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Chromosoma

, Volume 123, Issue 3, pp 303–312 | Cite as

ELCS in ice: cryo-electron microscopy of nuclear envelope-limited chromatin sheets

  • Mikhail Eltsov
  • Sergey Sosnovski
  • Ada L. Olins
  • Donald E. OlinsEmail author
Research Article

Abstract

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.

Keywords

Chromatin structure Cryo-electron microscopy High-pressure/freeze substitution Aldehyde fixation Envelope-limited chromatin sheets (ELCS) 30-nm chromatin fibers 

Notes

Acknowledgments

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.

Ethical standards

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.

Supplementary material

412_2014_454_MOESM1_ESM.mpg (4.9 mb)
Online Resource 1 Video of electron microscope tomographic computed X/Y slices from chemically fixed granulocytic HL-60/S4 cells, emphasizing cross-sections of ELCS. The video displays sequential slices (∼1.2 nm thick) along the Z-axis. The image frame size (i.e., x and y lengths) is 2.56 μm (MPG 5046 kb)
Online Resource 2

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)

412_2014_454_MOESM3_ESM.mpg (12.7 mb)
Online Resource 3 Video of an isosurface representation from a stack of tomographic slices of ELCS, first oriented to display a cross sectional view and then turned to display logitudinal views of the chromatin fibers running tangential to the inner nuclear membrane. Scale bar 100 nm (MPG 13000 kb)
412_2014_454_MOESM4_ESM.mpg (10.2 mb)
Online Resource 4 Video of an isosurface representation from a stack of tomographic slices of ELCS, oriented to display logitudinal views of the chromatin fibers running tangential to the inner nuclear membrane. Scale bar 100 nm (MPG 10461kb)
412_2014_454_MOESM5_ESM.pdf (1.1 mb)
Online Resource 5 Evaluation of the influence of compression on the INM to INM distance. a, The compression during vitreous sectioning affects the structure in an orientation dependent manner. While the dimension of the object perpendicular to the cutting direction is preserved, the dimensions tilted or parallel are shortened (Woodcock 1994; Al-Amoudi et al. 2005). To access the effect of compression on the INMs distance within ELCS, we measured the angle (α) between two vectors, with a pivot point located in one of the INM. The first vector points the direction of knife marks (km) from the pivot point, revealing the cutting direction. The second vector connects the pivot point with the closest point of the apposed INM, providing a local measurement of the distance between INMs (d). b, The distance between INMs was plotted as a function of sin (α). Fitting the distance measurements with a linear regression line reveals an average uncompressed INM distance, at sin (α) = 1, equal to 57.4 nm. (PDF 1076 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Mikhail Eltsov
    • 1
  • Sergey Sosnovski
    • 2
  • Ada L. Olins
    • 3
  • Donald E. Olins
    • 3
    Email author
  1. 1.Cell Biology and Biophysics UnitEuropean Molecular Biology LaboratoryHeidelbergGermany
  2. 2.Neurophysiology & New Microscopies LaboratoryINSERM U603 - CNRS UMR 8154ParisFrance
  3. 3.Department of Pharmaceutical Sciences, College of PharmacyUniversity of New EnglandPortlandUSA

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