81
using fluorescence titrations. ITC can also be used to accurately
measure the dissociation constant (K d ) of the sensor and the proportion of the sensor that is active.
The approach for in situ characterization of the FRET sensor
will depend on where the sensor will be used to measure the resting level of its ligand, its dynamic changes or distribution. For
instance, this could be intra- or extracellularly, in cell culture systems, or more complex tissue such as acute slices or in vivo. While
intracellular ligand measurements typically require the sensor to be
expressed by the cell of interest using an appropriate vector, extracellular sensor localization can be achieved by expression using
plasma membrane targeting sequences and anchoring motifs [4].
However, sensor expressed endogenously and targeted to the
membrane can be exposed to intracellular ligands during transit to
the membrane. Both sensor populations, surface-presented and
intracellular, will generate fluorescence that cannot be easily separated using diffraction-limited microscopy techniques, thus potentially limiting the interpretation of data. An alternative approach to
target FRET exclusively to extracellular space relies on a biotinstreptavidin anchoring strategy [6, 28]. In our previous work, a
biotin tag was incorporated into the FRET sensor using the
pDOTS10 plasmid. Extracellular immobilization of the sensor was
then achieved by linking the sensor to surface proteins, biotinylated using commercially available streptavidins and NHS-ester
activated biotinylation reagents, via streptavidin [6].
Classically, FRET sensor imaging is performed using single photon excitation and quantification of donor/acceptor fluorescence
intensity, donor lifetime, or emission spectra [4]. In thicker preparations like acute tissue slices and in vivo, two-photon excitation using
near-infrared pulsed lasers has superior performance and provides
good optical access to deeper structures [29] at the potential cost of
reduced spectral separation of FRET donor/acceptor excitation
(e.g., ECFP/EYFP) [30]. Using pulsed lasers, typically in the MHz
frequency range (e.g., Ti:sapphire lasers around 80 MHz), also
enables fluorescence lifetime imaging using time- correlated single
photon counting (TCSPC-FLIM) of FRET donor fluorescence, a
powerful tool to study binding of a ligand to a FRET sensor. Both
modes of excitation allow monitoring of sensor fluorescence at high
spatial and temporal, micrometer and millisecond, resolution.
4 Notes
1. pDOTS4 and pDOTS10 are mother plasmids for the cloning
of SBPs to generate FRET sensors [6]. The pDOTS4 backbone is based on a pRSET plasmid [4] (pRSET FLIPE-600n,
Addgene #13537, courtesy of Wolf Frommer) containing a
6× His-Tag at the N-terminus. For the construction of
Improving FRET Sensors by Ancestral Gene Resurrection
using fluorescence titrations. ITC can also be used to accurately
measure the dissociation constant (K d ) of the sensor and the proportion of the sensor that is active.
The approach for in situ characterization of the FRET sensor
will depend on where the sensor will be used to measure the resting level of its ligand, its dynamic changes or distribution. For
instance, this could be intra- or extracellularly, in cell culture systems, or more complex tissue such as acute slices or in vivo. While
intracellular ligand measurements typically require the sensor to be
expressed by the cell of interest using an appropriate vector, extracellular sensor localization can be achieved by expression using
plasma membrane targeting sequences and anchoring motifs [4].
However, sensor expressed endogenously and targeted to the
membrane can be exposed to intracellular ligands during transit to
the membrane. Both sensor populations, surface-presented and
intracellular, will generate fluorescence that cannot be easily separated using diffraction-limited microscopy techniques, thus potentially limiting the interpretation of data. An alternative approach to
target FRET exclusively to extracellular space relies on a biotinstreptavidin anchoring strategy [6, 28]. In our previous work, a
biotin tag was incorporated into the FRET sensor using the
pDOTS10 plasmid. Extracellular immobilization of the sensor was
then achieved by linking the sensor to surface proteins, biotinylated using commercially available streptavidins and NHS-ester
activated biotinylation reagents, via streptavidin [6].
Classically, FRET sensor imaging is performed using single photon excitation and quantification of donor/acceptor fluorescence
intensity, donor lifetime, or emission spectra [4]. In thicker preparations like acute tissue slices and in vivo, two-photon excitation using
near-infrared pulsed lasers has superior performance and provides
good optical access to deeper structures [29] at the potential cost of
reduced spectral separation of FRET donor/acceptor excitation
(e.g., ECFP/EYFP) [30]. Using pulsed lasers, typically in the MHz
frequency range (e.g., Ti:sapphire lasers around 80 MHz), also
enables fluorescence lifetime imaging using time- correlated single
photon counting (TCSPC-FLIM) of FRET donor fluorescence, a
powerful tool to study binding of a ligand to a FRET sensor. Both
modes of excitation allow monitoring of sensor fluorescence at high
spatial and temporal, micrometer and millisecond, resolution.
4 Notes
1. pDOTS4 and pDOTS10 are mother plasmids for the cloning
of SBPs to generate FRET sensors [6]. The pDOTS4 backbone is based on a pRSET plasmid [4] (pRSET FLIPE-600n,
Addgene #13537, courtesy of Wolf Frommer) containing a
6× His-Tag at the N-terminus. For the construction of
Improving FRET Sensors by Ancestral Gene Resurrection