acker, H.; Fandrey, Joachim

doi:10.1515/bc.2004.017

Cited by 20 publications

(16 citation statements)

References 37 publications

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“…Sites of interaction were heterogeneously distributed confi rming the speckle-like distribution of HIF-1α which was described for Fig. 17.1 but also in earlier work using 2-photon laser microscopy [ 2 ].…”

Section: Fluorescence Recovery After Photo-bleaching (Frap) U2os Celsupporting

confidence: 76%

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

Bernardini

Fandrey

2016

Advances in Experimental Medicine and Biology

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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.

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Section: Fluorescence Recovery After Photo-bleaching (Frap) U2os Celsupporting

confidence: 76%

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

Bernardini

Fandrey

2016

Advances in Experimental Medicine and Biology

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“…Within the hypoxic nucleus, endogenous HIF-1a and HIF-1b are colocalised in speckle-like structures, as observed by immunofluorescence combined with three-dimensional visualisation studies [47]. These speckle-like structures were also observed when ECFP-HIF-1a and EYFP-HIF-1b fusion proteins were coexpressed, but not when each construct was transfected alone, implying that these structures are the sites of HIF-1 complexes [48].…”

Section: Subcellular Distribution Of Hif Subunits and Interaction Witmentioning

confidence: 84%

Imaging of the hypoxia-inducible factor pathway: insights into oxygen sensing

Berchner‐Pfannschmidt¹,

Frede²,

Wotzlaw³

et al. 2008

Eur Respir J

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The transcription factor complex hypoxia-inducible factor (HIF)-1 controls the expression of most genes involved in adaptation to hypoxic conditions. HIF-1 is a heterodimer composed of oxygen-labile HIF-a and constitutively expressed HIF-b subunits. The oxygendependent regulation of HIF-a is a multistep process that includes degradation under normoxia but stabilisation, translocation into the nucleus and activation under hypoxic conditions.The present paper summarises the contributions of optical methods to the understanding of oxygen-dependent regulation of the HIF-1 pathway. The tissue-and cell-specific distribution of HIF-a was visualised immunohistochemically and by immunofluorescence. Transcriptional activity of HIF-1 was monitored using green fluorescent protein as a reporter under control of hypoxia response elements in living cells, spheroids and tumour tissues in living mice. With cyan and yellow variants of green fluorescent protein fused to HIF subunits and regulatory proteins, subcellular distribution, migration and interaction were imaged in vivo by means of fluorescence recovery after photo-bleaching and fluorescence resonance energy transfer. Noninvasive imaging of these cellular and molecular processes by laser scanning microscopy complements ex vivo molecular biology assays and provides an additional spatial and temporal dimension to the understanding of the HIF-1 pathway.KEYWORDS: Fluorescence recovery after photo-bleaching, fluorescence resonance energy transfer, green fluorescent protein fusion proteins, hypoxia-inducible factor-1, immunohistology, in vivo imaging

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“…The coverslips were mounted on the slides with Mowiol (Calbiochem, Bad Soden, Germany). Three-dimensional visualization was performed with laser scanning microscope (PCM2000; Nikon) equipped with a two photon Coherent Mira 900M laser as described before (34,35). Yellow fluorescence of Alexa-532 was collected through a 585-nm long pass filter.…”

Section: Methodsmentioning

confidence: 99%

Nuclear Oxygen Sensing: Induction of Endogenous Prolyl-hydroxylase 2 Activity by Hypoxia and Nitric Oxide

Berchner‐Pfannschmidt

Tug

Trinidad

et al. 2008

Journal of Biological Chemistry

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The abundance of the transcription factor hypoxia-inducible factor is regulated through hydroxylation of its ␣-subunits by a family of prolyl-hydroxylases (PHD1-3). Enzymatic activity of these PHDs is O 2 -dependent, which enables PHDs to act as cellular O 2 sensor enzymes. Herein we studied endogenous PHD activity that was induced in cells grown under hypoxia or in the presence of nitric oxide. Under such conditions nuclear extracts contained much higher PHD activity than the respective cytoplasmic extracts. Although PHD1-3 were abundant in both compartments, knockdown experiments for each isoenzyme revealed that nuclear PHD activity was only due to PHD2. Maximal PHD2 activity was found between 120 and 210 M O 2 . PHD2 activity was strongly decreased below 100 M O 2 with a half-maximum activity at 53 ؎ 13 M O 2 for the cytosolic and 54 ؎ 10 M O 2 for nuclear PHD2 matching the physiological O 2 concentration within most cells. Our data suggest a role for PHD2 as a decisive oxygen sensor of the hypoxia-inducible factor degradation pathway within the cell nucleus.The capability of mammalian cells to sense oxygen and adapt gene expression according to O 2 availability is critical for the maintenance of oxygen homoeostasis within the tissue. The heterodimeric transcriptional regulator hypoxia-inducible factor (HIF) 2 is central to the regulation of gene expression in response to decreased oxygen levels, i.e. hypoxia (1). HIFs are composed of the constitutive ␤-subunit and one of three O 2 -labile ␣-subunits HIF-1, -2, or -3␣ (2, 3). Both the stability and the activity of the HIF ␣-subunits are regulated by oxygen-dependent post-translational modifications.Under normoxic conditions HIF␣s are hydroxylated by a family of prolyl-4-hydroxylases (PHD1-3) at two conserved proline residues in HIF-1␣ and -2␣ (Pro 402/564 or Pro 405/531 , respectively) or a single proline residue in HIF-3␣ (Pro 490 ) (4 -6). Hydroxylated HIF␣ is recognized by the von HippelLindau protein (pVHL) E3 ubiquitin ligase complex and targeted for proteasomal degradation (7-9). Hypoxia reduces prolyl-hydroxylation by PHDs, resulting in stabilization of the ␣-subunit. This allows dimerization with the ␤-subunit and induces expression of about 100 genes involved in adaptation to hypoxia (10). Transcriptional activity of the HIF complex is regulated by hydroxylation of an asparagine residue within the C-terminal trans-activating domain of HIF-1␣ and -2␣ by an asparagyl-hydroxylase termed factor-inhibiting HIF; active factor-inhibiting HIF hydroxylates Asp 803 in HIF-1␣ under normoxia, which impedes binding of the transcriptional coactivatorp300/CBP(11,12).ByhydroxylatingHIF-␣sinanoxygen-dependent manner, PHDs and factor-inhibiting HIF function as oxygen-sensing enzymes of HIF activation.The three PHD isoenzymes belong to the superfamily of iron and 2-oxoglutarate-dependent dioxygenases. These enzymes need O 2 as cosubstrate, which provides the molecular basis for their O 2 -sensing function. Oxygen dependence of PHDs is reflected by their K m (O 2 ) va...

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Visualization of the three-dimensional organization of hypoxia-inducible factor-1α and interacting cofactors in subnuclear structures

Cited by 20 publications

References 37 publications

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

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

Imaging of the hypoxia-inducible factor pathway: insights into oxygen sensing

Nuclear Oxygen Sensing: Induction of Endogenous Prolyl-hydroxylase 2 Activity by Hypoxia and Nitric Oxide

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