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33 Janelia Publications

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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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    07/21/20 | 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 Jul 21:. 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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    07/20/20 | Microdomains form on the luminal face of neuronal extracellular vesicle membranes.
    Matthies D, Lee NY, Gatera I, Pasolli HA, Zhao X, Liu H, Walpita D, Liu Z, Yu Z, Ioannou MS
    Scientific Reports. 2020 Jul 20;10(1):11953. doi: 10.1038/s41598-020-68436-x

    Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology.

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    06/22/20 | Two-parameter mobility assessments discriminate diverse regulatory factor behaviors in chromatin.
    Lerner J, Gomez-Garcia PA, McCarthy RL, Liu Z, Lakadamyali M, Zaret KS
    Molecular Cell. 2020 Jun 22:. doi: 10.1016/j.molcel.2020.05.036

    Enzymatic probes of chromatin structure reveal accessible versus inaccessible chromatin states, while super-resolution microscopy reveals a continuum of chromatin compaction states. Characterizing histone H2B movements by single-molecule tracking (SMT), we resolved chromatin domains ranging from low to high mobility and displaying different subnuclear localizations patterns. Heterochromatin constituents correlated with the lowest mobility chromatin, whereas transcription factors varied widely with regard to their respective mobility with low- or high-mobility chromatin. Pioneer transcription factors, which bind nucleosomes, can access the low-mobility chromatin domains, whereas weak or non-nucleosome binding factors are excluded from the domains and enriched in higher mobility domains. Nonspecific DNA and nucleosome binding accounted for most of the low mobility of strong nucleosome interactor FOXA1. Our analysis shows how the parameters of the mobility of chromatin-bound factors, but not their diffusion behaviors or SMT-residence times within chromatin, distinguish functional characteristics of different chromatin-interacting proteins.

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    06/17/20 | Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells.
    Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y, Nakano K, Saitoh N, Liu Z, Yamamoto T, Okamura T, Ohkawa Y, Kimura H, Nikaido I
    Science Advances. 2020 Jun 17;6(25):eaaz6699. doi: 10.1126/sciadv.aaz6699

    Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

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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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    12/23/19 | MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions.
    Piccolo FM, Liu Z, Dong P, Hsu C, Stoyanova EI, Rao A, Tjian R, Heintz N
    eLife. 2019 Dec 23;8:. doi: 10.7554/eLife.51449

    Methyl-CpG-binding-Protein 2 (MeCP2) is an abundant nuclear protein highly enriched in neurons. Here we report live-cell single-molecule imaging studies of the kinetic features of mouse MeCP2 at high spatial-temporal resolution. MeCP2 displays dynamic features that are distinct from both highly mobile transcription factors and immobile histones. Stable binding of MeCP2 in living neurons requires its methyl-binding domain and is sensitive to DNA modification levels. Diffusion of unbound MeCP2 is strongly constrained by weak, transient interactions mediated primarily by its AT-hook domains, and varies with the level of chromatin compaction and cell type. These findings extend previous studies of the role of the MeCP2 MBD in high affinity DNA binding to living neurons, and identify a new role for its AT-hooks domains as critical determinants of its kinetic behavior. They suggest that limited nuclear diffusion of MeCP2 in live neurons contributes to its local impact on chromatin structure and gene expression.

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    12/18/19 | Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression.
    Cai D, Feliciano D, Dong P, Flores E, Gruebele M, Porat-Shliom N, Sukenik S, Liu Z, Lippincott-Schwartz J
    Nature Cell Biology. 2019 Dec;21(12):1578-1589. doi: 10.1038/s41556-019-0433-z

    Yes-associated protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a select set of enhancers for target gene activation. How YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized the YAP transcription factor TEAD1 and other YAP-related co-activators, including TAZ, and subsequently induced the transcription of YAP-specific proliferation genes. Super-resolution imaging using assay for transposase-accessible chromatin with photoactivated localization microscopy revealed that the YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA polymerase II, the accessible chromatin domains later acquired RNA polymerase II, transcribing RNA. The removal of the intrinsically-disordered YAP transcription activation domain prevented the formation of YAP condensates and diminished downstream YAP signalling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid-liquid phase separation.

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    09/01/19 | A neuron-glia Co-culture system for studying intercellular lipid transport.
    Ioannou MS, Liu Z, Lippincott-Schwartz J
    Curr Protoc Cell Biol. 2019 Sep 01;84(1):e95. doi: 10.1002/cpcb.95

    Neurons and glia operate in a highly coordinated fashion in the brain. Although glial cells have long been known to supply lipids to neurons via lipoprotein particles, new evidence reveals that lipid transport between neurons and glia is bidirectional. Here, we describe a co-culture system to study transfer of lipids and lipid-associated proteins from neurons to glia. The assay entails culturing neurons and glia on separate coverslips, pulsing the neurons with fluorescently labeled fatty acids, and then incubating the coverslips together. As astrocytes internalize and store neuron-derived fatty acids in lipid droplets, analyzing the number, size, and fluorescence intensity of lipid droplets containing the fluorescent fatty acids provides an easy and quantifiable measure of fatty acid transport. © 2019 The Authors.

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    08/13/19 | Bright and photostable chemigenetic indicators for extended in vivo voltage imaging.
    Abdelfattah AS, Kawashima T, Singh A, Novak O, Liu H, Shuai Y, Huang Y, Campagnola L, Seeman SC, Yu J, Zheng J, Grimm JB, Patel R, Friedrich J, Mensh BD, Paninski L, Macklin JJ, Murphy GJ, Podgorski K, Lin B, Chen T, Turner GC, Liu Z, Koyama M, Svoboda K, Ahrens MB, Lavis LD, Schreiter ER
    Science. 2019 Aug 13;365(6454):699-704. doi: 10.1126/science.aav6416

    Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, "Voltron", that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 min of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

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