"This paper has experienced three reviewers, and the evaluations are all positive. Because the probe we developed is one of the few people in the world whose sensitivity in cells is more than ten times, the first reviewer believes that this work has achieved a huge leap.
The second reviewer believes that the probe has good performance in all aspects and can provide powerful tools for biologists who study cyclic adenosine phosphate (cAMP, cyclic AMP) related signaling pathway , and start the research on cAMP "When talking about the reviewer's evaluation, Chu Jun, a researcher at the Institute of Biomedical Optics and Molecular Imaging Research Center of the Institute of Medical Engineering, Shenzhen Advanced Technology, Chinese Academy of Sciences, said confidently.
Figure | Chu Jun (Source: Data Picture)
With cutting-edge technologies such as protein rational design and directional evolution, Chu Jun and his team developed an cyclized rearrangement probe based on fluorescent protein. This gene-encoded probe, known as G-Flamp1, can have a fluorescence change of up to 12 times in living cells. It has the advantages of high sensitivity, high brightness, appropriate affinity and rapid response dynamics, and can monitor endogenous cAMP signal changes in the brain with high sensitivity.
On September 12, 2022, the relevant paper was published on Nature CommunicationsNature Communications[1].
Assistant researcher at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, and Wu Chunling, a postdoctoral fellow at Peking University, serve as the first author of the paper, and Chu Jun serves as the corresponding author of .
Figure | Related papers (Source: Nature Communications)
Use gene encoding probe to track the dynamic changes of space-time in cAMP
cAMP is a very critical class of molecules in cells and can play a role in regulating various functional activities in cells. It is also called " Second Messenger ".
"If we regard many molecules in the cell as a network, the second messenger is the node that transmits the network signal to other terminals, and cAMP is one of the nodes." Chu Jun explained.
cAMP is closely related to the physiological processes in the human body such as immunity, metabolism, drug addiction, etc. Therefore, in order to study these processes in depth, it is necessary to monitor changes in cAMP signals in real time in vivo.
To explore the changes in molecules, researchers mainly conduct research from the two dimensions of time and space. However, how to track changes in cAMP molecules in vivo is a key issue that needs to be solved.
Just develop optical molecular probe , and combined with advanced optical microscopy imaging technology, real-time dynamic non-invasive monitoring of molecular changes.
At present, about 50 similar cAMP probes have been developed around the world, but due to their respective limitations, the problem cannot be solved well. For example, the probe has a low sensitivity and a signal change of only 1.5 times, making it difficult to track molecular changes in living bodies; or, the probe is darker, making it difficult to help researchers understand the relationship between physiological or pathological processes and cAMP. It is in this context that the gene-encoding probe G-Flamp1 was developed. It does not have the disadvantages of high toxicity and non-heritability of probes based on nanomaterials, dyes, and other types. It can well locate any specific location in the cell and perfectly reflect the change process of cAMP under a certain behavior in the living body.
Figure | G-Flamp1 schematic and in vitro characterization (Source: Nature Communications)
Using G-Flamp1, Chu Jun's team tested the process of cAMP changes in fruit fly and mice under different behaviors.
First, under the condition of stimulation of odor, in order to explore whether the cAMP molecules in Kenyon cells in the mushroom body of the fruit fly brain can play a role, the researchers used two-photon imaging technology to find that the dynamic changes of cAMP in different sub-regions of the mushroom body of the fruit fly brain are different.
Figure | Through two-photon imaging technology, G-Flamp1 reveals the dynamics of cAMP induced by physiological stimulation in fruit flies (Source: Nature Communications)
Next, the team wanted to understand the utility of G-Flamp1 monitoring physiologically related cAMP changes in live organs, and co-expressing G-Flamp1 and red calcium ion probe jRGECO1a in mouse motor cortical neurons. With the help of two-photon imaging, G-Flamp1 demonstrates changes in cAMP signaling in mouse motor cortex neurons after being induced by movement.
Figure | (Source: Nature Communications)
Subsequently, in order to test the ability of G-Flamp1 to monitor cAMP changes in the base area of the brain, Chu Jun's team used fiber photometry to measure the cAMP levels in the nucleus accumben in the mouse brain. It first injects adeno-associated virus expressing G-Flamp1 into NAc and measures fluorescence signals using fiber optic photometry while the mice were trained to perform a conditioned reflex task.
results show that the G-Flamp1 signal in the water reward experiment showed characteristic dynamics during the learning process. Specifically, throughout the training, the amplitude of water-induced responses decreased, while the response to reward prediction sounds gradually increased. This dynamic change simulates the dopamine signal during the classical conditioning period, indicating that the increase in cAMP in NAc is driven primarily by dopamine release.
(Source: Nature Communications)
Solve basic Biomedical problems, actively promote the transformation of related results
This result has the following two applications.
From the perspective of basic research, scientists can use G-Flamp1 to solve many basic biomedical problems at present, such as exploring the changes of cAMP in drug addiction or learning and memory.
From the perspective of results transformation, using G-Flamp1, scientists can quickly and high-throughput screen drugs related to G protein-coupled receptors (GPCRs, G Protein-Coupled Receptors).
Among them, it is worth mentioning that GPCRs are currently the largest drug target. Among the drugs approved by U.S. Food and Drug Administration , more than one-third of the drugs target GPCR, a family protein. In addition, since cAMP is a downstream molecule of GPCR, the concentration of cAMP downstream changes when a drug activates or inhibits GPCR. In summary, this may become a very wide application.
On March 15, 2015, Chu Jun returned to Shenzhen from the United States. At that time, he looked forward to carrying out world-class work in the country and publishing high-quality papers. At the beginning, fluorescent proteins were still the main research direction. Later, after he found it difficult to achieve a major breakthrough, he focused on the cyclization rearrangement probe based on fluorescent proteins and determined the cAMP molecular probe as the main attack direction.
Chu Jun uses the most cutting-edge technology of cyclic rearrangement of fluorescent proteins to build a new probe with high performance. He said: "This principle is very simple, but it will be difficult to do. Because making this probe is a bit similar to screening drugs, it requires a lot of high-throughput screening and will also face more uncertainties."
After one and a half years of hard work, he and the research team developed a cAMP probe with a fluorescence change of about 20%, but this is still a big gap with the best probe in the world. After a long optimization process, it finally obtained the cAMP probe G-Flamp1 with a fluorescence change of 1200%.
Review of the entire study, Chu Jun was very unforgettable and deeply touched.He said that the reason why this paper was published is inseparable from the selfless help provided by many professors and teachers in the academy, the persistence of the research team members and the strong support of their families.
Regarding the follow-up plan for this research, Chu Jun and the research team will start from the following three aspects.
first, optimize or improve probe performance to meet the needs of different biomedical scenarios.
Second, develop long-wavelength cAMP probes, and combine other short-wavelength probes to monitor the changes of two different molecules under a specific behavior.
Third, give full play to the tool attributes of the probe and use probes to solve more unsolved biological problems.
At the same time, the research team also hopes to cooperate with domestic companies committed to developing original innovative drugs, use their probes to screen drugs, and carry out independent research and development of drugs in China.
Reference:
1.L.Wang, C.Wu, W.Peng, Z.Zhou.et al. A high-performance generally encoded fluorescent indicator for in vivo cAMP imaging. Nat. Commun.(2022). https://www.nature.com/articles/s41467-022-32994-7
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