Wolf Frommer Lab Research

GENA development: Genetically encoded FRET sensors

Genetically encoded sensors transduce the interaction of a target molecule with a recognition element into a macroscopic observable, via allosteric regulation of one or more signaling elements. These sensors provide a means to quantitatively determine steady state levels of analytes in living cells with high temporal and spatial resolution. The recognition element may bind the target, bind and enzymatically convert the target, or may serve as a substrate for the target, as in the case of a specific target sequence for the construction of protease activity sensors. The most common reporter element is a sterically separated donor-acceptor FRET pair of fluorescent proteins, although single fluorescent proteins or enzymes may be suitable as well. Some molecular sensors additionally employ a conformational actuator (most commonly a peptide which binds to one conformational state of the recognition element), to magnify the allosteric effect upon and resulting output of the reporter element. We recently published overviews over the field, e.g. Lalonde et al., 2005, Looger et al., 2005 and references therein.

A family of metabolite sensors

We recently constructed genetically encoded FRET sensors for a variety of important analytes such as glucose, maltose, ribose and glutamate. The centerpiece of these sensors is a recognition element derived from the superfamily of bacterial periplasmic binding protein (PBPs), proteins that in their native background function as primary receptors for chemotaxis and as high affinity scavengers (atto- to low micro-molar) for hundreds of different small molecules. PBPs undergo a large conformational change upon ligand binding and thus seem ideally suited for sensor construction. Fusion of individual PBPs with a pair of GFP variants produced a large set of sensors, e.g. for sugars like maltose, ribose and glucose or for the neurotransmitter glutamate. These sensors have been adopted for measurement of sugar uptake and homeostasis in living animal cells, and sub-cellular analyte levels were determined with nuclear-targeted versions and for monitoring glutamate release from neurons.

Next steps

Binding specificity: One of the foremost goals is the expansion of the molecular toolbox through the conversion of additional PBP superfamily members, as well as proteins with binding specificity not seen in the PBPs. Additionally, the complementary methods of computational protein redesign (Looger et al., 2003) and directed evolution have been used to alter the binding specificity of members of these protein families, demonstrating that the repertoire may be extended beyond that found in nature.

Detection range: Due to large vacuole and the largely unknown organellar compartmentalization of metabolites in plants, there is currently a great deal of uncertainty regarding cytosolic metabolite concentrations in plant cells. Thus it will be necessary to test a family of sensors with dynamic ranges from 100 nM to 10 mM. Such a series of sensors has been constructed for a number of PBPs (de Lorimier et al., 2002).

Signal-to-noise: The sensors developed thus far provide a saturating ratio change of up to 0.3, providing a sufficient signal-to-noise ratio to facilitate in vivo measurement. Both empirical and rational techniques (Nagai and Miyawaki, 2004) (Frommer et al., unpublished data) are capable of improving sensor signal change (mainly through fluorophore dipole re-orientation), allowing the sensors to be used in novel environments.

Sensitivity to other parameters: YFP is particularly sensitive to pH and halides, a property previously exploited to develop halide sensors (Kuner and Augustine, 2000), emphasizing the importance of control sensors to identify potential artifacts (Fehr et al., 2003). The use of FRET acceptor variants such as Venus or mKO, with lower pKa and halide sensitivity, may improve sensor robustness (Karasawa et al., 2004). The use of thermo- and acid-stable recognition elements should further enhance sensor applicability.

Calibration: Molecular sensors are indicators of change; because the complex cellular environment affects sensor response, the sensor has to be calibrated in situ (Fehr et al., 2003; Fehr et al., 2004). Stable plant lines: It has thus far been difficult to generate stably transformed plant lines expressing functional sensors. A complicating factor may be the simultaneous presence of two highly homologous GFP variant sequences, which may aggravate gene silencing. Problems were observed when attempting to generate cameleon-expressing mice (Hasan et al., 2004). This issue was overcome by testing a variety of constructs and by the use of regulatable promoters. Effect of nanosensors on cellular processes: It is conceivable that nanosensors may perturb metabolism when expressed in a living cell. This may be even more dramatic when using sensors for analytes present at low levels, e.g. signaling molecules. Programmed low-level expression may provide a remedy.

Temporal resolution: FRET imaging systems commonly use filter wheels and collect data at intervals of several seconds. Due to the high sensitivity of these instruments, it is possible to reduce excitation intensity to below levels which lead to quenching, permitting the use of parallel image acquisition using image splitters combined with video-rate streaming.

Spatial resolution: For most applications, even those requiring organellar resolution, standard epifluorescence FRET systems will suffice. For some purposes, though, it may be advantageous to follow changes in optical sections using a Nipkow spinning disc confocal or multiphoton microscope.

Tissue penetration: Both epifluorescence and conventional confocal microscopy are limited with respect to sample thickness; for better penetration, multiphoton microscopy or even endoscopy may be necessary. Multiplexing: Several donor-acceptor protein pairs are available for use in FRET imaging. This should permit the simultaneous visualization of multiple analytes, either in the same or different cellular compartments. Alternatively, independent sensor elements may be constructed using two copies of a single fluorescent protein, using techniques such as fluorescence anisotropy decay (Squire et al., 2004).