Biology subjects

Cohen, A. E.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Large-scale all-optical electrophysiology links excitability and connectivity to circuit dynamics in vivo

To understand neural computations, one would like to relate circuit dynamics to neuronal intrinsic excitability and connectivity. We developed tools for large-scale all-optical electrophysiology and applied them to approximately 300 layer 1 interneurons per mouse in vivo. Across two mice, targeted stimulation and voltage imaging identified 1,469 inhibitory and 5,752 directed electrical connections among 14,312 tested pairs. Presynaptic identity was more strongly associated with inhibitory post-synaptic potential (IPSP) amplitude, whereas postsynaptic identity was more strongly associated with IPSP decay time. Gap junctional and synaptic interactions had distinct spatiotemporal kernels, with corresponding signatures in spontaneous activity. Excitability measured under optogenetic stimulus predicted spiking responses to subthreshold voltage, depolarization rate, and spike history during spontaneous firing. Genetic subtypes also showed different behavioral-state-dependent voltage-to-spike relationships despite similar population-average subthreshold voltages. These measurements link cellular properties and pairwise interactions to spontaneous circuit dynamics, establishing an approach for functional circuit mapping in vivo.

neuroscience↗

bioRxiv2026

Graphene Device for High-Temperature Crystallography Visualizes How Enzymatic Reactivity is Rooted in Ensemble Probability

Life exists at temperatures ranging from -20 to 122 {degrees}C. However, the majority of high-resolution structural data in the Protein Data Bank (PDB) were obtained at cryogenic temperatures, where biological function is halted due to the lack of thermal fluctuations. To overcome this fundamental problem and directly link structure to biological function, we have created a graphene-based device that significantly extends the temperature range for high-resolution macromolecular X-ray diffraction data collection. Using the new device, we obtained models of the transition state ensembles for a psychrophilic, a mesophilic, and a thermophilic homolog of the enzyme orotidine 5-monophosphate decarboxylase from -173 to 65 {degrees}C. The data reveal how the active site ensemble structure at the transition state of each homolog changes with temperature, directly visualizing how the measured catalytic rates are rooted in the ensemble probabilities of reactive distances. The multi-temperature transition state ensembles further illuminate why cryogenic data, although useful, are inaccurate for describing biological processes.

biophysics↗

bioRxiv2026

Mapping absolute membrane voltage using dynamic photocycle control

Fluorescent voltage indicators are widely used to report relative changes in membrane potential, but mapping absolute voltages remains difficult. Here we present Voltage Measurement by Activated Photocycles (VMAP), a simple method for absolute voltage imaging based on a photophysical switch between voltage-insensitive and sensitive indicator states. VMAP requires no specialized hardware or additional labeling, and is applicable across species, sample preparations, and microscope configurations. Using VMAP, we quantified drug-induced shifts in neuronal resting potential, revealed the emergence of bioelectric patterns during multi-day recordings of human iPSC populations, and created 3D membrane-potential maps across whole live zebrafish embryos. By making absolute voltage imaging accessible from cellular to organismal scales and from milliseconds to days, VMAP opens a route to mapping bioelectrical organization in complex living systems.

biophysics↗

bioRxiv2026

CAGE-TRX expands the scope of time-resolved crystallography through genetically encoded active-site photocaging

We present CAGE-TRX, a broadly applicable time-resolved strategy for release-quench-probe cryocrystallography and release-probe room temperature serial crystallography. These workflows enable light-triggered control of enzyme activity via genetically encoded photocaged amino acids. By decoupling reaction initiation from substrate design, this approach allows synchronized catalysis in crystallo and the capture of transient intermediates. Using {beta}-lactamases as model systems, we demonstrate efficient decaging, restoration of activity, and structural visualization of reaction intermediates.

biochemistry↗

bioRxiv2026

A dendrite-resolved, in vivo transfer function from spike patterns to dendritic Ca2+

Dendrites transform local electrical activity into intracellular Ca2+ signals that drive plasticity1,2, yet the voltage[->]Ca2+ mapping during natural behavior remains poorly defined. Here, we measure this transfer function via simultaneous voltage and Ca2+ imaging throughout the dendritic arbors of hippocampal CA2 pyramidal neurons in behaving mice. Dendritic Ca2+ exhibited a hierarchical activation pattern dominated by back-propagating action potentials: simple spikes primarily drove somatic and proximal Ca2+, whereas complex spikes produced larger somatic Ca2+ signals and propagated farther into distal dendrites, sometimes in a branch-selective manner. Dendrite-restricted co-activation of voltage and Ca2+ without concurrent somatic events was rare. A biophysics-inspired model accurately predicted local Ca2+ transients from local voltage waveforms. Our data and model provide a quantitative understanding of when - and why - dendritic Ca2+ signals in CA2 pyramidal cells arise during behavior.

neuroscience↗

bioRxiv2026

Dendritic excitations primarily mediate back-propagation in CA1 pyramidal neurons during behavior

Dendrites integrate synaptic inputs to trigger action potentials, and dendrites carry back-propagating action potentials (bAPs) to synapses where these signals contribute to plasticity. Despite strong evidence for a rich repertoire of nonlinear dendritic excitations, the in vivo roles of these excitations in dendritic integration and back-propagation remain uncertain. Here, we used high-speed voltage imaging through a chronically implanted microprism to map membrane potential dynamics from basal to apical dendrites of CA1 neurons in mice navigating in a virtual reality environment. Despite complex dendritic branch morphology, the dynamics were largely captured by 2 or 3 electrical compartments: basal, soma, and apical. Fast dendritic spikes almost always started from bAPs, indicating that dendritic spikes are primarily a consequence rather than a cause of somatic spiking. These fast spikes sometimes triggered slower apical dendritic plateau depolarizations, which drove complex spikes at the soma. We found that the biophysics of dendritic excitability determined the distribution of simple and complex spikes across a place field. Our results show how CA1 pyramidal neurons convert synaptic inputs to spiking outputs and suggest a primary role of dendritic nonlinearities in mediating activity-dependent plasticity.

neuroscience↗

bioRxiv2025

DENDRO: Recovery and denoising of whole-tree dendritic voltagefrom 2D voltage movies

The ability to image voltage at high spatiotemporal resolution across an entire dendritic tree would represent a major advance in systems and circuit neuroscience. Recent advances in genetically encoded voltage indicators (GEVIs) have brought this possibility closer to reality. However, due to fundamental tradeoffs between imaging speed, resolution, SNR, and volume, this goal has remained out of reach. Here we develop a computational method that fuses 3D anatomical information with 2D voltage video data, yielding full time-varying 3D voltage estimates. Our method, termed DENDRO, comprises two steps. In step one, we use the anatomical data to build a microscope model which maps from voltages along the tree to observed fluorescence at the imaging plane. By exploiting local spatial smoothness of the voltage signal, we parameterize the voltage signal using a set of local basis functions, which reduces the dimensionality of the problem and allows us to approximately invert the microscope model. This step leverages spatial but not temporal smoothness of the underlying signal and yields noisy 3D estimates. In step two, we train a lightweight self-supervised neural network to perform spatiotemporal denoising of the inferred voltages. On simulated data, we find that DENDRO is able to recover voltages at high accuracy across an entire dendritic tree. On real voltage movies from hippocampal slices, DENDRO recovers known dendritic phenomena at single trial resolution and millisecond time-scales, and allows visualization of backpropagating action potentials in 3D.

neuroscience↗

bioRxiv2025

Electrophysiology in small compartments

Voltage-gated ion channels play important roles in many membrane-enclosed structures, including synaptic vesicles, endosomes, mitochondria, chloroplasts, viruses and bacteria. Here we study how compartment size and channel gating interact to shape voltage dynamics and ion content in sub-micron structures. In small compartments, assumptions underlying conductance-based (Hodgkin-Huxley type) models of membrane voltage must be relaxed: [1] stochastic gating of individual ion channels can quickly and substantially change membrane voltage; [2] these changes can equilibrate faster than channel state transitions; and [3] ionic currents, even through as few as two channels, can substantially alter ionic concentrations. We adapted conductance-based models to incorporate these effects, and we then simulated voltage dynamics of small vesicles as a function of vesicle radius and channel density. We identified regimes in this parameter space with qualitatively distinct dynamics. We then performed stochastic simulations to explore the role of NaV1.5 in maturation of macrophage endosomes. The stochastic model predicted dramatically different dynamics compared to a deterministic approach. Electrophysiology of nanoscale structures can be very different from larger structures, even when ion channel composition and density are preserved. SIGNIFICANCEWith tools of optical electrophysiology, one can measure and perturb membrane voltage in sub-micron structures. Recent experiments in organelles, dendritic spines, and bacteria motivate a re-examination of basic assumptions about bioelectrical phenomena in these compartments. This paper provides a framework for predicting and interpreting bioelectrical dynamics in small structures.

biophysics↗

bioRxiv2025

Time-Resolved Crystallography Reveals the Mechanisms of GTP hydrolysis for N-RAS and the Oncogenic Mutants G12C, G12V and Q61L

The RAS family of small GTPases are molecular switches that convey downstream signals regulating cell proliferation, differentiation, and apoptosis. The signaling competent GTP-bound RAS transitions to its inactive GDP-bound form through {gamma}-phosphate hydrolysis. Oncogenic RAS mutations hamper GTP hydrolysis and are present in up to 30% of all human cancers. Structural studies of RAS proteins bound to non-hydrolysable GTP analogs have revealed snapshots of the enzyme in its possibly active form. Yet, the mechanism of GTP hydrolysis has not been structurally resolved. To visualize this reaction in real time, we performed time-resolved crystallographic experiments employing a photolabile caged-GTP substrate. Fifty-seven distinctive reaction intermediates were captured during hydrolysis of a live GTP for N-RAS, the oncogenic mutants G12C, G12V and Q61L; and Y32R, a fast hydrolytic mutant. The reaction mechanisms and rates for the native and each of the mutants differed significantly; however, they shared common elements: an initially catalytically-defective open state, which transitions into the closed Michaelis complex state with solvent-assisted O3B-P{gamma} bond lengthening and breaking, followed by the release of the Mg2+ stabilized PO3-/PO4-3 species and unfolding of the switch loops. Given the conserved nature of GTP- and ATP-ases active sites, this structural work lays the basis to understand the universal mechanism of {gamma}-phosphate hydrolysis. Furthermore, search for cryptic binding sites during GTP hydrolysis in G12C, G12V, and Q61L mutants reveals the presence of distinctive state-dependent binding pockets that could be targets for structure-based drug discovery of experimentally resolved intermediates states.

cancer biology↗

bioRxiv2025

Optical single-channel recording via diffusional confinement in membrane tethers

Single-channel electrophysiology probes ion channel gating, but how can one probe membrane transport when the single-unit current is undetectable? We pulled membrane tethers from live cells to isolate individual transmembrane proteins. The tether constrained diffusion of transported substrate to the tether axis, leading to [~]1000-fold enhancement of substrate concentration and observation time compared to planar membranes. Fluorescent reporters inside the tether revealed individual transport events. We imaged unitary Ca2+ transport events in tethers containing the low-conductance T-type Ca2+ channel CaV3.2, and compared our results to ensemble electrophysiology and stochastic gating simulations. This work establishes tether-based single-channel recordings as a powerful tool to study dynamics of membrane transport. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/652649v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1ed0ac1org.highwire.dtl.DTLVardef@15d1b6eorg.highwire.dtl.DTLVardef@1b3ff91org.highwire.dtl.DTLVardef@4f99e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Structure of active methyl-CoM reductase, Earth's main methane producer

Our work reveals the structure of the active state of Methyl-Coenzyme M Reductase (MCR), the key and rate-limiting enzyme in biological methane formation. We find large differences between the active Ni(I) and inactive Ni(II) proteins and provide insight into how nature makes and breaks the C-H bond of methane. The Ni(II)-F430 center in inactive MCR contains four planar nitrogen ligands, a lower axial glutamine oxo, and an upper axial thiolate. The Ni(I)-enzyme replaces the axial ligands with a single water. The one-electron redox change results in movement of the Ni ion and upward swing of the {beta}-lactam ring in the tetrapyrrole coupled to a domino-like protein quake through second sphere residues, inter-subunit interactions, a substrate tunnel, affecting even the dimensions of the unit cell. These structural changes lead Ni(I)-MCR to release a charge clamp that, in the Ni(II) state, locks down substrate Coenzyme B. Determining the Ni(I)-MCR structure required development of rigorous anaerobic crystallographic techniques. Validation of the MCR redox state was accomplished by in-line and parallel spectroscopic and unit cell analyses. This structure has large implications for developing technologies to limit methane emissions and efficiently produce biofuels. Methodology described here will enhance structural biology for other oxygen-sensitive enzymes.

biochemistry↗

bioRxiv2025

Mechanism of giant magnetic field effect in fluorescence of mScarlet3, a red fluorescent protein

Several fluorescent proteins, when expressed in E. coli, are sensitive to weak magnetic fields1. We found that mScarlet3 fluorescence in E. coli reversibly decreased by 21% in the presence of a 60 mT magnetic field, the largest magnetic field effect (MFE) reported in any fluorescent protein. Purified mScarlet3 did not show an MFE, but addition of flavin mononucleotide (FMN) and simultaneous illumination with blue and yellow light restored the MFE. Through extensive photophysical experiments, we developed a quantitative model of the giant MFE in mScarlet3-FMN mixtures. The key reaction step involved electron transfer from fully reduced FMNH2 to triplet-state mScarlet3, to form a triplet spin-correlated radical pair. The magnetic field then controlled the branching ratio between singlet recombination vs. triplet separation. Our quantitative model of the mScarlet3-FMN photocycle provides a framework for design and optimization of magnetic field-sensitive proteins, opening possibilities in fluorescent protein-based magnetometry, magnetic imaging, and magnetogenetic control.

biophysics↗

bioRxiv2025

Luminos: open-source software for bidirectional microscopy

Bidirectional microscopy (BDM) combines simultaneous targeted optical perturbation and imaging of biophysical or biochemical signals (e.g. membrane voltage, Ca2+, or signaling molecules). A core challenge in BDM is precise spatial and temporal alignment of stimulation, imaging, and other experimental parameters. Here we present Luminos, an open-source MATLAB library for modular and precisely synchronized control of BDM experiments. The system supports hardware-triggered synchronization across stimulation, recording, and imaging channels with microsecond accuracy. Source code and documentation for Luminos are available online at https://www.luminosmicroscopy.com and https://github.com/adamcohenlab/luminos-microscopy. This library will facilitate development of bidirectional microscopy methods across the biological sciences.

bioengineering↗

bioRxiv2025

Swimming motions evoke Ca2+ events in vascular endothelial cells of larval zebrafish via mechanical activation of Piezo1

Calcium signaling in blood vessels regulates their growth1,2, immune response3, and vascular tone4. Vascular endothelial cells are known to be mechanosensitive5-7, and it has been assumed that this mechanosensation mediates calcium responses to pulsatile blood flow8-10. Here we show that in larval zebrafish, the dominant trigger for vascular endothelial Ca2+ events comes from body motion, not heartbeat-driven blood flow. Through a series of pharmacological and mechanical perturbations, we showed that body motion is necessary and sufficient to induce endothelial Ca2+ events, while neither neural activity nor blood circulation is either necessary or sufficient. Knockout and temporally restricted knockdown of piezo1 eliminated the motion-induced Ca2+ events. Our results demonstrate that swimming-induced tissue motion is an important driver of endothelial Ca2+ dynamics in larval zebrafish. HighlightsO_LISwimming motions in larval zebrafish evoke large, rapid, and pervasive Ca2+ transients in vascular endothelial cells. C_LIO_LIThese Ca2+ transients do not require neural firing, muscular electrical activity, endocrine factors, or heart-driven blood flow. C_LIO_LIMechanical forces are necessary and sufficient for endothelial Ca2+ transients. C_LIO_LIEndothelial Ca2+ transients require the mechanosensitive ion channel Piezo1. C_LI

physiology↗

bioRxiv2024

Tools for intersectional optical and chemical tagging on cell surfaces

We present versatile tools for intersectional optical and chemical tagging of live cells. Photocaged tetrazines serve as "photo-click" adapters between recognition groups on the cell surface and diverse chemical payloads. We describe two new functionalized photocaged tetrazine structures which add a light-gating step to three common cell-targeting chemical methods: HaloTag/chloroalkane labeling, nonspecific primary amine labeling, and antibody labeling. We demonstrate light-gated versions of these three techniques in live cultured cells. We then explore two applications: monitoring tissue flows on the surface of developing zebrafish embryos, and combinatorial multicolor labeling and sorting of optically defined groups of cells. Photo-click adapters add optical control to cell tagging schemes, with modularity in both tag and cell attachment chemistry.

molecular biology↗

bioRxiv2024

Scalable Fabrication of an Array-Type Fixed-Target Device for Automated Room Temperature X-ray Protein Crystallography

1.X-ray crystallography is one of the leading tools to analyze the 3-D structure, and therefore, function of proteins and other biological macromolecules. Traditional methods of mounting individual crystals for X-ray diffraction analysis can be tedious and result in damage to fragile protein crystals. Furthermore, the advent of serial crystallography methods explicitly require the mounting of large numbers of crystals. To address this need, we have developed a device that facilitates the straightforward mounting of protein crystals for diffraction analysis, and that can be easily manufactured at scale. Inspired by grid-style devices that have been reported in the literature, we have developed an X-ray compatible microfluidic device that can be used to trap protein crystals in an array configuration, while also providing excellent optical transparency, a low X-ray background, and compatibility with the robotic sample handling and environmental controls used at synchrotron macromolecular crystallography beamlines. At the Stanford Synchrotron Radiation Lightsource (SSRL), these capabilities allow for fully remote-access data collection at controlled humidity conditions. Furthermore, we have demonstrated continuous manufacturing of these devices via roll-to-roll fabrication to enable cost-effective and efficient large-scale production.

molecular biology↗

bioRxiv2024

All-optical mapping of Ca2+ transport and homeostasis in dendrites

Calcium mediates many important signals in dendrites. However, the basic transport properties of calcium in dendrites have been difficult to measure: how far and how fast does a local influx of calcium propagate? We developed an all-optical system for simultaneous targeted Ca2+ import and concentration mapping. We co-expressed a blue light-activated calcium selective channelrhodopsin, CapChR2, with a far-red calcium sensor, FR-GECO1c, in cultured rat hippocampal neurons, and used patterned optogenetic stimulation to introduce calcium into cells with user-defined patterns of space and time. We determined a mean steady-state length constant for Ca2+ transport{phi} [~] 5.8 m, a half-life for return to baseline t1/2 [~] 1.7 s, and an effective diffusion coefficient D [~] 20 m2/s, though there were substantial differences in Ca2+ dynamics between proximal and distal dendrites. At high Ca2+ concentration, distal dendrites showed nonlinear activation of Ca2+ efflux, which we pharmacologically ascribed to the NCX1 antiporter. Genetically encoded tools for all-optical study of Ca2+ transport and handling provide a powerful capability for studying this important messenger.

neuroscience↗

bioRxiv2024

Optogenetic control of Nodal signaling patterns

A crucial step in early embryogenesis is the establishment of spatial patterns of signaling activity. Tools to perturb morphogen signals with high resolution in space and time can help reveal how embryonic cells decode these signals to make appropriate fate decisions. Here, we present new optogenetic reagents and an experimental pipeline for creating designer Nodal signaling patterns in live zebrafish embryos. Nodal receptors were fused to the light-sensitive heterodimerizing pair Cry2/CIB1N, and the Type II receptor was sequestered to the cytosol. The improved optoNodal2 reagents eliminate dark activity and improve response kinetics, without sacrificing dynamic range. We adapted an ultra-widefield microscopy platform for parallel light patterning in up to 36 embryos and demonstrated precise spatial control over Nodal signaling activity and downstream gene expression. Patterned Nodal activation drove precisely controlled internalization of endodermal precursors. Further, we used patterned illumination to generate synthetic signaling patterns in Nodal signaling mutants, rescuing several characteristic developmental defects. This study establishes an experimental toolkit for systematic exploration of Nodal signaling patterns in live embryos.

developmental biology↗