ELCS in ice: cryo-electron microscopy of nuclear envelope-limited chromatin sheets
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Nuclear envelope-limited chromatin sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ∼30 nm, containing a layer of parallel chromatin fibers. In this study, the ultrastructure of ELCS was compared by three different methods: (1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining, (2) high-pressure freezing/freeze substitution into plastic/sectioning and staining, and (3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. ELCS could be clearly visualized by all three methods and, consequently, must exist in vivo and are not fixation artifacts. The ∼30-nm chromatin fibers could only be observed following aldehyde fixation; none were seen in cryo-sections. Electron microscopic tomography tangential views of aldehyde-fixed ELCS suggested an ordering of the separate chromatin fibers adjacent to the nuclear envelope. Possible mechanisms of this chromatin ordering are discussed.
KeywordsChromatin structure Cryo-electron microscopy High-pressure/freeze substitution Aldehyde fixation Envelope-limited chromatin sheets (ELCS) 30-nm chromatin fibers
The authors express their appreciation to the European Molecular Biology Laboratory (EMBL, Heidelberg), the German Cancer Research Center (DKFZ, Heidelberg), and the University of New England (Portland) for their support and encouragement of these studies. We particularly wish to thank Peter Lichter, Harald Herrmann (DKFZ), and Jörg Langowski (DKFZ), who generously opened their laboratories to ALO and DEO. The authors also express their gratitude to Mary Morphew and Andreas Hoenger (Boulder, CO), and Rachel Santarella-Mellwig (EMBL). The Boulder Laboratory for 3-D Electron Microscopy of Cells is supported by the National Center for Research Resources, NIH.
The authors of this manuscript declare that all experiments comply with the current laws of the country in which they were performed.
Conflict of interest
The authors declare that they have no conflict of interest.
Video of electron microscope tomographic computed X/Y slices from chemically fixed granulocytic HL-60/S4 cells, emphasizing cross-sections of ELCS. One slice of this tomogram is shown in Fig. 6a. The video displays sequential slices (∼1.2 nm thick) along the Z-axis. The image frame sizes (i.e., x and y lengths) are 1.50 and 0.79 μm (MPG 4319 kb)
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