Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.

Autor: Hoffman DP; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Shtengel G; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Xu CS; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Campbell KR; Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA., Freeman M; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Wang L; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.; Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA., Milkie DE; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Pasolli HA; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Iyer N; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Bogovic JA; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Stabley DR; Neuroimaging Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA., Shirinifard A; Bioimage Analysis Core, St. Jude Children's Research Hospital, Memphis, TN 38105, USA., Pang S; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Peale D; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Schaefer K; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Pomp W; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.; Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA., Chang CL; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Lippincott-Schwartz J; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA., Kirchhausen T; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.; Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA., Solecki DJ; Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA., Betzig E; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. hessh@janelia.hhmi.org betzige@janelia.hhmi.org.; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.; Department of Physics, University of California, Berkeley, CA 94720, USA.; Howard Hughes Medical Institute, Berkeley, CA 94720, USA.; Helen Wills Neuroscience Institute, Berkeley, CA 94720, USA.; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA., Hess HF; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. hessh@janelia.hhmi.org betzige@janelia.hhmi.org.
Jazyk: angličtina
Zdroj: Science (New York, N.Y.) [Science] 2020 Jan 17; Vol. 367 (6475).
DOI: 10.1126/science.aaz5357
Abstrakt: 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.
(Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
Databáze: MEDLINE
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