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98 Janelia Publications

Showing 1-10 of 98 results
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    03/29/22 | Bromodomains regulate dynamic targeting of the PBAF chromatin remodeling complex to chromatin hubs.
    Kenworthy CA, Haque N, Liou S, Chandris P, Wong V, Dziuba P, Lavis LD, Liu W, Singer RH, Coleman RA
    Biophysical Journal. 2022 Mar 29:. doi: 10.1016/j.bpj.2022.03.027

    Chromatin remodelers actively target arrays of acetylated nucleosomes at select enhancers and promoters to facilitate or shut down the repeated recruitment of RNA Pol II during transcriptional bursting. It is poorly understood how chromatin remodelers such as PBAF dynamically target different chromatin states inside a live cell. Our live-cell single molecule fluorescence microscopy study reveals chromatin hubs throughout the nucleus where PBAF rapidly cycles on and off the genome. Deletion of PBAF's bromodomains impairs targeting and stable engagement of chromatin in hubs. Dual color imaging reveals that PBAF targets both euchromatic and heterochromatic hubs with distinct genome binding kinetic profiles that mimic chromatin stability. Removal of PBAF's bromodomains stabilizes H3.3 binding within chromatin indicating that bromodomains may play a direct role in remodeling of the nucleosome. Our data suggests that PBAF's dynamic bromodomain mediated engagement of a nucleosome may reflect the chromatin remodeling potential of differentially bound chromatin states.

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    02/28/22 | 2,7-Diaminobenzopyrylium Dyes Are Live-Cell Mitochondrial Stains2,7-Diaminobenzopyrylium Dyes Are Live-Cell Mitochondrial Stains
    Banala S, Tkachuk AN, Patel R, Kumar P, Brown TA, Lavis LD
    ACS Bio & Med Chem Au. 2022 Feb 28:. doi: 10.1021/acsbiomedchemau.1c00068

    Small-molecule fluorescent stains enable the imaging of cellular structures without the need for genetic manipulation. Here, we introduce 2,7-diaminobenzopyrylium (DAB) dyes as live-cell mitochondrial stains excited with violet light. This amalgam of the coumarin and rhodamine fluorophore structures yields dyes with high photostability and tunable spectral properties.

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    02/28/22 | Melding Synthetic Molecules and Genetically Encoded Proteins to Forge New Tools for Neuroscience.
    Kumar P, Lavis LD
    Annual Review of Neuroscience. 2022 Feb 28:. doi: 10.1146/annurev-neuro-110520-030031

    Unraveling the complexity of the brain requires sophisticated methods to probe and perturb neurobiological processes with high spatiotemporal control. The field of chemical biology has produced general strategies to combine the molecular specificity of small-molecule tools with the cellular specificity of genetically encoded reagents. Here, we survey the application, refinement, and extension of these hybrid small-molecule:protein methods to problems in neuroscience, which yields powerful reagents to precisely measure and manipulate neural systems. Expected final online publication date for the , Volume 45 is July 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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    02/28/22 | Melding Synthetic Molecules and Genetically Encoded Proteins to Forge New Tools for Neuroscience.
    Kumar P, Lavis LD
    Annual Review Neuroscience. 2022 Feb 28:. doi: 10.1146/annurev-neuro-110520-030031

    Unraveling the complexity of the brain requires sophisticated methods to probe and perturb neurobiological processes with high spatiotemporal control. The field of chemical biology has produced general strategies to combine the molecular specificity of small-molecule tools with the cellular specificity of genetically encoded reagents. Here, we survey the application, refinement, and extension of these hybrid small-molecule:protein methods to problems in neuroscience, which yields powerful reagents to precisely measure and manipulate neural systems. Expected final online publication date for the , Volume 45 is July 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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    02/07/22 | The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes.
    Courtney KC, Wu L, Mandal T, Swift M, Zhang Z, Alaghemandi M, Wu Z, Bradberry MM, Deo C, Lavis LD, Volkmann N, Hanein D, Cui Q, Bao H, Chapman ER
    Nature Structural & Molecular Biology. 2022 Feb 07;29(2):97-107. doi: 10.1038/s41594-021-00716-0

    Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.

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    02/01/22 | Caveat fluorophore: an insiders' guide to small-molecule fluorescent labels.
    Grimm JB, Lavis LD
    Nature Methods. 2022 Feb 01;19(2):149-58. doi: 10.1038/s41592-021-01338-6

    The last three decades have brought a revolution in fluorescence microscopy. The development of new microscopes, fluorescent labels and analysis techniques has pushed the frontiers of biological imaging forward, moving from fixed to live cells, from diffraction-limited to super-resolution imaging and from simple cell culture systems to experiments in vivo. The large and ever-evolving collection of tools can be daunting for biologists, who must invest substantial time and effort in adopting new technologies to answer their specific questions. This is particularly relevant when working with small-molecule fluorescent labels, where users must navigate the jargon, idiosyncrasies and caveats of chemistry. Here, we present an overview of chemical dyes used in biology and provide frank advice from a chemist's perspective.

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    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.

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    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.

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    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.

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    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.

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