[173]. However, with recent progress in CRISPR/Cas9 genome engineering techniques, it has become possible to edit more precisely the genomes of diverse cell
types and organisms and routinely insert fluorescent protein tags into endogenous
genomic loci in some organisms. Hara et al. [173] reported expression of an
endoplasmic reticulum (ER) Ca
2+ biosensor in transgenic mouse pancreas. The
expression of a yellow cameleon3.3er (YC3.3er) transgene with mouse insulin
1 promoter was limited to pancreatic beta cells within islets of Langerhans and
absent in the exocrine pancreas and other tissues [173]. The study established that by
controlling transgene transcription with a cell-specific promoter, transgenic expression of FRET-based Ca
2+ sensors can be incorporated in mammals to facilitate realtime optical imaging of signal transduction events in living tissues. Yang et al.
developed a red fluorescent protein-based cAMP indicator named “Pink Flamindo”
which could effectively trace the spatiotemporal dynamics of intracellular cAMP
generated by photoactivated adenylate cyclase in response to light and in dual-color
imaging studies using a green Ca
2+ indicator.
The elevation of cAMP levels in vivo in cerebral cortical astrocytes was successfully monitored by two-photon imaging. The cAMP-PKA signaling pathway plays a
key role in the excitability of neurons. In order to study its role, protein kinase A
(PKA) expression in neurons has been mapped in whole-brain context in live
animals. In a study by Gervasi et al., in vivo multiphoton imaging was used to
measure the dynamics of PKA responses to dopamine and octopamine in the MB
neurons of living flies. The PKA activity was monitored on real-time basis by using
the genetically encoded FRET probe AKAR2 [133]. The AKAR2 probe is based on
yeast-derived phosphothreonine-binding domain (FHA1) and an optimized PKA
substrate domain. The substrate domain upon phosphorylation by PKA interacts
with the binding pocket of the FHA1 domain, increasing the FRET between two
GFP variants CFP and citrine [174]. A recent study by Sun et al. developed a
genetically encoded GPCR-activation-based-DA (GRAB DA) sensors to measure
DA changes reliably and specifically with high spatiotemporal precision in
Drosophila (Fig. 5ii) [175]. The GRAB DA sensor could resolve a single-electricalstimulus-evoked DA release in mouse brain slices and detect endogenous DA release
in living flies with subcellular resolution, sub-second kinetics, and excellent molecular specificity. Similarly, Portugues et al. studied the dynamics and spatial distribution of neuronal activities during optokinetic response in zebra fish larvae. The
whole-brain activity dynamics was assigned by specific hue based on the timing of
its response relative to the stimulus, which enabled categorization of brain regions
into distinct response-based functional modules (Fig. 5iii) [176]. Another recent
technique is called “Brainbow” that used the combinatorial expression of a series of
four different color fluorescent proteins resulting in at least 90 different colors of
cells in the brain such that the lineage of each neuron can be traced [177]. For
translational purposes, mammalian systems such as mouse are appealing targets for
similar in vivo studies. However, there are a number of challenges for in vivo kinase
studies, particularly in living mice. A RhoA-FRET biosensor-based transgenic
mouse was recently developed by Nobis et al., for real-time longitudinal, intravital
imaging of RhoA deregulation in invasive mammary and pancreatic cancers [178].
172
U. K. Sukumar et al.
types and organisms and routinely insert fluorescent protein tags into endogenous
genomic loci in some organisms. Hara et al. [173] reported expression of an
endoplasmic reticulum (ER) Ca
2+ biosensor in transgenic mouse pancreas. The
expression of a yellow cameleon3.3er (YC3.3er) transgene with mouse insulin
1 promoter was limited to pancreatic beta cells within islets of Langerhans and
absent in the exocrine pancreas and other tissues [173]. The study established that by
controlling transgene transcription with a cell-specific promoter, transgenic expression of FRET-based Ca
2+ sensors can be incorporated in mammals to facilitate realtime optical imaging of signal transduction events in living tissues. Yang et al.
developed a red fluorescent protein-based cAMP indicator named “Pink Flamindo”
which could effectively trace the spatiotemporal dynamics of intracellular cAMP
generated by photoactivated adenylate cyclase in response to light and in dual-color
imaging studies using a green Ca
2+ indicator.
The elevation of cAMP levels in vivo in cerebral cortical astrocytes was successfully monitored by two-photon imaging. The cAMP-PKA signaling pathway plays a
key role in the excitability of neurons. In order to study its role, protein kinase A
(PKA) expression in neurons has been mapped in whole-brain context in live
animals. In a study by Gervasi et al., in vivo multiphoton imaging was used to
measure the dynamics of PKA responses to dopamine and octopamine in the MB
neurons of living flies. The PKA activity was monitored on real-time basis by using
the genetically encoded FRET probe AKAR2 [133]. The AKAR2 probe is based on
yeast-derived phosphothreonine-binding domain (FHA1) and an optimized PKA
substrate domain. The substrate domain upon phosphorylation by PKA interacts
with the binding pocket of the FHA1 domain, increasing the FRET between two
GFP variants CFP and citrine [174]. A recent study by Sun et al. developed a
genetically encoded GPCR-activation-based-DA (GRAB DA) sensors to measure
DA changes reliably and specifically with high spatiotemporal precision in
Drosophila (Fig. 5ii) [175]. The GRAB DA sensor could resolve a single-electricalstimulus-evoked DA release in mouse brain slices and detect endogenous DA release
in living flies with subcellular resolution, sub-second kinetics, and excellent molecular specificity. Similarly, Portugues et al. studied the dynamics and spatial distribution of neuronal activities during optokinetic response in zebra fish larvae. The
whole-brain activity dynamics was assigned by specific hue based on the timing of
its response relative to the stimulus, which enabled categorization of brain regions
into distinct response-based functional modules (Fig. 5iii) [176]. Another recent
technique is called “Brainbow” that used the combinatorial expression of a series of
four different color fluorescent proteins resulting in at least 90 different colors of
cells in the brain such that the lineage of each neuron can be traced [177]. For
translational purposes, mammalian systems such as mouse are appealing targets for
similar in vivo studies. However, there are a number of challenges for in vivo kinase
studies, particularly in living mice. A RhoA-FRET biosensor-based transgenic
mouse was recently developed by Nobis et al., for real-time longitudinal, intravital
imaging of RhoA deregulation in invasive mammary and pancreatic cancers [178].
172
U. K. Sukumar et al.