Analysis of chromatin fibers in Hela cells with electron tomography

Biophys Rep 2015, 1(1):51–60 DOI 10.1007/s41048-015-0009-9 Biophysics Reports R E S E A RC H A RT I C L E Analysis of chromatin fibers in Hela cells with electron tomography Xiaomin Li1,3, Hongli Feng1, Jianguo Zhang2, Lei Sun2, Ping Zhu1& 1 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2 Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 3 University of Chinese Academy of Sciences, Beijing 100049, China Received: 9 March 2015 / Accepted: 12 April 2015 / Published online: 7 August 2015 Abstract The presence and folding pattern of chromatin in eukaryotic cells remain elusive and controversial. In this study, we prepared ultra-thin sections of Hela cells with three different fixation and sectioning methods, i.e., chemical fixation, high pressure freezing with freeze substitution, and cryo-ultramicrotomy with SEM-FIB (focused ion beam), and analyzed in vivo architecture of chromatin fibers in Hela nuclei with electron tomography technology. The results suggest that the chromatin fibers in eukaryotic Hela cells are likely organized in an architecture with a diameter of about 30 nm. Keywords Chromatin fiber, Hela cell, Electron tomography, Chemical fixation, High-pressure freezing, Cryoultramicrotomy, DualBeam-FIB INTRODUCTION The folding of chromatin in eukaryotic cells is closely related to the genetic transcription, replication and repair (Horn and Peterson 2002; Luger et al. 1997). Packaging of DNA in eukaryotic cells is hierarchical. The linear ‘‘beads-on-string’’ arrangement of nucleosomes, which is formed by histone octamers (H2A:H2B: H3:H4 = 2:2:2:2) (Luger et al. 1997) wrapped by DNA, is regarded as the first level arrangement of chromatin (Huynh et al. 2005). Although the nucleosome had been structurally characterized by X-ray crystallography at 1.9 Å (Davey et al. 2002), how polynucleosomes are folded into 30-nm chromatin fibers, which are typically regarded as the secondary structure of DNA, is inconclusive. Based on the early studies of chromatin in different cells using a variety of methods (Bednar et al. 1995; Daban 2011; Gerchman and Ramakrishnan 1987; Grigoryev and Woodcock 2012; Kruithof et al. 2009; Xiaomin Li and Hongli Feng have contributed equally to this work. & Correspondence: (P. Zhu) Robinson and Rhodes 2006; Schalch et al. 2005; Simpson and Stafford 1983; Widom et al. 1985; William et al. 1986; William and Langmore 1991), researchers had proposed two major types of model for the secondary chromatin structure, i.e., ‘‘one-start’’ solenoid model and ‘‘two-start’’ zig-zag model (Finch and Klug 1976; Horowitz et al. 1994; Robinson et al. 2006). Recently, using cryo-electron microscopy single particle analysis, we reconstructed the 3D structure of in vitro reconstituted 30-nm chromatin fibers at 11 Å resolution and found that chromatin fibers with two different nucleosome repeat lengths (NRLs, 12 9 177 and 12 9 187 bp) present a left-handed double helix structure (Song et al. 2014), which represents a considerable advance on the structure characteristics of chromatin fibers. However, the existence of 30-nm chromatin fibers in the nuclei of eukaryotic cells is still remained to be elucidated in vivo (Eltsov et al. 2008). Extensive studies have been made previously on the organization of native chromatin fibers, including those in starfish sperm nuclei (Giannasca et al. 1993; Horowitz et al. 1994), chicken erythrocyte nuclei (Langmore and Ó The Author(s) 2015. This article is published with open access at Springerlink.com 51 | August 2015 | Volume 1 | Issue 1 RESEARCH ARTICLE X. Li et al. Paulson 1983; Woodcock et al. 1984), Thyone briareus (sea cucumber) sperm, Necturus maculosus (mud-puppy) erythrocytes (Athey et al. 1990; William et al. 1986; Woodcock 1994), and in other cells (Davies et al. 1974; Derenzini et al. 2014; Eltsov et al. 2014; Everid et al. 1970; Fakan and van Driel 2007; Fussner et al. 2011; Konig et al. 2007; Matsuda et al. 2010). An electron tomography (ET) study showed that continuously variable zig-zag nucleosomal ribbons could be observed in chicken erythrocyte nuclei, both in the native form in situ and in the isolated form (Horowitz et al. 1994). Nevertheless, the samples used in that study were chemically fixed, dehydrated, embedded in resin, and stained by heavy metal. It was argued that the results could be attributed to the probable structure rearrangement and surrounding background staining artifacts (Eltsov et al. 2008). To visualize the close-to-native chromatin in vivo, techniques with a better preservation of the native status of the nuclei, i.e., high-pressure freezing, cryo-sectioning, and cryo-electron tomography, are necessary (Scheffer et al. 2011). However, even with a vitrified sectioning of cells and the contrast transfer function (CTF) correction on the electron microscopic images, it is difficult to visualize the high-order structure of 30-nm chromatin fibers in situ (Eltsov et al. 2008; McDowall et al. 1986). In this study, we performed ET analysis to visualize the native chromatin arrangement in vivo, by taking three different sample preparation methods, i.e., ultrathin-sectioning with chemical fixation, ultrathin-sectioning with high pressure freezing and freeze substitution, and plunge-freezing with focused ion beam (FIB) cryo-sectioning. Among them, the ultrathin-sectioning with chemical fixation, embedding in resin, and chemical staining provides good contrast for electron microscopy imaging. Both high-pressure freezing and plunge-freezing can preserve the frozen-hydrated sample at cryo-temperatures without dehydration and keep the sample in a close-to-native state (Scheffer et al. 2011). The FIB method is a novel alternative to cryoultramicrotomy for thinning of frozen-hydrated biological specimens, which has brought a lot of attentions due to its peculiar advantages (Rigort et al. 2010). ET is a useful technology that has the ability to obtain 3D architectures of both homogeneous and heterogeneous samples (Scheffer et al. 2011). In particular, cryo-electron tomography has the ability to visualize the molecular assemblies in the unaltered frozen-hydrated state at reasonably high resolution. Here, we tried to explore the architecture of chromatin fibers in Hela cells in situ by combining all of these technologies. The results suggest that chromatins are likely present in the nuclei of Hela cells with an architecture of fibers with a diameter of about 30 nm. 52 | August 2015 | Volume 1 | Issue 1 RESULTS AND DISCUSSION EM analysis of 30-nm chromatin fibers in Hela S3 cells and isolated nuclei It is well recognized that the isolated chromatins from chicken erythrocyte nuclei present a fiberic form in width of *30 nm (Scheffer et al. 2011). For the Hela S3 cells, the arrangement of 30-nm fibers had also been observed in the isolated chromatins (Langmore and Paulson 1983). 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