Main Menu (Mobile)- Block

Main Menu - Block

janelia7_blocks-janelia7_secondary_menu | block
janelia7_blocks-janelia7_fake_breadcrumb | block
Lavis Lab / Publications
general_search_page-panel_pane_1 | views_panes

11 Publications

Showing 1-10 of 11 results
Your Criteria:
    Lavis LabLooger Lab
    12/23/20 | Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.
    Unger EK, Keller JP, Altermatt M, Liang R, Matsui A, Dong C, Hon OJ, Yao Z, Sun J, Banala S, Flanigan ME, Jaffe DA, Hartanto S, Carlen J, Mizuno GO, Borden PM, Shivange AV, Cameron LP, Sinning S, Underhill SM, Olson DE, Amara SG, Temple Lang D, Rudnick G, Marvin JS, Lavis LD, Lester HA, Alvarez VA, Fisher AJ, Prescher JA, Kash TL, Yarov-Yarovoy V, Gradinaru V, Looger LL, Tian L
    Cell. 2020 Dec 23;183(7):1986-2002.e26. doi: 10.1016/j.cell.2020.11.040

    Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

    View Publication Page
    11/05/21 | Open Chemistry: What if we just give everything away?
    Lavis LD
    eLife. 2021 Nov 05;10:. doi: 10.7554/eLife.74981

    A group leader decided that his lab would share the fluorescent dyes they create, for free and without authorship requirements. Nearly 12,000 aliquots later, he reveals what has happened since.

    View Publication Page
    10/28/21 | Biosensors based on peptide exposure show single molecule conformations in live cells.
    Liu B, Stone OJ, Pablo M, Herron JC, Nogueira AT, Dagliyan O, Grimm JB, Lavis LD, Elston TC, Hahn KM
    Cell. 2021 Oct 28;184(22):5670-5685. doi: 10.1016/j.cell.2021.09.026

    We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.

    View Publication Page
    09/28/21 | A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility
    Shu-Hsien Sheu , Srigokul Upadhyayula , Vincent Dupuy , Song Pang , Andrew L. Lemire , Deepika Walpita , H. Amalia Pasolli , Fei Deng , Jinxia Wan , Lihua Wang , Justin Houser , Silvia Sanchez-Martinez , Sebastian E. Brauchi , Sambashiva Banala , Melanie Freeman , C. Shan Xu , Tom Kirchhausen , Harald F. Hess , Luke Lavis , Yu-Long Li , Séverine Chaumont-Dubel , David E. Clapham
    bioRxiv. 2021 Sep 28:

    Chemical synapses between axons and dendrites mediate much of the brain’s intercellular communication. Here we describe a new kind of synapse – the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam – scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

    View Publication Page
    09/02/21 | Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.
    Nguyen VQ, Ranjan A, Liu S, Tang X, Ling YH, Wisniewski J, Mizuguchi G, Li KY, Jou V, Zheng Q, Lavis LD, Lionnet T, Wu C
    Molecular Cell. 2021 Sep 02;81(17):3560-3575. doi: 10.1016/j.molcel.2021.07.022

    Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.

    View Publication Page
    09/01/21 | Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes.
    Timothy A. Brown , Katherine S. Schaefer , Arthur Tsang , Hyun Ah Yi , Jonathan B. Grimm , Andrew L. Lemire , Fadi M. Jradi , Charles Kim , Kevin McGowan , Kimberly Ritola , Derek T. Armstrong , Heba H. Mostafa , Wyatt Korff , Ronald D. Vale , Luke D. Lavis
    Journal of Biomolecular Techniques. 2021 Sep 01;32(3):121-133. doi: https://doi.org/10.1101/2020.12.26.20248878

    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 ‘LAMPshade’ 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.

    View Publication Page
    07/27/21 | Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin.
    Kim JM, Visanpattanasin P, Jou V, Liu S, Tang X, Zheng Q, Li KY, Snedeker J, Lavis LD, Lionnet T, Wu C
    eLife. 2021 Jul 27;10:. doi: 10.7554/eLife.69387

    Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending 'tug-of-war' that controls a temporally shifting window of accessibility for the transcription initiation machinery.

    View Publication Page
    07/21/21 | Enabling In Vivo Photocatalytic Activation of Rapid Bioorthogonal Chemistry by Repurposing Silicon-Rhodamine Fluorophores as Cytocompatible Far-Red Photocatalysts.
    Wang C, Zhang H, Zhang T, Zou X, Wang H, Rosenberger JE, Vannam R, Trout WS, Grimm JB, Lavis LD, Thorpe C, Jia X, Li Z, Fox JM
    Journal of the American Chemical Society. 2021 Jul 21;143(28):10793-10803. doi: 10.1021/jacs.1c05547

    Chromophores that absorb in the tissue-penetrant far-red/near-infrared window have long served as photocatalysts to generate singlet oxygen for photodynamic therapy. However, the cytotoxicity and side reactions associated with singlet oxygen sensitization have posed a problem for using long-wavelength photocatalysis to initiate other types of chemical reactions in biological environments. Herein, silicon-Rhodamine compounds (SiRs) are described as photocatalysts for inducing rapid bioorthogonal chemistry using 660 nm light through the oxidation of a dihydrotetrazine to a tetrazine in the presence of cyclooctene dienophiles. SiRs have been commonly used as fluorophores for bioimaging but have not been applied to catalyze chemical reactions. A series of SiR derivatives were evaluated, and the Janelia Fluor-SiR dyes were found to be especially effective in catalyzing photooxidation (typically 3%). A dihydrotetrazine/tetrazine pair is described that displays high stability in both oxidation states. A protein that was site-selectively modified by cyclooctene was quantitatively conjugated upon exposure to 660 nm light and a dihydrotetrazine. By contrast, a previously described methylene blue catalyst was found to rapidly degrade the protein. SiR-red light photocatalysis was used to cross-link hyaluronic acid derivatives functionalized by dihydrotetrazine and cyclooctenes, enabling 3D culture of human prostate cancer cells. Photoinducible hydrogel formation could also be carried out in live mice through subcutaneous injection of a Cy7-labeled hydrogel precursor solution, followed by brief irradiation to produce a stable hydrogel. This cytocompatible method for using red light photocatalysis to activate bioorthogonal chemistry is anticipated to find broad applications where spatiotemporal control is needed in biological environments.

    View Publication Page
    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.

    View Publication Page
    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.

    View Publication Page