Cryo-electron tomography of cells: connecting structure and function

Cryo-electron tomography (cryo-ET) allows the visualization of cellular structures under close-to-life conditions and at molecular resolution. While it is inherently a static approach, yielding structural information about supramolecular organization at a certain time point, it can nevertheless provide insights into function of the structures imaged, in particular, when supplemented by other approaches. Here, we review the use of experimental methods that supplement cryo-ET imaging of whole cells. These include genetic and pharmacological manipulations, as well as correlative light microscopy and cryo-ET. While these methods have mostly been used to detect and identify structures visualized in cryo-ET or to assist the search for a feature of interest, we expect that in the future they will play a more important role in the functional interpretation of cryo-tomograms.     

Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.     

冷冻电子断层扫描研究原代培养神经元中的线粒体膜动态变化（英文）

<正>Dear Editor,Mitochondria acts as a cellular organelle that produces ATP and buffers Ca²⁺, and plays an important role in neuronal growth, survival and function^[1]. Loss of mitochondria will make the ATP supply insufficient, resulting in synaptic transmission dysfunction^[2]. Further, presynaptic mitochondrial dysfunctions are often associated with severe neurological diseases^[3].     

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.     

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts. Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.     

Electron tomography of plant thylakoid membranes

For more than half a century, electron microscopy has been a main tool for investigating the complex ultrastructure and organization of chloroplast thylakoid membranes, but, even today, the three-dimensional relationship between stroma and grana thylakoids, and the arrangement of the membrane protein complexes within them are not fully understood. Electron cryo-tomography (cryo-ET) is a powerful new technique for visualizing cellular structures, especially membranes, in three dimensions. By this technique, large membrane protein complexes, such as the photosystem II supercomplex or the chloroplast ATP synthase, can be visualized directly in the thylakoid membrane at molecular (4-5 nm) resolution. This short review compares recent advances by cryo-ET of plant thylakoid membranes with earlier results obtained by conventional electron microscopy.     

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

Cryo-electron tomography (cryo-ET) has reached nanoscale resolution for in situ three-dimensional imaging of macromolecular complexes and organelles. Yet its current resolution is not sufficient to precisely localize or identify most proteins in situ; for example, the location and arrangement of components of the nexin-dynein regulatory complex (N-DRC), a key regulator of ciliary/flagellar motility that is conserved from algae to humans, have remained elusive despite many cryo-ET studies of cilia and flagella. Here, we developed an in situ localization method that combines cryo-ET/subtomogram averaging with the clonable SNAP tag, a widely used cell biological probe to visualize fusion proteins by fluorescence microscopy. Using this hybrid approach, we precisely determined the locations of the N and C termini of DRC3 and the C terminus of DRC4 within the three-dimensional structure of the N-DRC in Chlamydomonas flagella. Our data demonstrate that fusion of SNAP with target proteins allowed for protein localization with high efficiency and fidelity using SNAP-linked gold nanoparticles, without disrupting the native assembly, structure, or function of the flagella. After cryo-ET and subtomogram averaging, we localized DRC3 to the L1 projection of the nexin linker, which interacts directly with a dynein motor, whereas DRC4 was observed to stretch along the N-DRC base plate to the nexin linker. Application of the technique developed here to the N-DRC revealed new insights into the organization and regulatory mechanism of this complex, and provides a valuable tool for the structural dissection of macromolecular complexes in situ.     

Apo alpha-lactalbumin and lysozyme are colocalized in their subsequently formed spherical supramolecular assembly

We have reported previously that the calcium-depleted form of bovine alpha-lactalbumin (apo alpha-LA) interacts with hen egg-white lysozyme (LYS) to form spherical supramolecular structures. These supramolecular structures contain an equimolar ratio of the two proteins. We further explore here the organization of these structures. The spherical morphology and size of the assembled LYS/apo alpha-LA supramolecular structures were demonstrated using confocal scanning laser microscopy and scanning electron microscopy. From confocal scanning laser microscopy experiments with labelled proteins, it was found that LYS and apo alpha-LA were perfectly colocalized and homogeneously distributed throughout the entire three-dimensional structure of the microspheres formed. The spatial colocalization of the two proteins was also confirmed by the occurrence of a fluorescence resonance energy transfer phenomenon between labelled apo alpha-LA and labelled LYS. Polarized light microscopy analysis revealed that the microspheres formed differ from spherulites, a higher order semicrystalline structure. As the molecular mechanism initiating the formation of these microspheres is still unknown, we discuss the potential involvement of a LYS/apo alpha-LA heterodimer as a starting block for such a supramolecular assembly.     

Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4–6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.     

Cryo-electron tomography: gaining insight into cellular processes by structural approaches

Visualization of cellular processes at a resolution of the individual protein should involve integrative and complementary approaches that can eventually draw realistic functional and cellular landscapes. Electron tomography of vitrified but otherwise unaltered cells emerges as a central method for three-dimensional reconstruction of cellular architecture at a resolution of 2-6 nm. While a combination of correlative light-based microscopy with cryo-electron tomography (cryo-ET) provides medium-resolution insight into pivotal cellular processes, fitting high-resolution structural approaches, for example, X-ray crystallography, into reconstructed macromolecular assemblies provides unprecedented information on native protein assemblies. Thus, cryo-ET bridges the resolution gap between cellular and structural biology. In this article, we focus on the study of eukaryotic cells and macromolecular complexes in a close-to-life-state. We discuss recent developments and structural findings enabling major strides to be made in understanding complex physiological functions.     

Optical super-resolution microscopy in neurobiology

Understanding the highly plastic nature of neurons requires the dynamic visualization of their molecular and cellular organization in a native context. However, due to the limited resolution of standard light microscopy, many of the structural specializations of neurons cannot be resolved. A recent revolution in light microscopy has given rise to several super-resolution light microscopy methods yielding 2-10-fold higher resolution than conventional microscopy. We here describe the principles behind these techniques as well as their application to the analysis of the molecular architecture of the synapse. Furthermore, we discuss the potential for continued development of super-resolution microscopy as necessary for live imaging of neuronal structure and function in the brain.     

Digitalizing neuronal synapses with cryo-electron tomography and correlative microscopy

Synapses are the structural and functional joints of neuronal circuits, and brain function is fundamentally based on synaptic quantal transmission and plasticity. Precise mapping of key components within individual synapses in different states can reveal the principles governing synapse formation, transmission, and plasticity and improving understanding of the mechanisms of synapse-related diseases. Cryo-electron tomography (cryo-ET) and correlative microscopy are increasingly powerful tools that can dissect the molecular sociology of intact cells, including neuronal synapses. In this study, we discuss current progress made in cryo-ET studies assessing neuronal synapses, especially sample preparation, molecule identification, and correlative approaches for synaptic dynamics and functions.     

Cell surface expression of H2 antigens on primary sensory neurons in response to acute but not latent herpes simplex virus infection in vivo.

CD8(+) T lymphocytes and class I major histocompatibility complex (MHC-I) molecules profoundly influence the severity of neuronal herpes simplex virus (HSV) infection in experimentally infected mice. Paradoxically, neurons are classically regarded as MHC-I deficient. However, it is shown here that H2-encoded heavy chains (alphaCs) and their associated light chain, beta2 microglobulin, are present on the surfaces of primary sensory neurons recovered from sensory ganglia within 1 to 2 weeks of HSV infection. During this time, some neurons are found to be tightly associated with T cells in vivo. Prior data showed that termination of productive HSV infection in the peripheral nervous system is not dependent on cell-mediated lysis of infected neurons. Consistent with these data, immunogold electron microscopy showed that the density of cell surface H2 on neurons is an order of magnitude lower than on satellite glia, which is predicted to favor a noncytolytic CD8 cell response.     

Cytological evidence for activation of neuroendocrine cells in the parvocellular preoptic nucleus of the goldfish hypothalamus following pharmacological adrenalectomy

Summary The nucleus preopticus (NPO) of the goldfish hypothalamus is composed of parvocellular (NPOpc) and magnocellular (NPOmc) neurosecretory neurons. The cytology of NPOpc and NPOmc neurons was examined with light and electron microscopy following pharmacological adrenalectomy with the adrenocortical inhibitor, metopirone. After five days of metopirone administration, light microscopy revealed a significant increase in nuclear area of NPOpc, but not of NPOmc, neurons.Ultrastructural examination of NPOpc neurons revealed two cell types, PC 1 and PC 2 neurons, which could be distinguished by the relative abundance and the size of the neurosecretory granules in the cytoplasm. The ultrastructural appearance of the NPOmc neurons revealed a single cell type containing abundant neurosecretory granules. Following five days of metopirone administration, the ultrastructural appearance of the PC 1 neurons indicated a state of enhanced secretory activity. Metopirone had no observable effect on the appearance of the PC 2 or NPOmc neurons. These observations demonstrate that PC 1 neurons are activated under the conditions of pharmacological adrenalectomy and suggest that the secretory activity of these neurons is inhibited by adrenocorticosteroids.     

Automated tracing of filaments in 3D electron tomography reconstructions using Sculptor and Situs

The molecular graphics program Sculptor and the command-line suite Situs are software packages for the integration of biophysical data across spatial resolution scales. Herein, we provide an overview of recently developed tools relevant to cryo-electron tomography (cryo-ET), with an emphasis on functionality supported by Situs 2.7.1 and Sculptor 2.1.1. We describe a work flow for automatically segmenting filaments in cryo-ET maps including denoising, local normalization, feature detection, and tracing. Tomograms of cellular actin networks exhibit both cross-linked and bundled filament densities. Such filamentous regions in cryo-ET data sets can then be segmented using a stochastic template-based search, VolTrac. The approach combines a genetic algorithm and a bidirectional expansion with a tabu search strategy to localize and characterize filamentous regions. The automated filament segmentation by VolTrac compares well to a manual one performed by expert users, and it allows an efficient and reproducible analysis of large data sets. The software is free, open source, and can be used on Linux, Macintosh or Windows computers.     

Structure of the nuclear pore complex goes atomic

The nuclear pore complex (NPC) is a supra-molecular assembly that mediates substance and information flow across the nuclear envelope (NE). Due to its extraordinary size and complexity, the NPC remains one of the most challenging tasks in structural elucidation at atomic resolution. Recent breakthroughs in cryo-electron microscopy (cryo-EM) reconstruction, Machine Learning empowered structure prediction and biochemical reconstitution have combined to yield molecular models of the NPC at unprecedented accuracy. Furthermore, in cellulo cryo-electron tomography (cryo-ET) structures reveal substantial structural dynamics of the NPC. These advances shed light on the organizational principles and functions of the NPC.     

A Golgi-electron microscope method for insect nervous tissue.

Golgi's light microscope method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 2 1/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50 mum sections are made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

Multishot tomography for high-resolution in situ subtomogram averaging

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) can resolve protein complexes at near atomic resolution, and when combined with focused ion beam (FIB) milling, macromolecules can be observed within their native context. Unlike single particle acquisition (SPA), cryo-ET can be slow, which may reduce overall project throughput. We here propose a fast, multi-position tomographic acquisition scheme based on beam-tilt corrected beam-shift imaging along the tilt axis, which yields sub-nanometer in situ STA averages.     

A Golgi-Electron Microscope Method for Insect Nervous Tissue

Golgi's light microscopic method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 21/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50μm sections am made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.