17 research outputs found
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Engineering microbial rhodopsins to expand the optogenetic toolkit
Cellular lipid membranes can – and often do – support a transmembrane electric field, serving as biological capacitors that maintain a voltage difference between their two sides. It isn't hard to see why these voltage gradients matter; the electrical spiking of neurons gives rise to our thoughts and actions, and the voltage dynamics of cardiomyocytes keep our hearts beating. Studies of bioelectricity have historically relied on electrode-based techniques to perturb and measure membrane potential, but these techniques have inherent limitations. I present new optogenetic methods of studying membrane potential that will broaden the scope of electrophysiological investigations by complementing traditional approaches.
I introduce the microbial rhodopsin Archaerhodopsin-3 (Arch), a transmembrane protein from Halorubrum sodomense. The fluorescence of Arch is a function of membrane potential, allowing it to serve as an optical voltage reporter. We use time-dependent pump-probe spectroscopy to interrogate the light- and voltage- dependent conformational dynamics of this protein, to elucidate the mechanism of voltage-dependent fluorescence in Arch.
I then present two new methods for imaging voltage using engineered variants of Arch. Both techniques take advantage of the unique photophysical properties of Arch(D95X) mutants. The first method, Flash Memory, records a photochemical imprint of the activity state -- firing or not firing -- of a neuron at a user-selected moment in time. The Flash Memory technique decouples the recording of neural activity from its readout, and can potentially allow us to take large-scale snapshots of voltage (e.g. maps of activity in a whole mouse brain). The second method allows for the quantitative optical measurement of membrane potential. This technique overcomes the problems that typically hinder intensity-based measurements by encoding a measurement of voltage in the time domain.
Finally, I present a method to visualize cellular responses to changes in membrane potential. I engineer mutants of Channelrhodopsin-2 (ChR2), a light-gated cation channel from Chlamydomonas reinhardtii that is used for optical control of neural activity, and use these optogenetic actuators in conjunction with GFP-based sensors to study the activity-dependent behavior of cultured neurons
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Flash Memory: Photochemical Imprinting of Neuronal Action Potentials onto a Microbial Rhodopsin
We developed a technique, âflash memoryâ, to record a photochemical imprint of the activity stateâfiring or not firingâof a neuron at a user-selected moment in time. The key element is an engineered microbial rhodopsin protein with three states. Two nonfluorescent 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 λwrite, population transfers from D2 to F, at a rate determined by the D1 â 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 λread excites fluorescence of F, probing this record. An optional third flash at a wavelength λreset converts F back to D2, for a subsequent writeâread 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 presented here will need improvements in sensitivity, speed, brightness, and membrane trafficking before this goal can be realized
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Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells
Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators
Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control
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 precise control of firing patterns; green fluorescent protein-based reporters, e.g., the GCaMP6f Ca2ĂŸ reporter, enable highly sensitive probing of cellular physiology. However, for most actuator-reporter combinations, spectral overlap prevents straightforward combination within a single cell. Here we explore multiwavelength control of channelrhodopsins to circumvent this limitation. The ââstoplightââ technique described in this article 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 green fluorescent protein-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, although cell-to-cell variation in expression levels presents a challenge for quantification.Chemistry and Chemical Biolog
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Bringing Bioelectricity to Light
Any bilayer lipid membrane can support a membrane voltage. The combination of optical perturbation and optical readout of membrane voltage opens the door to studies of electrophysiology in a huge variety of systems previously inaccessible to electrode-based measurements. Yet, the application of optogenetic electrophysiology requires careful reconsideration of the fundamentals of bioelectricity. Rules of thumb appropriate for neuroscience and cardiology may not apply in systems with dramatically different sizes, lipid compositions, charge carriers, or protein machinery. Optogenetic tools are not electrodes; thus, optical and electrode-based measurements have different quirks. Here we review the fundamental aspects of bioelectricity with the aim of laying a conceptual framework for all-optical electrophysiology.Chemistry and Chemical BiologyPhysic
Temporal Dynamics of Microbial Rhodopsin Fluorescence Reports Absolute Membrane Voltage
Plasma membrane voltage is a fundamentally important property of a living cell; its value is tightly coupled to membrane transport, the dynamics of transmembrane proteins, and to intercellular communication. 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 human embryonic kidney 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.Chemistry and Chemical Biolog
Residual structure within the disordered C-terminal segment of p21 and its implications for molecular recognition
Abstract: Probably the most unusual class of proteins in nature is the intrinsically unstructured proteins (IUPs), because they are not structured yet play essential roles in protein-protein signaling. Many IUPs can bind different proteins, and in many cases, adopt different bound conformations. The p21 protein is a small IUP (164 residues) that is ubiquitous in cellular signaling, for example, cell cycle control, apoptosis, transcription, differentiation, and so forth; it binds to approximately 25 targets. How does this small, unstructured protein recognize each of these targets with high affinity? Here, we characterize residual structural elements of the C-terminal segment of p21 encompassing residues 145-164 using a combination of NMR measurements and molecular dynamics simulations. The N-terminal half of the peptide has a significant helical propensity which is recognized by calmodulin while the C-terminal half of the peptide prefers extended conformations that facilitate binding to the proliferating cell nuclear antigen (PCNA). Our results suggest that the final bound conformations of p21 (145-164) pre-exist in the free peptide even without its binding partners. While the conformational flexibility of the p21 peptide is essential for adapting to diverse binding environments, the intrinsic structural preferences of the free peptide enable promiscuous yet high affinity binding to a diverse array of molecular targets
A Switch in p53 Dynamics Marks Cells That Escape from DSB-Induced Cell Cycle Arrest
© 2020 The Author(s) Cellular responses to stimuli can evolve over time, resulting in distinct early and late phases in response to a single signal. DNA damage induces a complex response that is largely orchestrated by the transcription factor p53, whose dynamics influence whether a damaged cell will arrest and repair the damage or will initiate cell death. How p53 responses and cellular outcomes evolve in the presence of continuous DNA damage remains unknown. Here, we have found that a subset of cells switches from oscillating to sustained p53 dynamics several days after undergoing damage. The switch results from cell cycle progression in the presence of damaged DNA, which activates the caspase-2-PIDDosome, a complex that stabilizes p53 by inactivating its negative regulator MDM2. This work defines a molecular pathway that is activated if the canonical checkpoints fail to halt mitosis in the presence of damaged DNA