Direct visualization of HIV-1 with correlative live-cell microscopy and cryo-electron tomography

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection.     

Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells

Cryo-electron tomography (cryo-ET) allows for the visualization of biological material in a close-to-native state, in three dimensions and with nanometer scale resolution. However, due to the low signal-to-noise ratio inherent to imaging of the radiation-sensitive frozen-hydrated samples, it appears oftentimes impossible to localize structures within heterogeneous samples. Because a major potential for cryo-ET is thereby left unused, we set out to combine cryo-ET with cryo-fluorescence microscopy (cryo-FM), in order to facilitate the search for structures of interest. We describe a cryo-FM setup and workflow for correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) that can be easily implemented. Cells are grown on finder grids, vitally labeled with one or two fluorescent dyes, and vitrified. After a structure is located by cryo-FM (with 0.4 μm resolution), its image coordinates are translated to cryo-ET stage coordinates via a home-built software routine. We tested our workflow on whole mount primary human umbilical vein endothelial cells. The correlative routine enabled us to investigate mitochondrial ultrastructure for the first time on intact human mitochondria, and led us to find mitochondrial cristae that were connected to the intermembrane space via large slits, which challenges the current view that such connections are established exclusively via small circular pores. Taken together, this study emphasizes that cryo-CLEM can be a routinely used technique that opens up exciting new possibilities for cryo-ET.     

Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography

Cryo-electron tomography of frozen-hydrated biological samples offers a means of studying large and complex cellular structures in three-dimensions and with nanometer-scale resolution. The low contrast of unstained biological material embedded in amorphous ice and the need to minimise the exposure of these radiation-sensitive samples to the electron beam result in a poor signal-to-noise ratio. This poses problems not only in the visualisation and interpretation of such tomograms, it is also a problem in surveying the sample and in finding regions which contain the features of interest and which are suitable for recording tomograms. To address this problem, we have developed a correlative fluorescence light microscopy-electron microscopy approach, which guides the search for the structures of interest and allows electron microscopy to zoom in on them. With our approach, the total dose spent on locating regions of interest is negligible. A newly designed cryo-holder allows imaging of fluorescently labelled samples after vitrification. The absolute coordinates of structures identified and located by cryo-light microscopy are transferred to the electron microscope via a Matlab-based user interface. We have successfully tested the experimental setup and the whole procedure with two types of adherent fluorescently labelled cells, a neuronal cell line and keratinocytes, both grown directly on EM grids.     

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy

Gammaherpesviruses, including the human pathogens Epstein–Barr virus and Kaposi’s sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral–host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection.     

Immunological synapses and neuronal synapses

The interface between two cells from the immune system has recently been coined "immunological synapse". The authors review recent findings concerning the structure of the synapse formed between T lymphocytes and antigen-presenting cells. T cells can be part of different synapses, depending on the antigen-presenting cell (B cell hybridoma, proteo-lipid bilayer, macrophage, dendritic cell). The synapse formed with dendritic cells is discussed in more details. A comparison is made with the synapses from the nervous system. Several parallel questions are discussed: how receptors can be clustered, what is the influence of synapse functioning on the structure of the synapse. It is suggested that in both cases two modes of communication exist in parallel: direct cell-cell contacts and soluble mediators, neurotransmitters in one case, putative immunotransmitters in the other.     

Multiscale imaging of neurons grown in culture: from light microscopy to cryo-electron tomography

Cryo-electron tomography (cryo-ET) allows the visualization of supramolecular architecture in cells preserved in a close-to-physiological state. In order to supplement the structural information obtained by cryo-ET with the functional state of the molecules involved based on fluorescent labeling we developed a method of correlating light microscopy and cryo-ET. This method is suitable for investigating complicated cellular landscapes such as mature neurons grown in culture. It has the advantage that a correlation is obtained without exposing a feature of interest to additional electron irradiation, and that it does not rely on visual recognition of features. Different modes of correlation are presented here: a feature identified on a light microscopy image is used to guide the cryo-ET investigation, and cryo-tomograms are correlated to light microscopy images. Cryo-tomograms of a neuronal synapse and of an isolated presynaptic terminal are shown as examples of the correlative method. The correlation method presented here can be expected to provide new insights into the structure-function relationship of supramolecular organization in neurons.     

Ribosomal dynamics explored by cryo-electron microscopy

Cryo-electron microscopy has furnished direct evidence for conformational changes of the ribosome as it proceeds, in a cyclic manner, through different functional states. Strategies to explore the ribosome dynamics include trapping of particular functionally meaningful states by chemical, genetic, or physical means. The new atomic information obtained by X-ray crystallography should make it possible to track conformational changes observed by cryo-electron microscopy to changes of the underlying structural framework of the ribosome.     

Conformational changes studied by cryo-electron microscopy

Biological processes involving movement, such as muscle contraction or the opening of an ion channel through a membrane, are mediated through conformational changes. These changes often occur in large and flexible macromolecular complexes. Cryo-electron microscopy provides a means of capturing different conformational states of such assemblies. Even if the resulting density maps are at low resolution, they can be combined with atomic structures of subcomplexes or isolated components determined by X-ray crystallography or NMR. This review presents a brief summary of the principles and recent advances in macromolecular structure determination by cryo-electron microscopy.     

Dynamics of antibodies from cryo-electron tomography

The issue of protein dynamics and its implications in the biological function of proteins are arousing greater and greater interest in molecular biology. In cryo-electron tomography experiments one takes several snapshots of a given biological macromolecule. In principle, a large enough collection of snapshots may then be used to calculate its equilibrium configuration in terms of the experimentally accessible degrees of freedom, and hence estimate its potential energy. Consequently, one could analyze the biological functions of biomolecules by directly accessing their dynamics. In this work, we analyze the results of cryo-electron tomography experiments on monoclonal murine IgG2a antibodies. With the aid of a novel software for image processing, we measure the equilibrium distribution of the angles which describe the configuration of the molecule. This helps us shed some critical light on recent results from X-ray crystallography. We then build a model of the antibody dynamics, which enables us to use the measured angular distribution in order to derive an explicit expression of the IgG potential energy. Finally, as a preliminary application of our results, we investigate the dynamical effects in the rate of formation of the antigen-antibody encounter complex. In particular, we suggest that the dynamics of antibodies operates in the direction of decreasing anticooperativity of the two antigen binding arms.     

Golgi apparatus analyzed by cryo-electron microscopy

In 1898, the Golgi apparatus was discovered by light microscopy, and since the 1950s, the ultrastructure composition is known by electron microscopic investigation. The complex three-dimensional morphology fascinated researchers and was sometimes even the driving force to develop novel visualization techniques. However, the highly dynamic membrane systems of Golgi apparatus are delicate and prone to fixation artifacts. Therefore, the understanding of Golgi morphology and its function has been improved significantly with the development of better preparation methods. Nowadays, cryo-fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in amorphous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy and tomography. Even though the overall morphology of vitrified Golgi stacks is comparable to well-prepared and resin-embedded samples, previously unknown structural details can be observed solely based on their native density. At this point, any further improvement of sample preparation would gain novel insights, perhaps not in terms of general morphology, but on fine structural details of this dynamic organelle.     

Model-based particle picking for cryo-electron microscopy

We describe an algorithm for finding particle images in cryo-EM micrographs. The algorithm starts from a crude 3D map of the target particle, computed from a relatively small number of manually picked images, and then projects the map in many different directions to give synthetic 2D templates. The templates are clustered and averaged and then cross-correlated with the micrographs. A probabilistic model of the imaging process then scores cross-correlation peaks to produce the final picks. We give quantitative results on two quite different target particles: keyhole limpet hemocyanin and p97 AAA ATPase. On these particles our automatic particle picker shows human performance level, as measured by the Fourier shell correlations of 3D reconstructions.     

Dissecting tripartite synapses with STED microscopy

The concept of the tripartite synapse reflects the important role that astrocytic processes are thought to play in the function and regulation of neuronal synapses in the mammalian nervous system. However, many basic aspects regarding the dynamic interplay between pre- and postsynaptic neuronal structures and their astrocytic partners remain to be explored. A major experimental hurdle has been the small physical size of the relevant glial and synaptic structures, leaving them largely out of reach for conventional light microscopic approaches such as confocal and two-photon microscopy. Hence, most of what we know about the organization of the tripartite synapse is based on electron microscopy, which does not lend itself to investigating dynamic events and which cannot be carried out in parallel with functional assays. The development and application of superresolution microscopy for neuron–glia research is opening up exciting experimental opportunities in this regard. In this paper, we provide a basic explanation of the theory and operation of stimulated emission depletion (STED) microscopy, outlining the potential of this recent superresolution imaging modality for advancing our understanding of the morpho-functional interactions between astrocytes and neurons that regulate synaptic physiology.     

Recent advances and current trends in cryo-electron microscopy

All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and cryo-electron tomography (cryo-ET) have facilitated the determination of high-resolution biomolecular structures that are not tractable with other methods. However, challenges remain. For SPA, these include improved resolution in an additional dimension: time. For cryo-ET, these include accessing difficult-to-image areas of a cell and finding rare molecules. Finally, there is a need for automated and faster workflows, as many projects are limited by throughput. Here, we review current developments in SPA cryo-EM and cryo-ET that push these boundaries. Collectively, these advances are poised to propel our spatial and temporal understanding of macromolecular processes.     

Development of imaging scaffolds for cryo-electron microscopy

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Preparation of macromolecular complexes for cryo-electron microscopy

This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a three-dimensional reconstruction of the macromolecular complex.     

The ribosome-structure and functional ligand-binding experiments using cryo-electron microscopy

Cryo-electron microscopy has greatly advanced our understanding of the basic steps of protein synthesis in the bacterial ribosome. This article gives an overview of what has been achieved so far. Through three-dimensional visualization of complexes that represent the ribosome in defined binding states, locations were derived for the tRNA in A, P, and E sites, as well as the elongation factors. In addition, the pathways of messenger RNA and the exiting polypeptide chain could be inferred.