Biology subjects

Cohen, A. E.

Publications and source records attributed to Cohen, A. E..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Photophysics-informed two-photon voltage imaging using FRET-opsin voltage indicators

Microbial rhodopsin-derived genetically encoded voltage indicators (GEVIs) are powerful tools for mapping bioelectrical dynamics in cell culture and in live animals. Forster resonance energy transfer (FRET)-opsin GEVIs use voltage-dependent changes in opsin absorption to modulate the fluorescence of an atached fluorophore, achieving high brightness, speed, and voltage sensitivity. However, the voltage sensitivity of most FRET-opsin GEVIs has been reported to decrease or vanish under two-photon (2P) excitation. Here we investigated the photophysics of the FRET-opsin GEVIs Voltron1 and 2. We found that the voltage sensitivity came from a photocycle intermediate, not from the opsin ground state. The voltage sensitivities of both GEVIs were nonlinear functions of illumination intensity; for Voltron1, the sensitivity reversed sign under low-intensity illumination. Using photocycle-optimized 2P illumination protocols, we demonstrate 2P voltage imaging with Voltron2 in barrel cortex of a live mouse. These results open the door to high-speed 2P voltage imaging of FRET-opsin GEVIs in vivo. TeaserVoltage sensitivity in FRET-opsin indicators comes from a photocycle intermediate, reachable via optimized 2P excitation.

neuroscience↗

bioRxiv2024

Ether lipids influence cancer cell fate by modulating iron uptake

Cancer cell fate has been widely ascribed to mutational changes within proteincoding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with high metastatic ability and cancer stem celllike traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, resulting in significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro threedimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids from plasma membranes also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

cancer biology↗

bioRxiv2024

Mapping changes in uterine contractility during the ovulatory cycle with wide-area calcium imaging in live mice.

Uterine contraction patterns vary during the ovulatory cycle and throughout pregnancy but prior measurements have produced limited and conflicting information on these patterns. We combined a virally delivered genetically encoded calcium reporter (GCaMP8m) and ultra-widefield imaging in live nonpregnant mice to characterize uterine calcium dynamics at organ scale throughout the estrous cycle. Prior to ovulation (proestrus and estrus) uterine excitations primarily initiated in a region near the oviduct, but after ovulation (metestrus and diestrus), excitations initiated at loci homogeneously distributed throughout the organ. The frequency of excitation events was lowest in proestrus and estrus, higher in metestrus and highest in diestrus. These results establish a platform for mapping uterine activity, and show that the question of whether there is an anatomically localized trigger for uterine excitations depends on the estrous cycle phase.

physiology↗

bioRxiv2024

Solaris: a panel of bright and sensitive hybrid voltage indicators for imaging membrane potential in cultured neurons

Dynamic changes in the membrane potential underlie neuronal activities. Fluorescent voltage indicators allow optical recording of electrical signaling across a neuronal population with cellular precision and at millisecond-level temporal resolution. Here we report the design and characterization of a chemigenetic hybrid voltage indicator, Solaris, in which a circularly permuted HaloTag is inserted into the first extracellular loop of Acetabularia rhodopsin. Solaris is compatible with fluorogenic HaloTag ligands JF525, JF549, JF552, JF585, and JF635. The most sensitive conjugate, Solaris585, has more than 2-fold higher voltage sensitivity than the spectrally similar Voltron2585 ({Delta}F/F0 = -28.1 {+/-} 1.3% versus -12.3 {+/-} 0.7% per action potential in cultured neurons). Solaris585 supports the measurement of optogenetically evoked spike activity or dual-color imaging in conjunction with green-emitting calcium or glutamate indicators. Solaris indicators are also applicable to fluorescence lifetime imaging, which probes the absolute membrane potential. This new hybrid voltage indicator is a valuable tool for imaging neuronal electrophysiological activities in cultured cells with substantially improved dynamic range compared to previous hybrid indicators.

neuroscience↗

bioRxiv2023

Optical constraints on two-photon voltage imaging

SignificanceGenetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits and limitations of one-photon (1P) vs. two-photon (2P) voltage imaging are not well characterized. AimWe consider the optical and biophysical constraints particular to 1P and 2P voltage imaging and compare the imaging properties of commonly used GEVIs under 1P and 2P excitation. ApproachWe measure brightness and voltage sensitivity of voltage indicators from commonly used classes under 1P and 2P illumination. We also measure the decrease in fluorescence as a function of depth in mouse brain. We develop a simple model of the number of measurable cells as a function of reporter properties, imaging parameters, and desired signal-to-noise ratio (SNR). We then discuss how the performance of voltage imaging would be affected by sensor improvements and by recently introduced advanced imaging modalities. ResultsCompared to 1P excitation, 2P excitation requires [~]104-fold more illumination power per cell to produce similar photon count rates. For voltage imaging with JEDI-2P in mouse cortex with a target SNR of 10 (spike height:baseline shot noise), a measurement bandwidth of 1 kHz, a thermally limited laser power of 200 mW, and an imaging depth of > 300 m, 2P voltage imaging using an 80 MHz source can record from no more 12 cells simultaneously. ConclusionsDue to the stringent photon-count requirements of voltage imaging and the modest voltage sensitivity of existing reporters, 2P voltage imaging in vivo faces a stringent tradeoff between shot noise and tissue photodamage. 2P imaging of hundreds of neurons with high SNR at depth > 300 m will require either major improvements in 2P GEVIs or qualitatively new approaches to imaging.

neuroscience↗

bioRxiv2023

Optical segmentation-based compressed readout of neuronal voltage dynamics

Functional imaging of biological dynamics generally begins with acquiring time-series images, followed by quantifying spatially averaged intensity traces for the regions of interest (ROIs). The conventional pipeline discards a substantial portion of the acquired data when quantifying intensity traces, indicative of inefficient data acquisition. Here we propose a conceptually novel acquisition pipeline that assigns each ROI to a single pixel in the detector, enabling optimally compressed acquisition of the intensity traces. As a proof-of-principle, we implemented a detection module composed of a pair of spatial light modulators and a microlens array, which segments the original image into multiple subimages by introducing distinct angular shifts to each ROI. Each subimage exclusively encodes the signal for the corresponding ROI, facilitating the compressed readout of its intensity trace using a single pixel. This spatial compression allowed for maximizing the temporal information without compromising the spatial information on ROIs. Harnessing our novel approach, we demonstrate the recording of circuit-scale neuronal voltage dynamics at over 5 kHz sampling rate, revealing the individual action potential waveforms within subcellular structures, as well as their submillisecond-scale temporal delays.

neuroscience↗

bioRxiv2023

Structural basis of transcription: RNA Polymerase II substrate binding and metal coordination at 3.0 A using a free-electron laser

Catalysis and translocation of multi-subunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near atomic resolution and precise arrangement of key active site components have been elusive. Here we present the free electron laser (FEL) structure of a matched ATP-bound Pol II, revealing the full active site interaction network at the highest resolution to date, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structure indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/bridge helix (BH) interactions induce conformational changes that could propel translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the hyperactive Rpb1 T834P bridge helix mutant reveals rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

biophysics↗

bioRxiv2023

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

biochemistry↗

bioRxiv2023

All-optical mapping of cAMP transport reveals rules of sub-cellular localization

Cyclic adenosine monophosphate (cAMP) is a second messenger that mediates diverse intracellular signals. Studies of cAMP transport in cells have produced wildly different results, from reports of nearly free diffusion to reports that cAMP remains localized in nanometer-scale domains. We developed an all-optical toolkit, cAMP-SITES, to locally perturb and map cAMP transport. In MDCK cells and in cultured neurons, cAMP had a diffusion coefficient of [~]130 {micro}m2/s, similar to the diffusion coefficients of other small molecules in cytoplasm. In neuronal dendrites, a balance between diffusion and degradation led to cAMP domains with a length-scale of 27 {+/-} 11 {micro}m (mean {+/-} s.d.). Geometrical confinement by membranes led to subcellular variations in cAMP concentration, but we found no evidence of nanoscale domains or of distinct membrane-associated and cytoplasmic pools. We discuss the scaling relations which govern diffusible signaling in tube-shaped structures.

biophysics↗

bioRxiv2023

Dendritic voltage imaging reveals biophysical basis of associative plasticity rules

Dendrites on neurons support nonlinear electrical excitations, but the computational significance of these events is not well understood. We developed molecular, optical, and analytical tools to map sub-millisecond voltage dynamics throughout the dendritic trees of CA1 pyramidal neurons under diverse optogenetic and synaptic stimulus patterns, in acute brain slices. We observed history-dependent spike back-propagation in distal dendrites, driven by locally generated Na+ spikes (dSpikes). Dendritic depolarization created a transient window for dSpike propagation, opened by A-type KV channel inactivation, and closed by slow NaV inactivation. Collisions of dSpikes with synaptic inputs triggered calcium channel and N-methyl-D-aspartate receptor (NMDAR)-dependent plateau potentials, with accompanying complex spikes at the soma. This hierarchical ion channel network acts as a spike-rate accelerometer, providing an intuitive picture of how dendritic excitations shape associative plasticity rules.

neuroscience↗

bioRxiv2023

Voltage dynamics of dendritic integration and back-propagation in vivo

Neurons integrate synaptic inputs within their dendrites and produce spiking outputs, which then propagate down the axon and back into the dendrites where they contribute to plasticity. Mapping the voltage dynamics in dendritic arbors of live animals is crucial for understanding neuronal computation and plasticity rules. Here we combine patterned channelrhodopsin activation with dual-plane structured illumination voltage imaging, for simultaneous perturbation and monitoring of dendritic and somatic voltage in Layer 2/3 pyramidal neurons in anesthetized and awake mice. We examined the integration of synaptic inputs and compared the dynamics of optogenetically evoked, spontaneous, and sensory-evoked back-propagating action potentials (bAPs). Our measurements revealed a broadly shared membrane voltage throughout the dendritic arbor, and few signatures of electrical compartmentalization among synaptic inputs. However, we observed spike rate acceleration-dependent propagation of bAPs into distal dendrites. We propose that this dendritic filtering of bAPs may play a critical role in activity-dependent plasticity.

neuroscience↗

bioRxiv2023

Mapping memories: pulse-chase labeling reveals AMPA receptor dynamics during memory formation

A tool to map changes in synaptic strength during a defined time window could provide powerful insights into the mechanisms governing learning and memory. We developed a technique, Extracellular Protein Surface Labeling in Neurons (EPSILON), to map -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) insertion in vivo by pulse-chase labeling of surface AMPARs with membrane-impermeable dyes. This approach allows for single-synapse resolution maps of plasticity in genetically targeted neurons during memory formation. We investigated the relationship between synapse-level and cell-level memory encodings by mapping synaptic plasticity and cFos expression in hippocampal CA1 pyramidal cells upon contextual fear conditioning (CFC). We observed a strong correlation between synaptic plasticity and cFos expression, suggesting a synaptic mechanism for the association of cFos expression with memory engrams. The EPSILON technique is a useful tool for mapping synaptic plasticity and may be extended to investigate trafficking of other transmembrane proteins.

neuroscience↗

bioRxiv2023

Diminishing neuronal acidification by channelrhodopsins with low proton conduction

Many channelrhodopsins are permeable to protons. We found that in neurons, activation of a high-current channelrhodopsin, CheRiff, led to significant acidification, with faster acidification in the dendrites than in the soma. Experiments with patterned optogenetic stimulation in monolayers of HEK cells established that the acidification was due to proton transport through the opsin, rather than through other voltage-dependent channels. We identified and characterized two opsins which showed large photocurrents, but small proton permeability, PsCatCh2.0 and ChR2-3M. PsCatCh2.0 showed excellent response kinetics and was also spectrally compatible with simultaneous voltage imaging with QuasAr6a. Stimulation-evoked acidification is a possible source of disruptions to cell health in scientific and prospective therapeutic applications of optogenetics. Channelrhodopsins with low proton permeability are a promising strategy for avoiding these problems. Statement of SignificanceAcidification is an undesirable artifact of optogenetic stimulation. Low proton-permeability opsins minimize this artifact while still allowing robust optogenetic control.

biophysics↗

bioRxiv2022

A phenotypic screening platform for chronic pain therapeutics using all-optical electrophysiology

Chronic pain associated with osteoarthritis (OA) remains an intractable problem with few effective treatment options. New approaches are needed to model the disease biology and to drive discovery of therapeutics. Here, we present an in vitro model of OA pain, where dorsal root ganglion (DRG) sensory neurons were sensitized by a defined mixture of disease-relevant inflammatory mediators, here called Sensitizing PAin Reagent Composition or SPARC. OA-SPARC components showed synergistic or additive effects when applied in combination and induced pain phenotypes in vivo. To measure the effect of OA-SPARC on neural firing in a scalable format for drug discovery, we used a custom system for high throughput all-optical electrophysiology. This system enabled light-based membrane voltage recordings from hundreds of neurons in parallel with single cell resolution and a throughput of up to 500,000 neurons per day, with patch clamp-like single action potential resolution. A computational framework was developed to construct a multiparameter OA-SPARC neuronal phenotype and to quantitatively assess phenotype reversal by candidate pharmacology with different mechanisms of action. We screened ~3000 approved drugs and mechanistically focused compounds, yielding data from over 1.2 million individual neurons with detailed assessment of both functional OA-SPARC phenotype rescue and orthogonal "off-target" effects. Analysis of confirmed hits revealed diverse potential analgesic mechanisms including well-known ion channel modulators as well as less characterized mechanisms including MEK inhibitors and tyrosine kinase modulators, providing validation of the platform for pain drug discovery.

neuroscience↗

bioRxiv2022

A bioelectrical phase transition patterns the first beats of a vertebrate heart

The heart is among the first organs to function in vertebrate development, but its transition from silent to beating has not been directly characterized. Using all-optical electrophysiology, we captured the very first zebrafish heartbeat and analyzed the development of cardiac excitability around this singular event. The first beats appeared suddenly and propagated coherently across the primordial heart. Targeted optogenetic perturbations mapped the development of excitability and conduction before and after the first heartbeats. Measured bioelectrical dynamics support a noisy saddle-node on invariant circle (SNIC) bifurcation as the critical phase transition that starts the heart. Simple models of this bifurcation quantitatively capture cardiac dynamics in space and time through early development, including coherent beating before transcriptional specification of pacemakers. Our work shows how gradual and largely asynchronous development of single-cell bioelectrical properties produces a stereotyped and robust tissue-scale transition from quiescence to coordinated beating.

developmental biology↗

bioRxiv2022

Topological action potentials in engineered tissues

Due to the nonlinear current-voltage relations of ion channels, an interface between two tissues can have very different bioelectrical properties compared to either tissue on its own. Here we show experimentally that gap junction-coupled interfaces between non-excitable tissues can be electrically excitable. This topologically protected excitability occurs over a far larger range of ion channel expression levels than does excitability in the bulk. Topological excitations at tissue interfaces can cause local elevations in calcium concentration, possibly providing a bioelectrical mechanism for interface sensing. As in condensed matter physics, topological excitations in electrophysiology constitute a distinct class of phenomena which may show exotic and novel properties.

biophysics↗

bioRxiv2022

The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and X-ray diffraction at room temperature

The NSP3 macrodomain of SARS CoV 2 (Mac1) removes ADP-ribosylation post-translational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the COVID-19 pandemic. Here, we determined neutron and X-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site, and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a re-evaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

biochemistry↗

bioRxiv2022

Dendritic branch structure compartmentalizes voltage-dependent calcium influx in cortical layer 2/3 pyramidal cells

Back-propagating action potentials (bAPs) regulate synaptic plasticity by evoking voltage-dependent calcium influx throughout dendrites. Attenuation of bAP amplitude in distal dendritic compartments alters plasticity in a location-specific manner by reducing bAP-dependent calcium influx. However, it is not known if neurons exhibit branch-specific variability in bAP-dependent calcium signals, independent of distance-dependent attenuation. Here, we reveal that bAPs fail to evoke calcium influx through voltage-gated calcium channels (VGCCs) in a specific population of dendritic branches in cortical layer 2/3 pyramidal cells, despite evoking substantial VGCC-mediated calcium influx in sister branches. These branches contain VGCCs and successfully propagate bAPs in the absence of synaptic input; nevertheless, they fail to exhibit bAP-evoked calcium influx due to a branch-specific reduction in bAP amplitude. We demonstrate that these branches have more elaborate branch structure compared to sister branches, which causes a local reduction in electrotonic impedance and bAP amplitude. Finally, we show that bAPs still amplify synaptically-mediated calcium influx in these branches because of differences in the voltage-dependence and kinetics of VGCCs and NMDA-type glutamate receptors. Branch-specific compartmentalization of bAP-dependent calcium signals may provide a mechanism for neurons to diversify synaptic tuning across the dendritic tree.

neuroscience↗