Genetically Encoded Far-Red Fluorescent Zn2+ and Ca2+ Indicators
Wu, Tianchen, Biophysics - School of Medicine, University of Virginia
Ai, Huiwang, MD-MPHY Mole Phys & Biophysics, University of Virginia
The color palette of genetically encoded fluorescent protein indicators has expanded rapidly in recent years. Genetically encoded indicators 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.
We first explored the tolerance of two far-red fluorescent proteins (FPs), mMaroon1 and mCarmine, towards circular permutation. Five circularly permuted far-red FPs (cpFrFPs) with excitation and emission maxima longer than 600 nm were identified. The ones that displayed appreciable brightness and efficient chromophore maturation became the starting points for engineering far-red fluorescent indicators.
Secreted Zn2+ plays important pathophysiological roles. In the nervous system, labile Zn2+ has emerged as a key neuromodulator. We engineered the first far-red fluorescent indicator for secreted Zn2+ (FRISZ) and utilized the new indicator to image physiological Zn2+ release in the brain in awake mice. In parallel, a far-red fluorescent indicator for Ca2+ (FRIC), a ubiquitous second messenger, was also developed.
In summary, the study has successfully expanded the color palette of genetically encoded Zn2+ and Ca2+ indicators to the far-red spectral range, serving as first, critical steps to create high-performance genetically encoded fluorescence indicators for mammalian tissue imaging.
PHD (Doctor of Philosophy)
Genetically encoded fluorescent protein indicators , Zinc and calcium sensors, Circular permutation
NIHUniversity of Virginia