Highlightable Ca2+ Indicators for Live Cell Imaging
journal contributionposted on 09.01.2013, 00:00 by Hiofan Hoi, Tomoki Matsuda, Takeharu Nagai, Robert E. Campbell
Two of the most powerful implementations of fluorescent protein (FP) technology are “highlighters”, which can be converted from nonfluorescent to fluorescent or from one color to another by illumination, and calcium ion (Ca2+) indicators. Combining the properties of both of these FP classes into a single construct would produce a highlightable Ca2+ indicator that would enable researchers to mark a single cell spectrally in a transfected tissue and image its intracellular Ca2+ dynamics. In an effort to create such a hybrid tool, we explored three different protein design strategies. The strategy that ultimately proved successful involved the creation of a circularly permuted version of a green-to-red photoconvertible FP and its introduction into a G-CaMP-type single-FP-based Ca2+ indicator. Optimization by directed evolution led to the identification of two promising variants that exhibit excellent photoconversion properties and have an up to 4.6-fold increase in red fluorescence intensity upon binding of Ca2+. We demonstrate the utility of these variants in HeLa cells and rat hippocampal neurons.