Mikhail Eltsov

Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ

Although the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resolution imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resolution observation of the cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chemical treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally corrected. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt.

Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure

How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30‐nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30‐nm chromatin fibres. Our comprehensive and quantitative study using cryo‐electron microscopy and synchrotron X‐ray scattering resolved the long‐standing contradictions regarding the existence of 30‐nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.

Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei

Chromatin folding in eukaryotes fits the genome into the limited volume of the cell nucleus. Formation of higher-order chromatin structures attenuates DNA accessibility, thus contributing to the control of essential genome functions such as transcription, DNA replication, and repair. The 30-nm fiber is thought to be the first hierarchical level of chromatin folding, but the nucleosome arrangement in the compact 30-nm fiber was previously unknown. We used cryoelectron tomography of vitreous sections to determine the structure of the compact, native 30-nm fiber of avian erythrocyte nuclei. The predominant geometry of the 30-nm fiber revealed by subtomogram averaging is a left-handed two-start helix with approximately 6.5 nucleosomes per 11 nm, in which the nucleosomes are juxtaposed face-to-face but are shifted off their superhelical axes with an axial translation of approximately 3.4 nm and an azimuthal rotation of approximately 54°. The nucleosomes produce a checkerboard pattern when observed in the direction perpendicular to the fiber axis but are not interdigitated. The nucleosome packing within the fibers shows larger center-to-center internucleosomal distances than previously anticipated, thus excluding the possibility of core-to-core interactions, explaining how transcription and regulation factors can access nucleosomes.

Packaging the genome: the structure of mitotic chromosomes.

Mitotic chromosomes are essential structures for the faithful transmission of duplicated genomic DNA into two daughter cells during cell division. Although more than 100 years have passed since chromosomes were first observed, it remains unclear how a long string of genomic DNA is packaged into compact mitotic chromosomes. Although the classical view is that human chromosomes consist of radial 30 nm chromatin loops that are somehow tethered centrally by scaffold proteins, called condensins, cryo-electron microscopy observation of frozen hydrated native chromosomes reveals a homogeneous, grainy texture and neither higher-order nor periodic structures including 30 nm chromatin fibres were observed. As a compromise to fill this huge gap, we propose a model in which the radial chromatin loop structures in the classic view are folded irregularly toward the chromosome centre with the increase in intracellular cations during mitosis. Consequently, compact native chromosomes are made up primarily of irregular chromatin networks cross-linked by self-assembled condensins forming the chromosome scaffold.

Visualization of cell microtubules in their native state

Background information. Over the past decades, cryo‐electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy.

How to “Read” a Vitreous Section

Publisher Summary Cryoelectron microscopy concerns the observation of hydrated specimen at a temperature so low that water does not evaporate significantly in the microscopes vacuum. This chapter addressed various scientists confronted with CEMOVIS data. It explains the ways to read micrographs, to interpret them, and to understand what they can and cannot provide. It is only recently that cryoelectron microscopy of vitreous sections (CEMOVIS) has been established as a practical method. CEMOVIS results in images that are much closer to the native state, and they provide higher resolution than those obtained from conventional sections. In the absence of any staining and with all the water present as immobilized liquid, CEMOVIS gives a faithful representation of the native state. What is seen on the image is the real distribution of the biological material within the thickness of the section. The global contrast between different regions of the micrograph is proportional to the density difference in the corresponding regions of the specimen.

Fine Structure of the Deinococcus radiodurans Nucleoid Revealed by Cryoelectron Microscopy of Vitreous Sections

Transmission electron microscopy revealed that the nucleoid of the extremely radioresistant bacteria Deinococcus radiodurans may adopt an unusual ring shape. This led to the hypothesis that the tight toroidal package of the D. radiodurans genome might contribute to radioresistance by preventing diffusion of ends of double-stranded DNA breaks. The molecular arrangement of DNA in the nucleoid, which must be determined to test this hypothesis, is not discernible by conventional methods of electron microscopy. We have applied cryoelectron microscopy of vitreous sections and found that the DNA arrangement in D. radiodurans differs from toroidal spooling. Diffuse coralline nucleoids of exponentially growing D. radiodurans do not reveal any particular molecular order. Electron-dense granules are generally observed in the centers of nucleoids. In stationary-phase cells, the nucleoid segregates from cytoplasm and DNA filaments show locally parallel arrangements, with increasing aspects of cholesteric liquid crystalline phase upon prolonged starvation. The relevance of the observed nucleoid organization to the radiation resistance of D. radiodurans is discussed.

Heritable yeast prions have a highly organized three-dimensional architecture with interfiber structures

Yeast prions constitute a “protein-only” mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI+], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits. Two factors make these traits heritable: (i) the amyloid conformation is self-templating; and (ii) the protein-remodeling factor heat-shock protein (Hsp)104 (acting together with Hsp70 chaperones) partitions the template to daughter cells with high fidelity. Prions formed by several other yeast proteins create their own phenotypes but share the same mechanistic basis of inheritance. Except for the amyloid fibril itself, the cellular architecture underlying these protein-based elements of inheritance is unknown. To study the 3D arrangement of prion assemblies in their cellular context, we examined yeast [PSI+] prions in the native, hydrated state in situ, taking advantage of recently developed methods for cryosectioning of vitrified cells. Cryo–electron tomography of the vitrified sections revealed the prion assemblies as aligned bundles of regularly spaced fibrils in the cytoplasm with no bounding structures. Although the fibers were widely spaced, other cellular complexes, such as ribosomes, were excluded from the fibril arrays. Subtomogram image averaging, made possible by the organized nature of the assemblies, uncovered the presence of an additional array of densities between the fibers. We suggest these structures constitute a self-organizing mechanism that coordinates fiber deposition and the regulation of prion inheritance.

Quantitative analysis of cytoskeletal reorganization during epithelial tissue sealing by large-volume electron tomography

The closure of epidermal openings is an essential biological process that causes major developmental problems such as spina bifida in humans if it goes awry. At present, the mechanism of closure remains elusive. Therefore, we reconstructed a model closure event, dorsal closure in fly embryos, by large-volume correlative electron tomography. We present a comprehensive, quantitative analysis of the cytoskeletal reorganization, enabling separated epidermal cells to seal the epithelium. After establishing contact through actin-driven exploratory filopodia, cells use a single lamella to generate ‘roof tile’-like overlaps. These shorten to produce the force, ‘zipping’ the tissue closed. The shortening overlaps lack detectable actin filament ensembles but are crowded with microtubules. Cortical accumulation of shrinking microtubule ends suggests a force generation mechanism in which cortical motors pull on microtubule ends as for mitotic spindle positioning. In addition, microtubules orient filopodia and lamellae before zipping. Our 4D electron microscopy picture describes an entire developmental process and provides fundamental insight into epidermal closure.

Study of the Deinococcus radiodurans Nucleoid by Cryoelectron Microscopy of Vitreous Sections: Supplementary Comments

Recently, we reported results of the structural analysis of Deinococcus radiodurans nucleoid observed by cryoelectron microscopy of vitreous sections (CEMOVIS) (15). We were able to visualize the arrangement of DNA molecules in the nucleoid of this extraordinary radioresistant bacterium. In previous work, Minsky and colleagues proposed that the nucleoid of D. radiodurans is organized as a densely packed DNA toroid. According to them, this structure could play a crucial role in the radiation resistance of D. radiodurans and, potentially, other species of Deinococcaceae (16, 24). According to our observations, the DNA arrangement in the nucleoid of D. radiodurans Sark differed from a dense toroidal spooling, suggesting that the model of Minsky and colleagues is not generally correct for this bacterium. Soon after publication, our article faced criticism from Dr. Minsky. His major points, as we understand them, were the following: (i) the quality of the images obtained by our method is insufficient to draw any serious conclusions and (ii) there was inadequate discussion of some of our results. We attribute these criticisms to the newness of our technique and to the attractiveness of his toroidal model, which our results do not support. We see that supplementary explanation and discussion are necessary. Below we explain why the micrographs of vitreous sections we have presented are faithful and detailed representations of native biological material but must be interpreted in a manner different than that for conventionally fixed, stained, and embedded sections. Furthermore, we discuss several aspects of our results in relation to what has previously been published by other researchers. CEMOVIS Micrographs obtained by CEMOVIS must be viewed using criteria different from those we have been accustomed to using during 50 years of electron microscopy of stained and dry specimens. We will try to convince the reader that the micrographs we present contain more, and more reliable, information than that obtained from resin-embedded sections. We will also explain why the cutting artifacts inherent to CEMOVIS are well controlled in good sections; they do not impair image interpretation.