@article {68969, title = {iATPSnFR2: a high dynamic range fluorescent sensor for monitoring intracellular ATP}, journal = {Proc Natl Acad Sci U S A}, year = {2024}, month = {2024 May 21}, abstract = {

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\ ε-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 μM to 500 μM. 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.

}, doi = {10.1073/pnas.2314604121 }, author = {Jonathan S. Marvin and Alexandros C. Kokotos and Mukesh Kumar and Camila Pulido and Ariana N. Tkachuk and Jocelyn Shuxin Yao and Timothy A. Brown and Timothy A. Ryan} } @article {69217, title = {A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions.}, journal = {bioRxiv}, year = {2024}, month = {2024 Feb 16}, abstract = {

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.

}, doi = {10.1101/2024.02.15.580492}, url = {https://doi.org/10.1101/2024.02.15.580492}, author = {Ryan J. Farrell and Kirsten G. Bredvik and Michael B. Hoppa and S. Thomas Hennigan and Timothy A. Brown and Timothy A. Ryan} } @article {69227, title = {A series of spontaneously blinking dyes for super-resolution microscopy}, journal = {bioRxiv}, year = {2024}, month = {2024 Feb 24}, abstract = {

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.

}, doi = {10.1101/2024.02.23.581625}, url = {https://www.biorxiv.org/content/early/2024/02/24/2024.02.23.581625}, author = {Katie L. Holland and Sarah E. Plutkis and Timothy A. Daugird and Abhishek Sau and Jonathan B. Grimm and Brian P. English and Qinsi Zheng and Sandeep Dave and Fariha Rahman and Liangqi Xie and Peng Dong and Ariana N. Tkachuk and Timothy A. Brown and Robert H. Singer and Zhe Liu and Catherine G. Galbraith and Siegfried M. Musser and Wesley R. Legant and Luke D. Lavis} } @article {69144, title = {Transforming chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers using chemistry.}, journal = {bioRxiv}, year = {2024}, month = {2024 Jan 01}, abstract = {

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.

}, doi = {10.1101/2023.12.30.573644}, url = {https://doi.org/10.1101/2023.12.30.573644}, author = {Pratik Kumar and Alina Gutu and Amelia Waring and Timothy A. Brown and Luke D. Lavis and Alison G. Tebo} } @article {68503, title = {Brain-wide measurement of protein turnover with high spatial and temporal resolution}, journal = {bioRxiv}, year = {2022}, month = {2022 Nov 13}, abstract = {

Cells regulate function by synthesizing and degrading proteins. This turnover ranges from minutes to weeks, as it varies across proteins, cellular compartments, cell types, and tissues. Current methods for tracking protein turnover lack the spatial and temporal resolution needed to investigate these processes, especially in the intact brain, which presents unique challenges. We describe a pulse-chase method (DELTA) for measuring protein turnover with high spatial and temporal resolution throughout the body, including the brain. DELTA relies on rapid covalent capture by HaloTag of fluorophores that were optimized for bioavailability\ in vivo. The nuclear protein MeCP2 showed brain region- and cell type-specific turnover. The synaptic protein PSD95 was destabilized in specific brain regions by behavioral enrichment. A novel variant of expansion microscopy further facilitated turnover measurements at individual synapses. DELTA enables studies of adaptive and maladaptive plasticity in brain-wide neural circuits.

}, doi = {10.1101/2022.11.12.516226}, url = {https://doi.org/10.1101/2022.11.12.516226}, author = {Boaz Mohar and Jonathan B. Grimm and Ronak Patel and Timothy A. Brown and Paul Tillberg and Luke D. Lavis and Nelson Spruston and Karel Svoboda} } @article {65970, title = {Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes.}, journal = {Journal of Biomolecular Techniques}, volume = {32}, year = {2021}, month = {2021 Sep 01}, pages = {121-133}, abstract = {

The worldwide COVID-19 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce two pH sensitive {\textquoteleft}LAMPshade{\textquoteright} dyes as novel readouts in an isothermal RT- LAMP amplification assay for SARS-CoV-2 RNA. The resulting JaneliaLAMP (jLAMP) assay is robust, simple, inexpensive, has low technical requirements and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.

}, doi = {https://doi.org/10.1101/2020.12.26.20248878}, author = {Timothy A. Brown and Katherine S. Schaefer and Arthur Tsang and Hyun Ah Yi and Jonathan B. Grimm and Andrew L. Lemire and Fadi M. Jradi and Charles Kim and Kevin McGowan and Kimberly Ritola and Derek T. Armstrong and Heba H. Mostafa and Wyatt Korff and Ronald D. Vale and Luke D. Lavis} }