Flavin Mononucleotide-Based Fluorescent Proteins Function in Mammalian Cells without Oxygen Requirement

Abstract

Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions.

The photophysics of LOV-based fluorescent proteins — new tools for cell biology

“…Since in previous studies the extinction coefficient of free FMN was used for LOV-based FPs, we first measured the extinction coefficient of the LOV-FPs by comparing the absorption of the chromophore at 450 nm in the bound and unbound state (see materials and methods). This analysis revealed that the extinction coefficients of all tested LOV-based FPs only differ to a minor extent ranging from 13 ). In contrast, the fluorescence quantum yields vary considerably among the LOV-based FPs and can be almost twice as high as that of FMN (see below and Tab.…”

“…These cyan-green fluorescing proteins bind flavin mononucleotide (FMN) as the chromophore and are either derived from bacterial photoreceptors (FMN-binding fluorescent proteins; FbFP 8 ) or are derivatives of the Arabidopsis thaliana phototropin 2 LOV2 domain (iLOV, miniSOG; 9,12 ). In contrast to all members of the GFP family, this novel class of LOV-based FPs develops the corresponding fluorescence signal under both, aerobic and anaerobic conditions 8,13,14 . This unique property renders them valuable for in vivo applications where molecular oxygen is limited; such as the analysis of microbial pathogenesis, hypoxia induced inflammatory processes, tumor pathophysiology and microbial fermentation as well as for the monitoring and optimization of bioremediation and bacterial production processes (e.g.…”

LOV-based reporters for fluorescence imaging

“…This property makes the application of this technology very attractive for the study of anaerobic cellular processes. For example, studies have shown that EcFbFP could surpass yellow fluorescent protein (YFP) as a fluorescent reporter in anaerobically grown cultures of E. coli and Rhodobacter capsulatus [15 ,25,26], as well as yeast [27] and a range of mammalian cells [28]. In addition, LOVbased FPs have been used to track host cell infections by anaerobic pathogens under physiologically relevant conditions [29][30][31].…”

Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120

“…This delay could confound the interpretation of the timing of gene expression in certain experiments. Third, in work conducted on E. coli (Drepper et al, 2010), Saccharomyces cerevisiae (Tielker et al, 2009), Candida albicans, Rhodobacter capsulatus (Drepper et al, 2007), Porphyromonas gingivalis (Choi et al, 2011), Bacteroides fragilis (Lobo et al, 2011), Arabidopsis thaliana (Chapman et al, 2008) and mammalian cells (Walter et al, 2012), FbFPs have proven to be suitable fluorophores in anoxia. To date, studies utilizing FbFPs have focused on either aerobic or anaerobic conditions.…”