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2 Janelia Publications

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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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