Chinese researchers have developed a new highly sensitive calcium-signaled fluorescent protein probe

Recently, the team of Professor Zhang Xiaohui of the State Key Laboratory of Cognitive Neuroscience and Learning of Beijing Normal University, the team of Professor Wang Youjun of the School of Life Sciences of Beijing Normal University, and the team of Professor Tang Aihui of the University of Science and Technology of China developed and constructed a new fluorescent protein probe “Nemo” (NEMO) for detecting calcium signal, which has stronger and more accurate quantitative measurement performance. Recently, the results were published online in the journal Nature Method.

Many activities of living organisms are inseparable from calcium ion (Ca2+) signaling molecules. The spatiotemporal variation of calcium ion concentration in cells is called calcium signaling, which controls or regulates various cellular life activities. The development of sensitive and accurate calcium signal detection probe tools is essential to explore the signaling mechanisms and laws related to life activities.

Calcium probes widely used in related fields mainly include organic small molecule probes and genetically encoded (fluorescent) protein probes (GECIs). At present, the most widely used single fluorescence GECI tool is the GCaMPs series, which is assembled and constructed by two modules: calcium sensing and fluorescence reaction. Among them, the calcium-sensing module contains calcium-binding protein (such as calmodulin CaM) and its target peptide (such as M13/RS20), and the module that produces fluorescence changes is cyclically rearranged green fluorescent protein cpGFP.

The scientists found that the performance of GECIs can be improved by changing the way the three components of CaM, M13 and GFP are connected, connecting short peptides and key amino acids in the interaction interface. Therefore, after several iterations on the GCaMP1 version originally built in 2001, the newly developed GCaMP8 series by 2023 has significantly improved sensitivity and reaction speed, but their response amplitude, that is, the resolution and linear dynamic range of calcium signal size, always need to be improved.

In this regard, the collaborative team adopted a new highly sensitive calcium ion probe built with a new strategy. Starting from the fluorescence response to the change of calcium ion concentration of GECI, the collaborative team used a new fluorescent protein mNeoGreen (mNG) with higher brightness to replace the widely used cpGFP, combined with a variety of design and optimization strategy combinations, constructed a GECI library containing dozens of candidate composite molecules, and finally obtained a set of novel GECI probes named NEMO after systematic calcium ion imaging screening and in vitro identification.

Compared with the existing GCaMP series probes, the sensitivity and calcium response amplitude of NEMO probes have been significantly improved, and the response amplitude of GECI probes to intracellular calcium signals has been more than 100 times for the first time in the field. At the same time, it has better light quenching ability and pH stability, and can achieve absolute quantitative detection of calcium ion levels.


The assembly method of single fluorescence genetically encoded calcium probe (GECI) GCaMPs (A) and NEMO (B) is provided by the research group

The collaborative team further found in tests on non-excitatory cell lines, isolated and cultured rat neurons, mouse brain neurons in vivo two-photon laser imaging, and deep brain region fiber recording, that compared with the latest or most widely used GCaMP8s or GCaMP6s, NEMO series responds to intracellular calcium signals at the same speed, but is more sensitive and has a higher signal-to-noise ratio, and the response amplitude is increased by about 10 times. (Source: China Science News, Zhang Xiaohui, Chen Bin)

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