23:T452,Synaptic zinc ion (Zn²⁺) has emerged as a key neuromodulator in the brain. However, the lack of research tools for directly tracking synaptic Zn²⁺ in the brain of awake animals hinders our rigorous understanding of the physiological and pathological roles of synaptic Zn²⁺. In this study, we developed a genetically encoded far-red fluorescent indicator for monitoring synaptic Zn²⁺ dynamics in the nervous system. Our engineered far-red fluorescent indicator for synaptic Zn²⁺ (FRISZ) displayed a substantial Zn²⁺-specific turn-on response and low-micromolar affinity. We genetically anchored FRISZ to the mammalian extracellular membrane via a transmembrane (TM) ⍺ helix and characterized the resultant FRISZ-TM construct at the mammalian cell surface. We used FRISZ-TM to image synaptic Zn²⁺ in the auditory cortex in acute brain slices and awake mice in response to electric and sound stimuli, respectively. Thus, this study establishes a technology for studying the roles of synaptic Zn²⁺ in the nervous system.24:T462,The color palette of genetically encoded fluorescent protein indicators (GEFPIs) has expanded rapidly in recent years. GEFPIs with excitation and emission within the "optical window" above 600 nm are expected to be superior in many aspects, such as enhanced tissue penetration, reduced autofluorescence and scattering, and lower phototoxicity. Circular permutation of fluorescent proteins (FPs) is often the first step in the process of developing single-FP-based GEFPIs. This study explored the tolerance of two far-red FPs, mMaroon1 and mCarmine, towards circular permutation. Several initial constructs were built according to previously reported circularly permuted topologies for other FP analogs. Mutagenesis was then performed on these constructs and screened for fluorescent variants. As a result, five circularly permuted far-red FPs (cpFrFPs) with excitation and emission maxima longer than 600 nm were identified. Some displayed appreciable brightness and efficient chromophore maturation. These cpFrFPs variants could be intriguing starting points to further engineer far-red GEFPIs for in vivo tissue imaging.

#	Concept	H-Index	Publications	Citations
1	Medicine	3	3	95
2	Natural Sciences	2	2	55
3	Cell Biology	2	2	80
4	Engineering	1	1	40
5	Biochemistry	1	1	40

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