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90 Publications

Showing 1-10 of 90 results
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
01/09/20 | Bright and tunable far-red chemigenetic indicators.
Deo C, Abdelfattah AS, Bhargava HK, Berro AJ, Falco N, Moeyaert B, Chupanova M, Lavis LD, Schreiter ER
bioRxiv. 2020 Jan 9:
01/06/20 | Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging.
Zhang Y, Schroeder LK, Lessard MD, Kidd P, Chung J, Song Y, Benedetti L, Li Y, Ries J, Grimm JB, Lavis LD, De Camilli P, Rothman JE, Baddeley D, Bewersdorf J
Nature Methods. 2020 Jan 06;17(2):225-231. doi: 10.1038/s41592-019-0676-4

Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.

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09/25/19 | Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging.
Zheng Q, Ayala AX, Chung I, Weigel AV, Ranjan A, Falco N, Grimm JB, Tkachuk AN, Wu C, Lippincott-Schwartz J, Singer RH, Lavis LD
ACS Central Science. 2019 Sep 25;5(9):1602-1613. doi: 10.1021/acscentsci.9b00676

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant () is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

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09/04/19 | Isomeric tuning yields bright and targetable red Ca indicators.
Deo C, Sheu S, Seo J, Clapham DE, Lavis LD
Journal of the American Chemical Society. 2019 Sep 04;141(35):13734-13738. doi: 10.1021/jacs.9b06092

Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca indicator by systematically exploring the relative configuration of dye and chelator, which can be targeted using the HaloTag self-labeling tag system. Our "isomeric tuning" approach is generalizable, yielding a far-red targetable indicator to visualize Ca fluxes in the primary cilium.

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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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07/11/19 | Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding.
Boersma S, Khuperkar D, Verhagen BM, Sonneveld S, Grimm JB, Lavis LD, Tanenbaum ME
Cell. 2019 Jul 11;178(2):458-72. doi: 10.1016/j.cell.2019.05.001

mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous, as multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a new fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real-time. We find that start site selection is largely stochastic, but that the probability of using a particular start site differs among mRNA molecules, and can be dynamically regulated over time. Together, this study provides key insights into translation start site selection heterogeneity, and provides a powerful toolbox to visualize complex translation dynamics.

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