Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive α-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1α prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1α accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation.

SummaryTwo-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580-1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two-photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two-photon and the one-photon emission spectra. However, a blue-shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non-linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.

It has been proposed that hydroxyl radicals (⅐OH) generated in a perinuclear iron-dependent Fenton reaction are involved in O 2-dependent gene expression. Thus, it was the aim of this study to localize the cellular compartment in which the Fenton reaction takes place and to determine whether scavenging of ⅐OH can modulate hypoxia-inducible factor 1 (HIF-1)-dependent gene expression. The Fenton reaction was localized by using the nonfluorescent dihydrorhodamine (DHR) 123 that is irreversibly oxidized to fluorescent rhodamine 123 while scavenging ⅐OH together with gene constructs allowing fluorescent labeling of mitochondria, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes, or lysosomes. A 3D two-photon confocal laser scanning microscopy showed ⅐OH generation in distinct hot spots of perinuclear ER pockets. This ER-based Fenton reaction was strictly pO 2-dependent.

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Two human neuroblastoma (NB) cell lines, SH-SY5Y and Kelly, were found to express the gene for erythropoietin (EPO) in an oxygen (O 2 )-dependent manner. However, NB cells had maximal production of EPO with lower partial pressure of O 2 values than the well-characterized hepatoma cell line HepG2. This maximal EPO expression was preceded by accumulation of the O 2 -sensitive ␣ subunit of the heterodimeric transcription-factor complex hypoxia-inducible factor 1 (HIF-1). Western blot analysis revealed that the amount of the ␤ subunit of HIF-1, identical to aryl hydrocarbon receptor nuclear translocator 1 (ARNT1), and the homolog ARNT2 increased in nuclear extracts from SH-SY5Y cells exposed to anoxia. In neuronal cells, ARNT1 and ARNT2 can form a heterodimer with HIF-1␣, generating a functional HIF-1 complex. Using the hypoxia response element of the human EPO enhancer, we conducted electrophoretic mobility shift assays that showed accumulation and binding of HIF-1 complexes containing both ARNT1 and ARNT2 in NB cells. In addition to the HIF-1 complex, hepatocyte nuclear factor 4␣ (HNF4␣) was found to be indispensable for hypoxiainduced EPO gene expression in hepa- IntroductionHypoxia-inducible expression of the glycoprotein hormone erythropoietin (EPO) is part of the body's response to hypoxia, which includes up-regulation of oxygen (O 2 )-dependent genes involved in vascular tone and growth, metabolic adaptation, and O 2 delivery. 1,2 EPO is produced primarily by the kidneys and liver, but EPO gene expression has also been found in several other tissues, including brain tissue, 3 breast cancer cells, 4 female genital tract tissues, 5 and rat Sertoli cells. 6 EPO gene expression is regulated by the heterodimeric transcription-factor complex hypoxia-inducible factor 1 (HIF-1), which is composed of a 120-kDa O 2 -regulated ␣ subunit and a 91-to 94-kDa constitutively expressed ␤ subunit. 7 Under normoxic conditions, HIF-1␣ is posttranslationally hydroxylated at proline residues 402 and 564, 8 which tags the protein for ubiquitination by the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor-suppressor protein. 9,10 Subsequently, HIF-1␣ protein is rapidly degraded by the proteasome system. 11 Under hypoxic conditions, the ␣ subunit is stabilized because of the lack of proline hydroxylation and accumulates. Stabilized HIF-1␣ translocates into the nucleus and forms an HIF-1 complex with the almost ubiquitously expressed HIF-1␤ (identical to aryl hydrocarbon receptor nuclear translocator 1 [ARNT1]). The HIF-1 complex binds to hypoxia response elements (HREs) found in enhancers or promoters of hypoxia-inducible genes. 12 In addition to ARNT1, kidney and neuronal cells express the ARNT1 homolog ARNT2, which can form a functional HIF complex with HIF-1␣. 13 The HIF-1 binding site (HBS) in the 3Ј EPO enhancer is one of 3 sites that are important for hypoxia-induced EPO gene transcription. 14 The HBS is the most upstream element and is followed by a 4-base-pair (bp) CACA repeat and finally a direct repeat of ...

Hypoxia-inducible factor-1 (HIF-1) coordinates the cellular response to a lack of oxygen by controlling the expression of hypoxia-inducible genes that ensure an adequate energy supply. Assembly of the HIF-1 complex by its oxygen-regulated subunit HIF-1alpha and its constitutive beta subunit also known as ARNT is the key event of the cellular genetic response to hypoxia. By two-photon microscopy, we studied HIF-1 assembly in living cells and the mobility of fluorophore-labeled HIF-1 subunits by fluorescence recovery after photobleaching. We found a significantly slower nuclear migration of HIF-1alpha than of HIF-1beta, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between alpha and beta subunits of the transcriptionally active HIF-1 complex bound to DNA. Both N termini of the fluorophore-labeled HIF-1 subunits were localized as close as 6.2 nm, but even the N and C terminus of the HIF-1 complex were not further apart than 7.4 nm. Our data suggest a more compact 3-dimensional organization of the HIF complex than described so far by 2-dimensional models.

SummaryTwo-photon (TP) excitation (820 -1150 nm) and emission (280 -700 nm) spectra for the fluorescent proteins (FPs) ECFP 3 , EGFP 3 and EYFP 3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their onephoton (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue-shifted and emission red-shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out-of-maximum excitation, common practice in TP excitation microscopy, can be used for routine application.

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