Cohen Lab Research

Spectroscopy and voltage-dependent nonlinear control with microbial rhodopsins

Microbial rhodopsin proteins have wondrously complex and fascinating optical responses. These proteins have a multitude of conformational states, each with unique spectral and electrical properties. Optical or thermal excitations drive transitions between these states. By mapping the conformational landscapes of microbial rhodopsin proteins, we have learned about basic mechanisms which then allow us to engineer new kinds of molecular logic into these versatile tools.

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Electrochromic FRET for multicolored voltage indicators

Genetically encoded fluorescent reporters of membrane potential promise to reveal aspects of neural function not detectable by other means. We developed a palette of multi-colored brightly fluorescent genetically encoded voltage indicators with sensitivities from 8 – 13% DF/F per 100 mV, and half-maximal response times from 4 – 7 ms. A fluorescent protein is fused to an Archaerhodopsin-derived voltage sensor. Voltage-induced shifts in the absorption spectrum of the rhodopsin lead to voltage-dependent nonradiative quenching of the appended fluorescent protein. Through a library screen, we identify linkers and fluorescent protein combinations which report neuronal action potentials in cultured rat hippocampal neurons with a single-trial signal-to-noise ratio from 7 to 9 in a 1 kHz imaging bandwidth at modest illumination intensity. The freedom to choose a voltage indicator from an array of colors facilitates multicolor voltage imaging, as well as combination with other optical reporters and optogenetic actuators.

 

See: P. Zou*, Y. Zhao*, A.D. Doughlass, D.R. Hochbaum, D. Brinks, C.A. Werley, D.J. Harrison, R.E. Campbell, A.E. Cohen (*Co-first authors), Bright and fast multicoloured voltage reporters via electrochromic FRET, Nature Communications, 5, 2014.


Stoplight rhodopsins for simultaneous optogenetic control and GFP imaging

To study the impact of neural activity on cellular physiology, one would like to combine precise control of firing patterns with highly sensitive probes of cellular physiology. Light-gated ion channels, e.g. Channelrhodopsin-2, enable the former, while GFP-based reporters, e.g. the GCaMP6f Ca2+ reporter, enable the latter. However, for most actuator-reporter combinations, spectral overlap prevents straightforward combination within a single cell. We explored multi-wavelength control of channelrhodopsins to circumvent this limitation. The “stoplight” technique uses channelrhodopsin variants that are opened by blue light and closed by orange light. Cells are illuminated with constant blue light to excite fluorescence of a GFP-based reporter. Modulated illumination with orange light negatively regulates activation of the channelrhodopsin. We performed detailed photophysical characterization and kinetic modeling of four candidate “stoplight” channelrhodopsins. The variant with the highest contrast, sdChR(C138S,E154A), enabled all-optical measurements of activity-induced calcium transients in cultured rat hippocampal neurons, though cell-to-cell variation in expression levels presents a challenge for quantification.

See:V. Venkatachalam, A.E. Cohen, Imaging GFP-based reporters in neurons with multiwavelength optogenetic control, Biophys. J., 107, 1554-1563, 2014.


Nonequilibrium fluorescence dynamics report absolute membrane voltage

Accurate measurement of the membrane voltage could elucidate subtle changes in cellular physiology, but existing genetically encoded fluorescent voltage reporters are better at reporting relative changes than absolute numbers. We developed an Archaerhodopsin-based fluorescent voltage sensor whose time-domain response to a stepwise change in illumination encodes the absolute membrane voltage. We validated this sensor in HEK cells. Measurements were robust to variation in imaging parameters and in gene expression levels, and reported voltage with an absolute accuracy of 10 mV. With further improvements in membrane trafficking and signal amplitude, time-domain encoding of absolute voltage could be applied to investigate many important and previously intractable bioelectric phenomena.

See: J. Hou, V. Venkatachalam, A.E. Cohen, Temporal dynamics of microbial rhodopsin fluorescence reports absolute membrane voltage, Biophysical Journal, 106.3, 639-648, 2014.


Flash Memory: a light-gated voltage integrator

We developed a technique, “Flash Memory”, to record a photochemical imprint of the activity state – firing or not firing – of a neuron at a user-selected moment in time. The key element is an engineered microbial rhodopsin protein with three states. Two non-fluorescent states, D1 and D2, exist in a voltage-dependent equilibrium. A stable fluorescent state, F, is reached by a photochemical conversion from D2. When exposed to light of a wavelength lambda_write, population transfers from D2 to F, at a rate determined by the D1 <--> D2 equilibrium. The population of F maintains a record of membrane voltage which persists in the dark. Illumination at a later time at a wavelength lambda_read excites fluorescence of F, probing this record. An optional third flash at a wavelength lambda_reset converts F back to D2, for a subsequent write-read cycle. The Flash Memory method offers the promise to decouple the recording of neural activity from its readout. In principle, the technique may enable one to generate snapshots of neural activity in a large volume of neural tissue, e.g. a complete mouse brain, by circumventing the challenge of imaging a large volume with simultaneous high spatial and high temporal resolution. The proof-of-principle Flash Memory sensors we developed will need improvements in sensitivity, speed, brightness and membrane trafficking before this goal can be realized.

 

See: V. Venkatachalam, D. Brinks, D. Maclaurin, D. Hochbaum, J.M. Kralj, A.E. Cohen, Flash Memory: Photochemical Imprinting of Neuronal Action Potentials onto a Microbial Rhodopsin, J. Am. Chem. Soci., 136, 2529-2537 2013.


Mechanism of voltage-sensitive fluorescence in Arch

Microbial rhodopsins were recently introduced as genetically encoded fluorescent indicators of membrane voltage. An understanding of the mechanism underlying this function would aid in the design of improved voltage indicators. We asked: what states can the protein adopt, and which states are fluorescent? How does membrane voltage affect the photostationary distribution of states? Here we present a detailed spectroscopic characterization of Archaerhodopsin 3 (Arch). We performed fluorescence spectroscopy on Arch and its photogenerated intermediates in E. coli and in single HEK 293 cells under voltage-clamp conditions. These experiments probed the effects of time-dependent illumination and membrane voltage on absorption, fluorescence, membrane current, and membrane capacitance. The fluorescence of Arch arises through a sequential three-photon process. Membrane voltage modulates protonation of the Schiff base in a 13-cis photocycle intermediate (M <--> N equilibrium); not in the ground state as previously hypothesized. We present experimental protocols for optimized voltage imaging with Arch and we discuss strategies for engineering improved rhodopsin-based voltage indicators.

See: D. Maclaurin*, V. Venkatachalam*, H. Lee, A.E. Cohen (*Co-first authors), Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin, PNAS, 110, 5939-5944, 2013.


Photochromic FRET as a probe of the photocycle

Photochromic fluorescence resonance energy transfer (pcFRET) provides an ultrasensitive readout of the progress through the photocycle in microbial rhodopsin proteins. A small-molecule fluorophore is covalently attached to the microbial rhodopsin. The emission spectrum of the fluorophore is chosen to overlap with the absorption spectrum of one of the intermediates in the photocycle of the rhodopsin. Changes in the absorption spectrum of the retinal chromophore lead to changes in the emission intensity the fluorophore through nonradiative energy transfer.

We used pcFRET to observe the photocycle in samples of microbial rhodopsins as small as 2 or 3 molecules. We saw dramatically different photocycle kinetics in Blue Proteorhodopsin and Sensory Rhodopsin II.

 

See: H. Bayraktar, A. P. Fields, J. M. Kralj, J. L. Spudich, K. J. Rothschild, A. E. Cohen, “Ultrasensitive measurements of microbial rhodopsin photocycles using photochromic FRET,” Photochem. & Photobiol., 88, 90-97 (2012)

 

 

©2012 Adam E. Cohen