Nanoscopy of the cellular response to hypoxia by means of fluorescence resonance energy transfer (FRET) and new FRET software

Wotzlaw, Christoph; Gneuss, Silke; Konietzny, Rebecca; Fandrey, Joachim

doi:10.1186/1757-5036-3-5

Cited by 10 publications

(15 citation statements)

References 37 publications

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“…In line with this data Wotzlaw and colleagues demonstrated that PHD1 and HIF-1a interact within the nuclear compartment (Wotzlaw et al, 2010). Recently, the expression patterns, as well as the subcellular localisation of PHDs, have been linked with tumorigenesis.…”

Section: Introductionsupporting

confidence: 49%

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Oxygen sensing by Prolyl-4-Hydroxylase PHD2 within the nuclear compartment and the influence of compartimentalisation on HIF-1 signalling

Pientka

Schindler

et al. 2012

Journal of Cell Science

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SummaryHypoxia-inducible factors (HIFs) regulate more than 200 genes involved in cellular adaptation to reduced oxygen availability. HIFs are heterodimeric transcription factors that consist of one of three HIF-a subunits and a HIF-b subunit. Under normoxic conditions the HIFa subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1, PHD2 and PHD3, resulting in recognition by von-Hippel-Lindau protein, ubiquitylation and proteasomal degradation. It has been suggested that PHD2 is the key regulator of HIF-1a stability in vivo. Previous studies on the intracellular distribution of PHD2 have provided evidence for a predominant cytoplasmic localisation but also nuclear activity of PHD2. Here, we investigated functional nuclear transport signals in PHD2 and identified amino acids 196-205 as having a crucial role in nuclear import, whereas amino acids 6-20 are important for nuclear export. Fluorescence resonance energy transfer (FRET) showed that an interaction between PHD2 and HIF-1a occurs in both the nuclear and cytoplasmic compartments. However, a PHD2 mutant that is restricted to the cytoplasm does not interact with HIF-1a and shows less prolyl hydroxylase activity for its target HIF-1a than wild-type PHD2 located in the nucleus. Here, we present a new model by which PHD2-mediated hydroxylation of HIF-1a predominantly occurs in the cell nucleus and is dependent on very dynamic subcellular trafficking of PHD2.

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Section: Introductionsupporting

confidence: 49%

“…PHD2 activity was inhibited by addition of 2 mM DMOG for 24 hours resulting in stabilisation of ECFP-HIF-1a or ECFP-HIF-1a-mut. 48 hours after transfection FRET was monitored as described previously and FRET signals were analysed according to the sensitised FRET method using FRET software (Wotzlaw et al, 2010).…”

Section: Plasmids and Dna Modificationmentioning

confidence: 99%

Oxygen sensing by Prolyl-4-Hydroxylase PHD2 within the nuclear compartment and the influence of compartimentalisation on HIF-1 signalling

Pientka

Schindler

et al. 2012

Journal of Cell Science

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“…In line with this, fluorescence resonance energy transfer (FRET) measurements have shown that PHD1 and HIF-1α interact within the nuclear compartment [79]. The nuclear import of PHD1 relies on an NLS between aa Pro-117 and Glu-123.…”

Section: Intracellular Distribution Of the Hif-α Hydroxylasesmentioning

confidence: 79%

Nuclear-cytoplasmatic shuttling of proteins in control of cellular oxygen sensing

2015

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In order to pass through the nuclear pore complex, proteins larger than ∼40 kDa require specific nuclear transport receptors. Defects in nuclear-cytoplasmatic transport affect fundamental processes such as development, inflammation and oxygen sensing. The transcriptional response to O2 deficiency is controlled by hypoxia-inducible factors (HIFs). These are heterodimeric transcription factors of each ∼100-120 kDa proteins, consisting of one out of three different O2-labile α subunits (primarily HIF-1α) and a more constitutive 1β subunit. In the presence of O2, the α subunits are hydroxylated by specific prolyl-4-hydroxylase domain proteins (PHD1, PHD2, and PHD3) and an asparaginyl hydroxylase (factor inhibiting HIF-1, FIH-1). The prolyl hydroxylation causes recognition by von Hippel-Lindau tumor suppressor protein (pVHL), ubiquitination, and proteasomal degradation. The activity of the oxygen sensing machinery depends on dynamic intracellular trafficking. Nuclear import of HIF-1α and HIF-1β is mainly mediated by importins α and β (α/β). HIF-1α can shuttle between nucleus and cytoplasm, while HIF-1β is permanently inside the nucleus. pVHL is localized to both compartments. Nuclear import of PHD1 relies on a nuclear localization signal (NLS) and uses the classical import pathway involving importin α/β receptors. PHD2 shows an atypical NLS, and its nuclear import does not occur via the classical pathway. PHD2-mediated hydroxylation of HIF-1α occurs predominantly in the cell nucleus. Nuclear export of PHD2 involves a nuclear export signal (NES) in the N-terminus and depends on the export receptor chromosome region maintenance 1 (CRM1). Nuclear import of PHD3 is mediated by importin α/β receptors and depends on a non-classical NLS. Specific modification of the nuclear translocation of the three PHD isoforms could provide a promising strategy for the development of new therapeutic substances to tackle major diseases.

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“…The Förster distance, i.e., the distance at which the energy transfer effi ciency is 50 %, is specifi c for the donor acceptor pair and is 4.92 nm for the ECFP and EYFP pair. Of note, FRET effi ciency was found to strongly depend on the acceptor/donor ratio [ 34 ]. It was therefore important to scan many cells with a range of acceptor/donor ratios and to determine FRET effi ciency until it reached its maximum at a plateau (Fig.…”

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

confidence: 99%

“…The effi ciency of the energy transfer (FRET-effi ciency) depends on the distance between the two chromophores which is the higher the closer the two proteins are. FRET effi ciency can then be used to calculate distances between proteins and prove interaction [ 34 ] calculated from the maximum FRET-effi ciency using the formula…”

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

confidence: 99%

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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Nanoscopy of the cellular response to hypoxia by means of fluorescence resonance energy transfer (FRET) and new FRET software

Cited by 10 publications

References 37 publications

Oxygen sensing by Prolyl-4-Hydroxylase PHD2 within the nuclear compartment and the influence of compartimentalisation on HIF-1 signalling

Oxygen sensing by Prolyl-4-Hydroxylase PHD2 within the nuclear compartment and the influence of compartimentalisation on HIF-1 signalling

Nuclear-cytoplasmatic shuttling of proteins in control of cellular oxygen sensing

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

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