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116 Publications

Showing 21-30 of 116 results
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    06/07/21 | Live and Let Dye.
    Lavis LD
    Biochemistry. 2021 Jun 07:. doi: 10.1021/acs.biochem.1c00299

    The measurement of ion concentrations and fluxes inside living cells is key to understanding cellular physiology. Fluorescent indicators that can infiltrate and provide intel on the cellular environment are critical tools for biological research. Developing these molecular informants began with the seminal work of Racker and colleagues ( (1979) 18, 2210), who demonstrated the passive loading of fluorescein in living cells to measure changes in intracellular pH. This work continues, employing a mix of old and new tradecraft to create innovative agents for monitoring ions inside living systems.

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    05/24/21 | A general method to improve fluorophores using deuterated auxochromes.
    Grimm JB, Xie L, Casler JC, Patel R, Tkachuk AN, Falco N, Choi H, Lippincott-Schwartz J, Brown TA, Glick BS, Liu Z, Lavis LD
    JACS Au. 2021 May 24;1(5):690-6. doi: 10.1021/jacsau.1c00006

    Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways—thereby increasing the brightness and photostability of fluorophores—are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.

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    04/01/21 | The HaloTag as a general scaffold for far-red tunable chemigenetic indicators.
    Deo C, Abdelfattah AS, Bhargava HK, Berro AJ, Falco N, Farrants H, Moeyaert B, Chupanova M, Lavis LD, Schreiter ER
    Nature Chemical Biology. 2021 Apr 01:. doi: 10.1038/s41589-021-00775-w

    Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

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    09/14/20 | Novel fluorescent ligands enable single-molecule localization microscopy of the dopamine transporter.
    Guthrie D, Klein Herenbrink C, Lycas M, Ku T, Bonifazi A, DeVree B, Mathiasen S, Javitch J, Grimm JB, Lavis LD, Gether U, Newman AH
    ACS Chemical Neuroscience. 2020 Sep 14:. doi: 10.1021/acschemneuro.0c00397

    The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [3H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF549-conjugated DG3-80 (3) and JF646-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.

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    08/20/20 | Rational design of bioavailable photosensitizers for manipulation and imaging of biological systems.
    Binns TC, Ayala AX, Grimm JB, Tkachuk AN, Castillon GA, Phan S, Zhang L, Brown TA, Liu Z, Adams SR, Ellisman MH, Koyama M, Lavis LD
    Cell Chemical Biology. 2020 Aug 20;27(8):1063-72. doi: 10.1016/j.chembiol.2020.07.001

    Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in vivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.

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    07/27/20 | A general method to optimize and functionalize red-shifted rhodamine dyes.
    Grimm JB, Tkachuk AN, Xie L, Choi H, Mohar B, Falco N, Schaefer K, Patel R, Zheng Q, Liu Z, Lippincott-Schwartz J, Brown TA, Lavis LD
    Nature Methods. 2020 Jul 27:. doi: 10.1038/s41592-020-0909-6

    Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing 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 framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.

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    04/27/20 | Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction.
    Ranjan A, Nguyen VQ, Liu S, Wisniewski J, Kim JM, Tang X, Mizuguchi G, Elalaoui E, Nickels TJ, Jou V, English BP, Zheng Q, Luk E, Lavis LD, Lionnet T, Wu C
    eLife. 2020 Apr 27;9:. doi: 10.7554/eLife.55667

    The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

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    04/01/20 | 3D ATAC-PALM: super-resolution imaging of the accessible genome.
    Xie L, Dong P, Chen X, Hsieh TS, Banala S, De Marzio M, English BP, Qi Y, Jung SK, Kieffer-Kwon K, Legant WR, Hansen AS, Schulmann A, Casellas R, Zhang B, Betzig E, Lavis LD, Chang HY, Tjian R, Liu Z
    Nature Methods. 2020 Apr 01;17(4):430-6. doi: 10.1038/s41592-020-0775-2

    To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA–fluorescence in situ hybridization (FISH), RNA–FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

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    01/22/20 | Accurate measurement of fast endocytic recycling kinetics in real time.
    Jonker CT, Deo C, Zager PJ, Tkachuk AN, Weinstein AM, Rodriguez-Boulan E, Lavis LD, Schreiner R
    Journal of Cell Science. 2020 Jan 22;133(2):. doi: 10.1242/jcs.231225

    The fast turnover of membrane components through endocytosis and recycling allows precise control of the composition of the plasma membrane. Endocytic recycling can be rapid with some molecules returning to the plasma membrane with a <5 minutes. Existing methods to study these trafficking pathways utilize chemical, radioactive, or fluorescent labeling of cell surface receptors in pulse-chase experiments, which require tedious washing steps and manual collection of samples. Here, we introduce a live-cell endocytic recycling assay, based on a newly designed cell-impermeable, fluorogenic ligand for HaloTag: 'Janelia Fluor 635i' (JFi; i=impermeant) which allows real-time detection of membrane receptor recycling at steady state. We used this method to study the effect of iron depletion on transferrin receptor (TfR) recycling using the chelator desferrioxamine. We found this perturbation significantly increases the TfR recycling rate. The high temporal resolution and simplicity of this assay provides a clear advantage over extant methods and makes it ideal for large scale cellular imaging studies. This assay can be adapted to examine other cellular kinetic parameters such as protein turnover and biosynthetic trafficking.

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    01/14/20 | Improved HaloTag Ligand Enables BRET Imaging With NanoLuc
    Thirukkumaran OM, Wang C, Asouzu NJ, Fron E, Rocha S, Hofkens J, Lavis LD, Mizuno H
    Frontiers in Chemistry. 2020 Jan 14;7:. doi: 10.3389/fchem.2019.0093810.3389/fchem.2019.00938.s001