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4 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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    01/01/20 | Aurora B functions at the apical surface after specialized cytokinesis during morphogenesis in C. elegans.
    Bai X, Melesse M, Sorensen Turpin CG, Sloan D, Chen C, Wang W, Lee P, Simmons JR, Nebenfuehr B, Mitchell D, Klebanow LR, Mattson N, Betzig E, Chen B, Cheerambathur D, Bembenek JN
    Development. 2020 Jan;147(1):1-16. doi: 10.1242/dev.181099

    While cytokinesis has been intensely studied, the way it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant embryonic divisions and found several reproducibly altered parameters at different stages. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we found several unexpected alterations to cytokinesis including apical midbody migration in polarizing epithelial cells of the gut, pharynx and sensory neurons. Aurora B kinase, which is essential for several aspects of cytokinesis, remains apically localized in each of these tissues after internalization of midbody ring components. Aurora B inactivation disrupts cytokinesis and causes defects in apical structures, even if inactivated post-mitotically. Therefore, cytokinesis is implemented in a specialized way during epithelial polarization and Aurora B has a new role in the formation of the apical surface.

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    01/17/20 | Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.
    Hoffman DP, Shtengel G, Xu CS, Campbell KR, Freeman M, Wang L, Milkie DE, Pasolli HA, Iyer N, Bogovic JA, Stabley DR, Shirinifard A, Pang S, Peale D, Schaefer K, Pomp W, Chang C, Lippincott-Schwartz J, Kirchhausen T, Solecki DJ, Betzig E, Hess HF
    Science. 2020 Jan 17;367(6475):. doi: 10.1126/science.aaz5357

    Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

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    01/11/20 | Tiled reconstruction improves structured illumination microscopy.
    Hoffman DP, Betzig E
    bioRxiv. 2020 Jan 11:. doi: 10.1101/2020.01.06.895318

    Structured illumination microscopy (SIM) is widely used for fast, long-term, live-cell super-resolution imaging. However, SIM images can contain substantial artifacts if the sample does not conform to the underlying assumptions of the reconstruction algorithm. Here we describe a simple, easy to implement, process that can be combined with any reconstruction algorithm to alleviate many common SIM reconstruction artifacts and briefly discuss possible extensions.

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