Biology subjects

Hayward, R. F.

Publications and source records attributed to Hayward, R. F..

7 recordsLinked to original sources

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↗

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

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

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

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

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↗