As electrical signals travel along chains of neurons, each cell undergoes
a dramatic shift in its internal calcium ion (Ca2+) concentration because
specialized channels allow ions to flood into the cytoplasm. This shift
provides a valuable indicator for tracking neural activity in real time, so
scientists have developed several fluorescent protein-based Ca2+
indicators that are genetically encoded and can therefore be expressed
directly in cells of interest.
Generally these indicators do not perform as well in live animals as in vitro.
Takeharu Nagai of Hokkaido University and Katsuhiko Mikoshiba of the
RIKEN Institute, suspected that indicators with higher affinity for Ca2+ might
work better. However, their approach was risky. “It was generally believed
that extremely high-affinity Ca2+ indicators would result in low cell viability
due to disturbed Ca2+ homeostasis, and show no signal changes due to
saturation by resting Ca2+,” say Nagai and Mikoshiba. “From this point of
view, our attempt was totally against common sense.”
Nevertheless, the indicators, dubbed YC-Nano, developed by Nagai and his
colleagues proved to be a remarkable success1. The indicators were derived
from yellow cameleon (YC), a genetically encoded indicator consisting of two
fluorescent proteins, a ‘donor’ and an ‘acceptor’, connected by a Ca2+-binding
domain. In the presence of Ca2+, the structure of YC rearranges such that the
two come close together in a manner that allows energy from the excited donor
to induce a readily detectable signal from the acceptor; in the absence
of Ca2+, only a minimal signal is produced.
The researchers introduced various modifications that lengthened the Ca2+
-binding segment between the two fluorescent domains, introducing additional
flexibility that considerably improved indicator sensitivity. The best-performing
versions exhibited five-fold greater Ca2+ affinity than YC and a high dynamic
range. “We were quite surprised that we managed to systematically produce a
series of indicator variants with different affinity by a very simple protein
engineering trick,” says Nagai.
YC-Nano accurately tracked the complex patterns of Ca2+ activation seen in the
aggregating process of social amoeba Dictyostelium, revealing propagating
waves throughout the aggregates in a rotating spiral. These indicators
also performed well in monitoring neuronal activity in the brains of mice, and
Mikoshiba foresees numerous experimental applications in the near future.
“Since YC-Nano can be stably expressed in specific types of neurons for a
long range of time,” he says, “we expect to perform chronic in vivo
imaging and analyze the modifications of neuronal network activities
underlying learning, development or diseases of the brain.”