Abstract: Our goal is to introduce a new class of genetically encoded optical indicators, based on the huge diversity of environmentally sensitive spectral shifts that occur naturally in microbial rhodopsin proteins. We recently created a microbial rhodopsin-based fluorescent indicator of pH, with a sensitive range from pH 6.8 to 8.8. We have preliminary results on a microbial rhodopsin-based indicator of membrane potential, which shows greater sensitivity than any existing optical sensor of membrane potential. Just as GFP revolutionized biology through its ability to track the positions of proteins in cells, we believe that microbial rhodopsins will have a broad impact through their ability to transduce the physical and chemical environment into an optical signal. Sensing voltage is our first target. Neuroscientists have long dreamed of a genetically encoded sensor that gives an optical signal in response to a change in membrane potential, with the goal of imaging electrical activity of neurons in vivo. Such a molecule could also be used to probe membrane potentials in mitochondria, cardiac cells, or in other non-neuronal cells. Our strategy is completely different from previous approaches to optical voltage sensing, and has already shown promising results. The technical implementation involves a) protein engineering and directed evolution to optimize an electrochromic response, and b) design and construction of an ultrasensitive laser imaging system capable of detecting this response in living cells. The methodology developed for sensing pH and voltage will later be applied to other sensing modalities, such as chloride and membrane tension. Public Health Relevance: We are working to develop a new class of molecules that allow us to see changes in voltage or pH inside of single cells. Neurons use voltage to communicate, so the ability to see neuronal activity will provide insights into brain function.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
NIH Director’s New Innovator Awards (DP2)
Project #
1DP2OD007428-01
Application #
7981713
Study Section
Special Emphasis Panel (ZGM1-NDIA-O (01))
Program Officer
Basavappa, Ravi
Project Start
2010-09-30
Project End
2015-06-30
Budget Start
2010-09-30
Budget End
2015-06-30
Support Year
1
Fiscal Year
2010
Total Cost
$2,520,000
Indirect Cost
Name
Harvard University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Kiskinis, Evangelos; Kralj, Joel M; Zou, Peng et al. (2018) All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS. Stem Cell Reports 10:1991-2004
Zhang, Hongkang; Cohen, Adam E (2017) Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond. Trends Biotechnol 35:625-639
Xu, Yongxian; Zou, Peng; Cohen, Adam E (2017) Voltage imaging with genetically encoded indicators. Curr Opin Chem Biol 39:1-10
Abdelfattah, Ahmed S; Farhi, Samouil L; Zhao, Yongxin et al. (2016) A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices. J Neurosci 36:2458-72
Chien, Miao-Ping; Werley, Christopher A; Farhi, Samouil L et al. (2015) Photostick: a method for selective isolation of target cells from culture. Chem Sci 6:1701-1705
Emiliani, Valentina; Cohen, Adam E; Deisseroth, Karl et al. (2015) All-Optical Interrogation of Neural Circuits. J Neurosci 35:13917-26
Brinks, Daan; Klein, Aaron J; Cohen, Adam E (2015) Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage. Biophys J 109:914-21
Cohen, Adam E; Venkatachalam, Veena (2014) Bringing bioelectricity to light. Annu Rev Biophys 43:211-32
Hou, Jennifer H; Venkatachalam, Veena; Cohen, Adam E (2014) Temporal dynamics of microbial rhodopsin fluorescence reports absolute membrane voltage. Biophys J 106:639-48
Hou, Jennifer H; Kralj, Joel M; Douglass, Adam D et al. (2014) Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents. Front Physiol 5:344

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