Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.

Abstract

Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.

0100200'05'06'07'08'09'10'11'12'13'14'15'16'17
Citations per Year

1,483 Citations

Semantic Scholar estimates that this publication has 1,483 citations based on the available data.

See our FAQ for additional information.

Cite this paper

@article{Nagai2004ExpandedDR, title={Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.}, author={Takeharu Nagai and Shuichi Yamada and Takashi Tominaga and Michinori Ichikawa and Atsushi Miyawaki}, journal={Proceedings of the National Academy of Sciences of the United States of America}, year={2004}, volume={101 29}, pages={10554-9} }