Nat Commun-Chu Jun's research group has developed a new high-performance genetically encoded cAMP fluorescent probe

Source—RWD Institute for Life Sciences

Responsible editor—Wang Sizhen, Fang Yiyi

Editor—Summer Leaf

Cyclic adenosine monophosphate (cAMP) is a key intracellular second messenger that integrates signals from a variety of G protein-coupled receptors (GPCRs) in applications such as learning and memory, drug addiction, motor control, immunity, tumors, Metabolism and other processes play an important role
.
High spatiotemporal resolution fluorescence imaging of cAMP molecular concentration changes at the live cell and in vivo levels is an important basis for
elucidating the cAMP signaling pathway and its biological functions.
Therefore, the development of highly sensitive cAMP fluorescent probes has become key
to studying complex biological processes.

Compared with non-genetically coded probes (dyes and materials), gene-coding probes have the advantages of low toxicity, low background, heritability, and can locate specific cell substructures or specific cells, which has unparalleled advantages
in basic research in life sciences.
However, existing cAMP fluorescent probes with more than 50 genes encoded either have low sensitivity (up to a factor of 1.
5 fluorescence variations) or have low fluorescence brightness, making it difficult to monitor weak endogenous cAMP in vivo The changes have greatly limited the study of the molecular regulatory mechanism and function of
cAMP in physiological and pathological conditions.

Recently, the Chu Jun research group of the Institute of Medical Engineering of the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences published a report entitled in the internationally renowned journal Nature Communications
The latest work of "A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging" demonstrates a high-performance genetically encoded one cAMP fluorescent probe
.
In this work, the researchers developed a "circularly permuted" based on rational design and protein-directed evolution based on crystal structure-mediated rational design and protein-directed evolution fluorescent protein) high-performance genetically encoded cAMP green fluorescent probe (named G-Flamp1).

In live cells, the probe achieved a 12-fold maximum fluorescence change under both single- and two-photon excitation, and the binding and dissociation times reached 0.
2 and 0.
087 sec
.
Using two-photon microscopy and fiber optic recorders, the probe monitors in real time the spatiotemporal dynamics of cAMP signals of specific neurons during specific animal behavior within model organisms such as fruit flies and mice.

To develop a highly sensitive probe suitable for in vivo detection, the researchers inserted cyclized rearranged green fluorescent protein (cpGFP) into the cAMP-binding domain of the bacterial MlotiK1 channel (mlCNBD
).
After insertion site screening, ligation peptide optimization, fluorescent protein and sensing module optimization, a high-performance gene-coding cAMP green fluorescent probe G-Flamp1 with high brightness, high sensitivity, suitable affinity and fast response speed were obtained.

Very interestingly, the crystal structure shows that the linker peptides of the G-Flamp1 probe have a unique structure: one of the linkage peptides is a very rigid β-strand structure.
This does not exist in other cyclized rearranged fluorescent protein probes with known crystal structures, providing new ideas and methods
for the development of other high-performance probes.

In vitro experiments, G-Flamp1 with and without cAMP has a different chromophore environment
.
G-Flamp1 has a maximum dynamic range of 450 nm (single photon) or 900-920 nm (double photon), which is about ΔF/F₀ 13
。 G-Flamp1 has a moderate affinity for cAMP and its dissociation constant _Kd value is 2.
17 μM
.
G-Flamp1 detects cAMP dynamics at sub-second time resolution
.
In cultured cells, the probe is evenly distributed in the cytoplasm and nucleus, with background fluorescence brightness between
comparable probes cAMPr and Flamindo2.
The G-Flamp1 probe has a dynamic range of 12 times in living cells, making it one of the few fluorescent protein probes with a dynamic range of
more than 10 times.
At the same time, the probe has good specificity and reversibility (Figure 1).

Figure 1 Characterization of G-Flamp1 probes in vitro and in cultured cells

(Source: Liang Wang, et al.
, Nat Commun, 2022).

The researchers then applied the G-Flamp1 probe to the model organism of
Drosophila.
The cAMP signaling pathway in Kenyon cells of the mushroom body of the fruit fly brain plays a key role
in odor-related memory.
The authors first obtained transgenic flies expressing G-Flamp1 probes in Kenyon cells, and then used two-photons for imaging, and when the flies were stimulated by odor or electric shock, different subregions of the mushroom body showed different spatiotemporal changes in cAMP signal (Figure 2), suggesting that different subregions may play relatively independent roles
in associative learning.

Fig.
2 Changes in cAMP signaling in Kenyon cells of Drosophila under different stimuli

(Source: Liang Wang, et al.
, Nat Commun, 2022).

To verify the utility of the G-Flamp1 probe for detecting cAMP dynamics in live animals, the researchers used adeno-associated virus to co-express green G-Flamp1 probe and red jRGECO1a in the mouse motor cortex Calcium probe.

In vivo two-photon imaging revealed cell-specific cAMP signaling in running with no significant correlation with calcium signaling (Figure 3).

This reflects the heterogeneity of responses of M1 neurons in the cerebral cortex during mouse exercise.

Fig.
3 Changes in cAMP signaling in mouse cortical neurons during exercise

(Source: Liang Wang, et al.
, Nat Commun, 2022).

Finally, the researchers expressed the G-Flamp1 probe in a deep nucleus accumbens (NAc) brain region deep in the mouse brain, and used optical fiber to record changes in cAMP signals in that brain region during the auditory Pavlov conditioned task.

The results showed that with the proficiency of training, the cAMP signal amplitude decreased when the mice were rewarded, and the cAMP signal amplitude increased when the corresponding audio signal was heard (Figure 4).
This property is similar to dopamine signaling, suggesting that dopamine release causes cAMP signaling
.
In summary, the high signal-to-noise ratio and high temporal resolution of the G-Flamp1 probe can detect the dynamic changes of endogenous cAMP signals in live mice with high sensitivity.

Fig.
4 Changes in cAMP signal in mouse NAc brain region during Pavlovian conditioned task

(Source: Liang Wang, et al.
, Nat Commun, 2022).