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119 Publications

Showing 21-30 of 119 results
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    06/15/22 | 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 Jun 15;2(3):307-12. 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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    05/03/22 | 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 May 3;121(9):1738-1752. 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 | 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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    02/01/22 | Organization of translating secretome mRNAS on endoplasmic reticulum
    Choi H, Liao Y, Yoon YJ, Grimm J, Lavis LD, Singer RH, Lippincott-Schwartz J
    Biophysical Journal. 2022 Feb 01;121(3):33a. doi: 10.1016/j.bpj.2021.11.2550

    The endoplasmic reticulum (ER) has a complex morphology comprised of stacked sheets, tubules, and three-way junctions, which together function as a platform for protein synthesis of membrane and secretory proteins. Specific ER subdomains are thought to be spatially organized to enable protein synthesis activity, but precisely where these domains are localized is unclear, especially relative to the plethora of organelle interactions taking place on the ER. Here, we use single-molecule tracking of ribosomes and mRNA in combination with simultaneous imaging of ER to assess the sites of membrane protein synthesis on the ER. We found that ribosomes were widely distributed throughout different ER morphologies, but the synthesis of membrane proteins (including Type I, II, and multi-spanning) and an ER luminal protein (Calreticulin) occurred primarily at three-way junctions. Lunapark played a key role in stabilizing transmembrane protein mRNA at three-way junctions. We additionally found that translating mRNAs coding for transmembrane proteins are in the vicinity of lysosomes and translate through a cap-independent but eIF2-dependent mechanism. These results support the idea that discrete ER subdomains co-exist with lysosomes to support specific types of protein synthesis activities, with ER-lysosome interactions playing an important role in the translation of secretome mRNAs.

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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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    02/01/22 | Molecular cartography: charting the sea of molecular organization in live synapses with nanoscale precision
    Nelson AJ, Zheng Q, Lavis LD, Ryan TA
    Biophysical Journal. 2022 Feb 01;121(3):302a. doi: 10.1016/j.bpj.2021.11.1246

    Understanding live-cell behavior in part requires high precision mapping of molecular species in 3-D dynamic environments. Approaches like single-molecule localization microscopy (SMLM) offer high promise for challenges posed by molecular cartography. Effectively, the precision of these approaches is dependent on the how many photons / second a fluorescent marker is capable of emitting. For this reason, many SRLM experiments are typically done using fluorescent organic dyes (such as Alexa Fluors) in reducing chemical environments which cause some organic dyes to stochastically cycle through dark states, allowing single-molecule localization (e.g. (d)STORM). The need to couple these dyes to antibodies and the harsh reducing conditions makes their application to live cell work problematic. To overcome these limitations, we made use of modifications to Janelia Fluor-based dyes which make them spontaneously cycle through dark states (blink) under physiological imaging conditions. The dyes are spectrally compatible with photo-activatable fluorescent proteins such as mEos and allow for simultaneous 2-color superresolution microscopy. When conjugated to a HaloTag, these artificial dyes can bind genetically encodable targets in live samples, allowing subsequent measurement in a live-cell environment. To correct for nanoscale chromatic aberrations we developed a new machine-learning based approach with reconstruction errors below achievable localization precisions. We show that these methods allow the reconstruction of live synapse surfaces and a variety of the associated molecular machineries with up to 50 nm accuracy in 3 dimensions.

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    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.

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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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