TomoFlow: Analysis of Continuous Conformational Variability of Macromolecules in Cryogenic Subtomograms based on 3D Dense Optical Flow.

Cryogenic Electron Tomography (cryo-ET) allows structural and dynamics studies of macromolecules in situ. Averaging different copies of imaged macromolecules is commonly used to obtain their structure at higher resolution and discrete classification to analyze their dynamics. Instrumental and data processing developments are progressively equipping cryo-ET studies with the ability to escape the trap of classification into a complete continuous conformational variability analysis. In this work, we propose TomoFlow, a method for analyzing macromolecular continuous conformational variability in cryo-ET subtomograms based on a three-dimensional dense optical flow (OF) approach. The resultant lower-dimensional conformational space allows generating movies of macromolecular motion and obtaining subtomogram averages by grouping conformationally similar subtomograms. The animations and the subtomogram group averages reveal accurate trajectories of macromolecular motion based on a novel mathematical model that makes use of OF properties. This paper describes TomoFlow with tests on simulated datasets generated using different techniques, namely Normal Mode Analysis and Molecular Dynamics Simulation. It also shows an application of TomoFlow on a dataset of nucleosomes in situ, which provided promising results coherent with previous findings using the same dataset but without imposing any prior knowledge on the analysis of the conformational variability. The method is discussed with its potential uses and limitations.

HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes.

Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (in situ). Its potential of providing information on the dynamics of macromolecular complexes in cells is still largely unexploited, due to the challenges of the data analysis. The crowded cell environment and continuous conformational changes of complexes make difficult disentangling the data heterogeneity. We present HEMNMA-3D, which is, to the best of our knowledge, the first method for analyzing cryo electron subtomograms in terms of continuous conformational changes of complexes. HEMNMA-3D uses a combination of elastic and rigid-body 3D-to-3D iterative alignments of a flexible 3D reference (atomic structure or electron microscopy density map) to match the conformation, orientation, and position of the complex in each subtomogram. The elastic matching combines molecular mechanics simulation (Normal Mode Analysis of the 3D reference) and experimental, subtomogram data analysis. The rigid-body alignment includes compensation for the missing wedge, due to the limited tilt angle of cryo-ET. The conformational parameters (amplitudes of normal modes) of the complexes in subtomograms obtained through the alignment are processed to visualize the distribution of conformations in a space of lower dimension (typically, 2D or 3D) referred to as space of conformations. This allows a visually interpretable insight into the dynamics of the complexes, by calculating 3D averages of subtomograms with similar conformations from selected (densest) regions and by recording movies of the 3D reference's displacement along selected trajectories through the densest regions. We describe HEMNMA-3D and show its validation using synthetic datasets. We apply HEMNMA-3D to an experimental dataset describing in situ nucleosome conformational variability. HEMNMA-3D software is available freely (open-source) as part of ContinuousFlex plugin of Scipion V3.0 (http://scipion.i2pc.es).

Correlative Light and Electron Microscopy (CLEM) Analysis of Nuclear Reorganization Induced by Clustered DNA Damage Upon Charged Particle Irradiation.

Chromatin architecture plays major roles in gene regulation as well as in the repair of DNA damaged by endogenous or exogenous factors, such as after radiation. Opening up the chromatin might provide the necessary accessibility for the recruitment and binding of repair factors, thus facilitating timely and correct repair. The observed formation of ionizing radiation-induced foci (IRIF) of factors, such as 53BP1, upon induction of DNA double-strand breaks have been recently linked to local chromatin decompaction. Using correlative light and electron microscopy (CLEM) in combination with DNA-specific contrasting for transmission electron microscopy or tomography, we are able to show that at the ultrastructural level, these DNA damage domains reveal a chromatin compaction and organization not distinguishable from regular euchromatin upon irradiation with carbon or iron ions. Low Density Areas (LDAs) at sites of particle-induced DNA damage, as observed after unspecific uranyl acetate (UA)-staining, are thus unlikely to represent pure chromatin decompaction. RNA-specific terbium-citrate (Tb) staining suggests rather a reduced RNA density contributing to the LDA phenotype. Our observations are discussed in the view of liquid-like phase separation as one of the mechanisms of regulating DNA repair.

Nucleosome conformational variability in solution and in interphase nuclei evidenced by cryo-electron microscopy of vitreous sections.

In Eukaryotes, DNA is wound around the histone octamer forming the basic chromatin unit, the nucleosome. Atomic structures have been obtained from crystallography and single particle cryo-electron microscopy (cryoEM) of identical engineered particles. But native nucleosomes are dynamical entities with diverse DNA sequence and histone content, and little is known about their conformational variability, especially in the cellular context. Using cryoEM and tomography of vitreous sections we analyse native nucleosomes, both in vitro, using purified particles solubilized at physiologically relevant concentrations (25-50%), and in situ, within interphase nuclei. We visualize individual nucleosomes at a level of detail that allows us to measure the distance between the DNA gyres wrapped around. In concentrated solutions, we demonstrate a salt-dependent transition, with a high salt compact conformation resembling the canonical nucleosome and an open low salt one, closer to nuclear nucleosomes. Although further particle characterization and cartography are needed to understand the relationship between this conformational variability and chromatin functional states, this work opens a route to chromatin exploration in situ.

Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope.

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM.

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.

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

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.

Facilitated aggregation of FG nucleoporins under molecular crowding conditions.

Intrinsically disordered and phenylalanine-glycine-rich nucleoporins (FG Nups) form a crowded and selective transport conduit inside the NPC that can only be transited with the help of nuclear transport receptors (NTRs). It has been shown in vitro that FG Nups can assemble into two distinct appearances, amyloids and hydrogels. If and how these phenomena are linked and if they have a physiological role still remains unclear. Using a variety of high-resolution fluorescence and electron microscopic (EM) tools, we reveal that crowding conditions mimicking the NPC environment can accelerate the aggregation and amyloid formation speed of yeast and human FG Nups by orders of magnitude. Aggregation can be inhibited by NTRs, providing a rationale on how the cell might control amyloid formation of FG Nups. The superb spatial resolving power of EM also reveals that hydrogels are enlaced amyloid fibres, and these findings have implications for existing transport models and for NPC assembly.

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.

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.

Nucleosomes stacked with aligned dyad axes are found in native compact chromatin in vitro.

In this study, electron tomograms of plunge-frozen isolated chromatin in both open and compacted form were recorded. We have resolved individual nucleosomes in these tomograms in order to provide a 3D view of the arrangement of nucleosomes within chromatin fibers at different compaction states. With an optimized template matching procedure we obtained accurate positions and orientations of nucleosomes in open chromatin in "low-salt" conditions (5 mM NaCl). The mean value of the planar angle between three consecutive nucleosomes is 70°, and the mean center-to-center distance between consecutive nucleosomes is 22.3 nm. Since the template matching approach was not effective in crowded conditions, for nucleosome detection in compact fibers (40 mM NaCl and 1 mM MgCl(2)) we developed the nucleosome detection procedure based on the watershed algorithm, followed by sub-tomogram alignment, averaging, and classification by Principal Components Analysis. We find that in compact chromatin the nucleosomes are arranged with a predominant face-to-face stacking organization, which has not been previously shown for native isolated chromatin. Although the path of the DNA cannot be directly seen in compact conditions, it is evident that the nucleosomes stack with their dyad axis aligned in forming a "double track" conformation which is a consequence of DNA joining adjacent nucleosome stacks. Our data suggests that nucleosome stacking is an important mechanism for generating chromatin compaction in vivo.

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.

Chromatin structure: does the 30-nm fibre exist in vivo?

A long strand of DNA is wrapped around the core histone and forms a nucleosome. Although the nucleosome has long been assumed to be folded into 30-nm chromatin fibres, their structural details and how such fibres are organised into a nucleus or mitotic chromosome remain unclear. When we observed frozen hydrated (vitrified) human mitotic cells using cryo-electron microscopy, which enables direct high-resolution imaging of the cellular structures in a close-to-native state, we found no higher order structures including 30-nm chromatin fibres in the chromosome. Therefore, we propose that the nucleosome fibres exist in a highly disordered, interdigitated state like a 'polymer melt' that undergoes dynamic movement. We postulate that a similar state exists in active interphase nuclei, resulting in several advantages in the transcription and DNA replication processes.

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.

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. RESULTS: We used cryo-electron microscopy and cryo-electron tomography of vitreous sections to investigate the ultrastructure of microtubules in their cellular context. Vitreous sections were obtained from organotypic slices of rat hippocampus and from Chinese-hamster ovary cells in culture. Microtubules revealed their protofilament ultrastructure, polarity and, in the most favourable cases, molecular details comparable with those visualized in three-dimensional reconstructions of microtubules reassembled in vitro from purified tubulin. The resolution of the tomograms was estimated to be approx. 4 nm, which enabled the detection of luminal particles of approx. 6 nm in diameter inside microtubules. CONCLUSIONS: The present study provides a first step towards a description of microtubules, in addition to other macromolecular assemblies, in an unperturbed cellular context at the molecular level. As the resolution appears to be similar to that obtainable with plunge-frozen samples, it should allow for the in vivo identification of larger macromolecular assemblies in vitreous sections of whole cells and tissues.

Transmission electron microscopy of the bacterial nucleoid.

Water-containing biological material cannot withstand the vacuum of the transmission electron microscope. The classical solution to this problem has been to dehydrate chemically fixed biological samples and then embed them in resin. During such treatment, the bacterial nucleoid is especially prone to aggregation, which affects its global shape and fine structure. Initial attempts to deal with aggregation by optimizing chemical fixation yielded contradictory results. Two decades ago, the situation improved with the introduction of freeze-substitution. This method is based on dehydration of unfixed cryo-immobilized samples at low temperature, which substantially reduces aggregation. As a result, the global shape of the nucleoid can be fairly well defined. Overall, in actively growing bacteria, the nucleoids are dispersed and "coralline" but become more confined when growth ceases. However, it is usually impossible to determine the molecular arrangement of DNA in the nucleoids of freeze-substituted bacteria because crystallization and the subsequent removal of water during substitution result in unavoidable distortions at the ultrastructural level. Recently, cryo-electron microscopy of vitreous sections has enabled the fully hydrated bacterial nucleoid to be studied close to the native state. Such studies have revealed aspects of bacterial nucleoid organization that are not preserved by freeze-substitution, including locally parallel or twisted bundles of DNA filaments, which are more frequently observed once bacterial growth has stopped, whereas in actively growing bacteria, the DNA is seen to be in a mostly disordered pattern.

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.