Biology subjects

Brown, T. A.

Publications and source records attributed to Brown, T. A..

15 recordsLinked to original sources

bioRxiv2026

iG6PSnFR: A genetically encoded fluorescent sensor for observing glucose-6-phosphate dynamics in living preparations

Glucose-6-phosphate (G6P) is a key intermediate in multiple energetic and anabolic pathways, and quantifying its dynamics is essential for understanding cellular physiology. We have previously developed a syndicate of intensity-based, genetically encoded sensors based on the insertion of circularly permuted GFP into a Venus-flytrap-like analyte-binding protein. Here we use the same approach to develop an intensity-based G6P Sensing Fluorescent Reporter (iG6PSnFR). We present two variants: a G6P-activated sensor that increases fluorescence and a G6P-inactivated sensor that decreases fluorescence. We validate performance across progressively more complex preparations, including purified protein in vitro, immortalized and primary neuronal cultures, isolated pancreatic islets, in an intravital liver model, and finally in vivo in C. elegans neurons. In each context, iG6PSnFR reports G6P changes consistent with expected responses to physiological perturbations.

biochemistry↗

bioRxiv2025

A sensitive orange fluorescent calcium ion indicator for imaging neural activity

Genetically encoded calcium indicators (GECIs) are vital tools for fluorescence-based visualization of neuronal activity with high spatial and temporal resolution. However, current highest-performance GECIs are predominantly green or red fluorescent, limiting multiplexing options and efficient excitation with fixed-wavelength femtosecond lasers operating at 1030 nm. Here, we introduce OCaMP (also known as O-GECO2), an orange fluorescent GECI engineered from O-GECO1 through targeted substitutions to improve calcium affinity while retaining the favorable photophysical properties of mOrange2. OCaMP exhibits improved two-photon cross-section, responsiveness, photostability, and calcium affinity relative to O-GECO1. In cultured neurons, zebrafish, and mouse cortex, OCaMP outperforms the red GECIs jRCaMP1a and jRGECO1a in sensitivity, kinetics, and signal-to-noise ratio. These properties establish OCaMP as a robust tool for high-fidelity neural imaging optimized for 1030 nm excitation and a compromise-free option within the spectral gap between existing green and red GECIs.

neuroscience↗

bioRxiv2024

The loss of nuptial gifts in sclerosomatid Opiliones coincides with increased behavioural sexual conflict

Nuptial gifts serve to increase donor fitness through a variety of pathways. Some nuptial gifts deliver benefits similar to those of antagonistic male behaviours: functioning to secure additional copulations, increase sperm transfer or storage, or increase paternity share. These commonalities may result in evolutionary transitions between solicitous and coercive strategies, wherein behavioural sexual conflict could function to secure mates in lieu of nuptial gifts. In temperate leiobunine harvesters (Arachnida: Opiliones), nuptial gifts have been repeatedly lost, resulting in two primary mating syndromes: an ancestral, sacculate state in which males endogenously produce high-investment nuptial gifts and females lack pregenital barriers, and a derived, nonsacculate state in which females have pregenital barriers and males produce significantly reduced, low-investment nuptial gifts. In this study, we investigated whether behavioural sexual conflict compensates for reduced nuptial gifts in nonsacculate harvesters by comparing the intensity of pre-, peri-, and postcopulatory mating behaviour between the nonsacculate species Leiobunum vittatum and L. euserratipalpe and the sacculate species L. aldrichi and L. bracchiolum. We additionally sought to establish an automated behavioural analysis pipeline by developing analogues for metrics traditionally scored manually. Our results revealed significantly different, potentially coercive, behaviour in nonsacculate species, indicating that the loss and reduction of pre- and pericopulatory nuptial gifts may contribute to increased behavioural antagonism. Mating behaviour also differed significantly between L. vittatum and L. euserratipalpe, indicating there are multiple suites of potentially antagonistic behaviours. Together, these results suggest that multiple behavioural strategies may be effective substitutes for nuptial gifts in leiobunine Opiliones, although the mechanisms through which male fitness is increased requires further research. HighlightsO_LITemperate Opiliones have repeatedly evolved reduced nuptial gift investment C_LIO_LIReduced-gift (nonsacculate) species possess morphology indicative of antagonism C_LIO_LIWe tested if mating behaviour differed between nonsacculate and sacculate species C_LIO_LINonsacculates displayed significantly higher behavioural antagonism than sacculates C_LIO_LINonsacculate species differed in their suites of antagonistic behaviours C_LI

animal behavior and cognition↗

bioRxiv2024

Coumarin as a general switching auxiliary to prepare photochromic and spontaneously blinking fluorophores

Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of "glitter bomb" labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho-carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.

cell biology↗

bioRxiv2024

A series of spontaneously blinking dyes for super-resolution microscopy

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

cell biology↗

bioRxiv2024

A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions

The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.

cell biology↗

bioRxiv2024

Transforming chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers using chemistry

Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.

biochemistry↗

bioRxiv2023

iATPSnFR2: a high dynamic range fluorescent sensor for monitoring intracellular ATP

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted super-folder GFP inserted between the ATP-binding helices of the{varepsilon} -subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a [~] 5-6 fold improvement in the dynamic range compared to the previous generation sensor, with excellent discrimination against other analytes and affinity variants varying from 4 M to 500 M. A chimeric version of this sensor fused to either the HaloTag protein or a suitably spectrally separated fluorescent protein, provides a ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting of the sensor to nerve terminals reveals previously uncharacterized single synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics. Significance StatementAdenosine triphosphate (ATP) is a key metabolite necessary for cellular life. Here we develop a next-generation genetically encoded ratiometric fluorescent ATP sensor that allows subcellular tracking of ATP levels in living cells. The large dynamic range makes it possible to follow the dynamics of this metabolite across cells and subcellular regions under different metabolic stressors. We expect that iATPSnFR2 will provide researchers with exciting new opportunities to study ATP dynamics with temporal and spatial resolution that has, until now, been unavailable.

biochemistry↗

bioRxiv2023

An engineered biosensor enables dynamic aspartate measurements in living cells

Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a GFP-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by mass spectrometry and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high throughput applications of variables that affect aspartate levels.

biochemistry↗

bioRxiv2022

Brain-wide measurement of protein turnover with high spatial and temporal resolution

Synaptic plasticity underlies learning and memory by altering neuronal connections in response to experiences1. However, the loci of learning-induced synaptic plasticity, and the degree to which plasticity is localized or distributed, remain largely unknown2. We developed a method (DELTA) for mapping brain-wide changes in synaptic protein turnover with single-synapse resolution, based on Janelia Fluor dyes and HaloTag knock-in mice. During associative learning, the turnover of the ionotropic glutamate receptor GluA2, an indicator of synaptic plasticity, was enhanced in several brain regions, most markedly in the hippocampal area CA1. More broadly distributed increases in turnover of synaptic proteins were observed in response to environmental enrichment. In CA1, GluA2 stability was regulated in an input specific manner, with more turnover in layers containing input from CA3 compared to entorhinal cortex. DELTA will facilitate exploration of the molecular and circuit basis of learning and memory and other forms of adaptive and maladaptive plasticity at scales ranging from single synapses to the entire brain.

neuroscience↗

bioRxiv2020

Deuteration improves small-molecule fluorophores

Fluorescence microscopy relies on dyes that absorb short-wavelength photons and emit longer-wavelength light. In addition to this fluorescence process, dyes can undergo other photochemical reactions that result in spectral shifts and irreversible photobleaching. Increases in brightness, chromostability, and photostability of fluorescent dyes are therefore crucial for advancing the frontier of bioimaging. Here, we describe a general approach to improve small-molecule fluorophores using deuteration. Incorporating deuterium into the alkylamino substituents of rhodamines and other dyes improves fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching. These compounds are easily synthesized and show improved performance in cellular imaging experiments.

cell biology↗

bioRxiv2019

Optimization and functionalization of red-shifted rhodamine dyes

Expanding the palette of fluorescent dyes is vital for pushing the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore due to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups.

cell biology↗

bioRxiv2018

Comment on ‘Multiple Origin but Single Domestication Led to Oryza sativa’

In 2015, we published an analysis of rice genomic data and showed that the japonica, indica and aus groups of cultivated rice were independently domesticated. Our conclusions were controversial as they contradicted a previous, high-profile analysis of the same dataset, which had suggested that all of cultivated rice derives from a single origin. Although there have been attempts since 2015 to bolster the single-origin hypothesis, until recently there has been no direct rebuttal of the methodology that we used to infer multiple origins. Such a rebuttal has now been published (Choi J.Y., Purugganan M.D., 2018 Multiple origin but single domestication led to Oryza sativa. G3 8:797-803), but the reanalysis that is presented only supports the single origin hypothesis if phylogenetic trees that are clearly paraphyletic are interpreted as monophyletic, and furthermore addresses only one component of the evidence that we presented for multiple domestications. We caution against accepting these analyses uncritically.

evolutionary biology↗

bioRxiv2017

A general method to fine-tune fluorophores for live-cell and in vivo imaging

AO_SCPCAPBSTRACTC_SCPCAPPushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. Here, we refine and extend this strategy, showing that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties with unprecedented precision. This strategy yields a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red with utility in live cells, tissue, and animals.

cell biology↗

bioRxiv2016

Diversity patterns across 1,800 chloroplast genomes of wild (Oryza rufipogon Griff.) and cultivated rice (O. sativa L.)

Cultivated Asian rice (O. sativa L.) comprises several groups with distinct ecological requirements and culinary uses. While the two subspecies of O. sativa - indica and japonica - have been subjected to a multitude of genetic and genomic analyses, less is known about the origins and diversity of the agronomically marginal groups - aus and aromatic rice. Here we reconstructed complete chloroplast genomes of over 1,800 accessions of wild and cultivated rice, including 240 aus and 73 aromatic varieties, and analysed the haplotype diversity of the taxonomic groups. We confirm the deep phylogenetic divergence between the main chloroplast haplotypes of japonica and indica, and reveal unique profiles of chloroplast diversity in aus and aromatic rice. Our results indicate that the latter two groups are not simple derivatives of indica and japonica, respectively, but originated from independent and/or reticulate domestication processes. Absence of phylogeographic patterns in the wild distribution of chloroplast haplogroups did not allow firm conclusions about geographic origins and the role of inter-group gene flow. Nonetheless, our results suggest that the domestication of indica, japonica, aus and aromatic rice operated on genetically different gene pools and followed different dynamics.

genomics↗