Cohen Lab Research

Microbial rhodopsins as fluorescent voltage-indicating proteins

Microbial rhodopsin proteins are found in aquatic organisms throughout the world and in all kingdoms of life. In the wild, these proteins transduce sunlight into changes in membrane potential, which their host organisms use for phototaxis and solar energy capture. Recently, several labs have expressed microbial rhodopsins in other cell types, enabling optical control of membrane potential. We engineered several microbial rhodopsins to run in reverse: to transduce changes in membrane potential into an optical signal. These voltage-indicating proteins provide a fast and sensitive tool for monitoring electrical dynamics in cells.

Requests for voltage-indicating proteins

Please see our Resources page

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Simultaneous voltage and calcium imaging in the zebrafish heart

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

The cardiac action potential (AP) and the consequent cytosolic Ca2+ transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. We applied a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 Ė 102 hours post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

See: J. Hou, J.M. Kralj, A.D. Douglass, F. Engert, A.E. Cohen, Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific development transitions in ionic currents, Front. Physiol., 5, 344, 2014. Supporting videos.


All-optical electrophysiology with microbial rhodopsins

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and 2, which show improved brightness and voltage sensitivity, microsecond response times, and produce no photocurrent. We engineered a novel channelrhodopsin actuator, CheRiff, which shows improved light sensitivity and kinetics, and spectral orthogonality to the QuasArs. A co-expression vector, Optopatch, enabled crosstalk-free genetically targeted all-optical electrophysiology. In cultured neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials in dendritic spines, synaptic transmission, sub-cellular microsecond-timescale details of action potential propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In brain slice, Optopatch induced and reported action potentials and subthreshold events, with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without use of conventional electrodes.

See: D.R. Hochbaum*, Y. Zhao*, S.L. Farhi, N. Klapoetke, C.A. Werley, V. Kapoor, P. Zou, J.M. Kralj, D. Maclaurin, N. Smedemark-Margulies, J.L. Saulnier, G.L. Boulting, C. Straub, Y.K. Cho, M. Melkonian, G.K-S. Wong, D.J. Harrison, V.N. Murthy, B.L. Sebatini, E.S. Boyden, R.E. Campbell, A.E. Cohen (*Co-first authors), All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins, Nature Methods, 11, 825-833, 2014.


Optical recordings in mammalian neurons

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. We achieved this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately 10-fold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear two-fold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio > 10. The mutant Arch(D95N) lacked endogenous proton pumping and showed 50% greater sensitivity than wild-type, but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin-based voltage indicators promise to enable optical interrogation of complex neural circuits, and electrophysiology in systems for which electrode-based techniques are challenging.

See: J. Kralj*, A. D. Douglass*, D. R. Hochbaum*, D. Maclaurin, A. E. Cohen (*Co-first authors), “Optical recording of action potentials in mammalian neurons using a microbial rhodopsin,” Nature Methods, 9, 90-95 (2012)


Electrical spiking in E. coli

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

The Proteorhodopsin Optical Proton Sensor (PROPS) is a probe of membrane potential in bacteria. Expression of PROPS in E. coli revealed electrical spiking at up to 1 Hz. Spiking was sensitive to chemical and physical perturbations, and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated.

The appearance of electrical spiking in E. coli raises many questions.  What channels or pores regulate spiking?  Do the different spiking waveforms indicate different biological processes?  Elucidation of the electrophysiology of E. coli will require observing the effects of additional pharmacological, genetic, and physical perturbations.  These studies may yield insights into the biology of a wide range of medically, industrially, and environmentally significant microorganisms.  At present PROPS does not express in eukaryotes, but modified forms of related microbial rhodopsins may enable optogenetic studies of electrical activity in eukaryotic organelles and cells, including mitochondria and neurons.

See: J. Kralj, D. R. Hochbaum, A. D. Douglass, A. E. Cohen, Electrical Spiking in Escherichia coli Probed with a Fluorescent Voltage-Indicating Protein, Science, 333, 345-348, 2011

©2012 Adam E. Cohen