Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent.pdf
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ExpandeddynamicrangeoffluorescentindicatorsforCa2bycircularlypermutedyellowfluorescent
Expanded dynamic range of fluorescent indicators
for Ca2 by circularly permuted yellow
fluorescent proteins
Takeharu Nagai*?, Shuichi Yamada?, Takashi Tominaga§, Michinori Ichikawa§, and Atsushi Miyawaki*?
*Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, and §Laboratory for Brain-Operative Devices, Brain Science
Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; ?Structure and Function of Biomolecules, Precursory Research for Embryonic Science and
Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 351-0198, Japan; and ?Laboratory of Signal
Transduction, Institute for Virus Research, Kyoto University, Kawaharacho 53, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
Edited by Roger Y. Tsien, University of California at San Diego, La Jolla, CA, and approved May 19, 2004 (received for review January 29, 2004)
Fluorescence resonance energy transfer (FRET) technology has been
used to develop genetically encoded fluorescent indicators for vari-
ous 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 Ca2 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 Ca2-saturated form, thereby increasing the
Ca2-dependent change in the ratio of YFPCFP by nearly 600%. Both
in cultured cells and in the nervous system of transgenic mice, the new
YC enables visualization of subcellular Ca2 dynamics with better
spatial and temporal resolution than before. Our study provides an
important guide for the development and improvement of indicators
using GFP-
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