Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

1-POS Board 1 Expanding the Toolbox of Genetically Encoded Voltage Indicators Ahmed S. Abdelfattah 1 , Samouil L. Farhi 2 , Yongxin Zhao 1 , Daan Brinks 2 , Adam E. Cohen 2 , Robert E. Campbell 1 . 1 University of Alberta, Edmonton, AB, Canada, 2 Harvard University, Cambridge, MA, USA. Simultaneous recording of neuronal activity from many locations is key to understanding how brain function emerges from neuronal circuits. Optical imaging using voltage indicators based on green fluorescent proteins (FPs) has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. To expand the toolbox of genetically encoded voltage indicators, we engineered two FP-based voltage indicators: A bright red fluorescent voltage indicator (FlicR1) and a green to red highlightable voltage indicator (FlicGR). We used directed protein evolution to screen libraries of thousands of variants to identify clones with sufficient brightness and response amplitude that would report membrane potential changes in mammalian cells. FlicR1 has voltage sensing properties that are comparable to the best available green indicators. It reports single action potentials in neurons in single trial recordings. Furthermore, FlicR1 can be imaged with wide field fluorescence microscopy using a typical mercury arc lamp, and faithfully reports spontaneous activity in cultured hippocampal neurons and rat brain organotypic slices. We also demonstrate that FlicR1 can be used in conjunction with PsChR, a blue-shifted channelrhodopsin, for all-optical neuronal activation and activity recording, although blue light photoactivation of the FlicR1 chromophore remains a challenge. FlicGR is the first example of a highlightable voltage indicator. A powerful application of fluorescent proteins is their use as highlighters where they can be converted from one color to another by light. Using blue light, we can photoconvert FlicGR from green to red. We demonstrate that both the green and red forms are sensitive to membrane potential changes in mammalian cells.

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