Genetically Encoded ROS Sensors

Fluorescence proteins, in particular the green fluorescent protein (GFP) and its variants are widely used tools to study a large variety of cellular processes (Tsien, 2008). They are used as novel biosensors for the local chemical environment in cells and cell organelles. Highly resolved fluorescence nanoscopy (Klar et al., 2000; Westphal et al., 2005) was boosted by the development of photo-switchable derivatives of GFP (Andresen et al., 2005, 2007; Hofmann et al., 2005; Dedecker et al., 2007, Eggeling et al., 2007; Brakemann et al., 2011).

Table 4. Spin traps suitable for imaging ROS.

Compound

Specificity

Further information/localizability

DMPO (Davies, 2002)

Spin trapping of Ю2, superoxide and hydroxyl radicals,

Transient EPR spectra specific for trapped radicals but spontaneous decay of DMPO- superoxide adduct with 45 sec. half lifetime

alpha-phenyl N-tertiary-butyl nitrone (PBN) (Davies, 2002)

EPR spectra rather unspecific for trapped radicals.

3,5-Dibromo-4-nitro- sobenzenesulfonic acid (DBNBS) (Davies, 2002)

Used for H2O2 sensing, specific EPR spectra

5-Diisopropoxyphosphoryl-5- methyl-1-pyrroline-N-oxide (DIPPMPO) (Zoia and Argyropoulosm, 2010)

Used in mitochondria, strongly applied for detecting superoxide

TEMPO-9-AC (Cohn et al., 2008)

Fluorogenic spin trap specific for hydroxyl radicals and superoxide

BODIPY® 665/676 (Pap et al., 1999)

Sensitive fluorescent reporter for lipid peroxidation

DanePy

(Hideg et al., 1998, 2000)

Specific to 1O2

Fluorescent spin trap - Fluorescence is quenched in presence of 'O2

GFP can directly be targeted or fused to specific target proteins for precise sub-cellular localization and analysis in vivo.

The application of genetically encoded fluorescent proteins (see Figure 65) in fluorescence microscopy provided new insights into the complicated and fascinating world of life on the microscopic scale. Superresolution microscopy was developed by Stephan Hell who was awarded the Nobel Prize in chemistry in 2014. As fluorescent proteins are specific markers that can be fused as tags to selected proteins, it became possible to follow the dynamics of a certain protein or enzyme in the living cell with minimal disturbance of the cell and its metabolism. Novel microscopic techniques like laser switching contrast microscopy (LSCM) allows for the imaging of the dynamics of optically switchable proteins in single cell compartments. We recently present an application for the monitoring of diffusive properties of single molecules of the photo- switchable fluorescent protein Dreiklang (Schmitt et al., 2016; Junghans et al., 2016), see Figure 66.

CHO cells expressing red fluorescent protein (RFP) in the cytosol and green fluorescent protein (GFP) localized in cell membranes

Figure 65. CHO cells expressing red fluorescent protein (RFP) in the cytosol and green fluorescent protein (GFP) localized in cell membranes.

High resolution image of a CHO cell expressing Dreiklang

Figure 66. High resolution image of a CHO cell expressing Dreiklang (Brakemann et al., 2011) after OFF photo-switching in the cell nucleus. The image shows a sum of 20 images captured after off-switching of Dreiklang by a 405 nm laser due to diffusion of the single molecules into the laser focus (Schmitt et al., 2016). Image reproduced with permission.

Genetically encoded ROS sensors are therefore one approach to overcome problems regarding the specificity of localization when monitoring ROS. To exploit the potential of GFP for sensing the local chemical matrix, extensive studies have been undertaken to develop GFP-based in vivo sensors by targeted mutations and generalized approaches like directed evolution. The optical properties of these biosensors depend on selective binding of protons, oxygen atoms, water molecules and/or cofactors or are induced by electron transfer (Heim et al., 1995; Yang et al., 1996; Brakemann et al., 2011; Kremers et al., 2011).

Fluorescent proteins which are sensitive to the microenvironment like pH (Miesenbock et al., 1998; Campbell and Choy, 2001; Hanson et al., 2002; Bizzarri et al., 2009; Schmitt et al., 2014b), ROS (Ostergaard et al., 2001; Schwarzlander et al., 2009; Belousov et al., 2006) or NADH (Hung et al., 2011; Tejwani et al., 2017; Wilkening et al., 2017) are used as standard tools for the selective imaging of physiological parameters and their dynamics. Often the GFP-based ROS sensor variants contain pairs of redox-active cysteines forming a disulfide bridge as redox switch. These proteins can be selectively expressed as fluorescence markers, fused to specific target proteins or to organelle-specific targeting sequences, thus enabling a specific and localized monitoring (and manipulation) of ROS at a molecular level (for a review, see Swanson et al., 2011).

Progress in engineering of ROS-sensitive fluorescence proteins led to the development of several derivatives of GFP containing the mentioned redox-active cysteines forming a disulfide bridge as redox switch (Jimenez-Banzo et al., 2008). One example is roGFP (Hanson et al., 2004; Schwarzlander et al., 2008). Derivatives of the yellow fluorescent protein (YFP) have also been described, which are modified by introduction of redox-active cysteines in constructs termed rxYFP149202 (Ostergaard et al.,

2001) or HyPer (Belousov et al., 2006). Chromophore transformations in red-fluorescent proteins offer tools for designing suitable red-shifted probes, which are advantageous for imaging studies due to the strong absorption in the green spectral range, in which chlorophylls exhibit only very low absorption. Excitation with longer wavelengths also leads to reduced autofluorescence (for a review, see Subach and Verkhusha, 2012).

The disulfide bridge in the oxidized rxYFP leads to a distortion of the typical beta-barrel structure of GFP derivatives, thus changing the fluorescence properties of rxYFP (Ostergaard, 2001). The mitochondrially- targeted redox sensitive GFP termed roGFP-mito does not specifically react in response to a certain species of ROS, but it is used to selectively label mitochondria in plants (Schwarzlander et al., 2009).

In an alternative approach, the H2O2-sensitive probe HyPer was constructed by fusing the regulatory domain of the H2O2-sensitive transcription factor OxyR from E.coli to a cyclically permuted YFP (Belousov et al., 2006). For applications of the genetically encoded ROS sensors in studies on ROS effects, see (Maulucci et al., 2008; Meyer and Dick, 2010; Mullineaux and Lawson, 2009).

Table 5 gives an overview on genetically encoded fluorescence proteins and their basic properties of selectivity and applicability in plants.

Table 5. Genetically encoded fluorescence proteins applicable for ROS monitoring.

Compound/reference

Specificity

Further information/localizability

rxYFP (Ostergaard et al., 2001)

Unspecific

roGFP (Schwarzlander et al., 2008, 2009)

Unspecific, applied to label plant mitochondria

HyPer (Belousov et al., 2006)

H2O2 sensitive by fusing the regulatory domain of the H2O2-sensitive transcription factor OxyR to YFP, not yet expressed in plant cells

GFP redox sensor (Niethammer et al., 2009)

Specific to H2O2, successfully applied in Zebrafish larvae to detect H2O2 patterns after wounding

The application of fluorescence markers for ROS sensing is generally complicated by photobleaching. In addition, fluorophores often act as :AgO2 sensitizers themselves. This problem is especially important for GFP derivates as ROS sensors. However, the generation of new GFP mutants that produce reduced amounts of ROS is a promising approach to overcome this problem, which again votes for the importance of developing improved genetically encoded fluorescence proteins for ROS sensing for future studies.

 
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