In situ cryoET (cryoelectron tomography) and cryo-FIB/SEM (cryo-focused ion beam/scanning electron microscopy) volume-EM (electron microscopy) imaging provide spatiotemporal snapshots of biological systems in their near-native aqueous environment. Freezing and subsequent thinning of thick biological specimens prior to cryo-imaging is a time-consuming and challenging task that requires state-of-art methodology. As a result, cryo-imaging reports obtained from non-trivial specimens including mammalian brain tissues are scarce and their physiological relevance remains to be determined. Here, we benchmarked plunge freezing with a variety of cryoprotectants that allow for mouse brain tissue vitrification of up to about 100 microns thick and across several brain regions while keeping the tissue functional. By utilizing the knock-in (KI) mouse model with fluorescent astrocytes we have performed targeted cryo-FIB/SEM volume-EM imaging as well as targeted high-resolution cryoET imaging. Prior to cryoET, we have successfully generated lamellae in a semi-automated fashion on both LMIS (liquid metal ion source)- and plasma-based cryo-FIB/SEM instrumentation thus expanding applicability of our pipeline. We visualized the NVU (neurovascular unit) and astrocyte's processes and validated the physiological relevance of our outputs based on the morphology of the corresponding cellular and subcellular features. The pipeline utilizes common vitrification setups and can be potentially extended toward alternative tissue specimens. Ultimately, we expect our approach to become an important step towards democratization of physiologically relevant in situ cryo-imaging studies.

Clusterin is a heterodimeric glycoprotein (α- and β-chain), which has been described as an extracellular molecular chaperone. In humans, clusterin is an amyloid-associated protein, co-localizing with fibrillar deposits in several amyloidoses, including Alzheimer's disease. To clarify its potential implication in amyloid formation, we located aggregation-prone regions within the sequence of clusterin α-chain, via computational methods. We had peptide-analogues, which correspond to each of these regions, chemically synthesized and experimentally demonstrated that all of them can form amyloid-like fibrils. We also provide evidence that the same peptide-analogues can inhibit amyloid-β fibril formation, potentially making them appropriate drug candidates for Alzheimer's disease. At the same time, our findings hint that the respective aggregation-prone clusterin regions may be implicated in the molecular mechanism in which clusterin inhibits amyloid formation. Furthermore, we suggest that molecular chaperones with amyloidogenic properties might have a role in the regulation of amyloid formation, essentially acting as functional amyloids.

RATIONALE:The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates. OBJECTIVE:We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid. METHODS AND RESULTS:Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics. CONCLUSIONS:Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.

Using correlative light and electron microscopy (CLEM), we studied the intracellular organization by of glucose-starved fission yeast cells (Schizosaccharomyces pombe) with regards to the localization of septin proteins throughout the cytoplasm. Thereby, we found that for cells carrying a deletion of the gene encoding septin-2 (spn2Δ), starvation causes a GFP-tagged version of septin-3 (spn3-GFP) and family members, to assemble into a single, prominent filamentous structure. It was previously shown that during exponential growth, spn2Δ cells form septin-3 polymers. However, the polymers we observed during exponential growth are different from the spn3p-GFP structure we observed in starved cells. Using CLEM, in combination with anti-GFP immunolabeling on plastic-sections, we could assign spn3p-GFP to the filaments we have found in EM pictures. Besides septin-3, these filamentous assemblies most likely also contain septin-1 as an RFP-tagged version of this protein forms a very similar structure in starved spn2Δ cells. Our data correlate phase-contrast and fluorescence microscopy with electron micrographs of plastic-embedded cells, and further on with detailed views of tomographic 3D reconstructions. Cryo-electron microscopy of spn2Δ cells in vitrified sections revealed a very distinct overall morphology of the spn3p-GFP assembly. The fine-structured, regular density pattern suggests the presence of assembled septin-3 filaments that are clearly different from F-actin bundles. Furthermore, we found that starvation causes substantial mitochondria fission, together with massive decoration of their outer membrane by ribosomes.

ABSTRACT Cardiomyocytes both cause and experience continual cyclic deformation. The exact effects of this deformation on the properties of intracellular organelles are not well characterized, although they are likely to be relevant for cardiomyocyte responses to active and passive changes in their mechanical environment. In the present study we provide three‐dimensional ultrastructural evidence for mechanically induced mitochondrial deformation in rabbit ventricular cardiomyocytes over a range of sarcomere lengths representing myocardial tissue stretch, an unloaded “slack” state, and contracture. We also show structural indications for interaction of mitochondria with one another, as well as with other intracellular elements such as microtubules, sarcoplasmic reticulum and T‐tubules. The data presented here help to contextualize recent reports on the mechanosensitivity and cell‐wide connectivity of the mitochondrial network and provide a structural framework that may aide interpretation of mechanically‐regulated molecular signaling in cardiac cells. Anat Rec, 302:146–152, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.

BACKGROUND:Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC) is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.METHODOLOGY/PRINCIPAL FINDINGS:We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.CONCLUSIONS/SIGNIFICANCE:The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

The field of cardiovascular research has benefitted from rapid developments in imaging technology over the last few decades. Accordingly, an ever growing number of large, multidimensional data sets have begun to appear, often challenging existing pre-conceptions about structure and function of biological systems. For tissue and cell structure imaging, the move from 2D section-based microscopy to true 3D data collection has been a major driver of new insight. In the sub-cellular domain, electron tomography is a powerful technique for exploration of cellular structures in 3D with unparalleled fidelity at nanometer resolution.Electron tomography is particularly advantageous for studying highly compartmentalised cells such as cardiomyocytes, where elaborate sub-cellular structures play crucial roles in electrophysiology and mechanics. Although the anatomy of specific ultra-structures, such as dyadic couplons, has been extensively explored using 2D electron microscopy of thin sections, we still lack accurate, quantitative knowledge of true individual shape, volume and surface area of sub-cellular domains, as well as their 3D spatial interrelations; let alone of how these are reshaped during the cycle of contraction and relaxation. Here we discuss and illustrate the utility of ET for identification, visualisation, and analysis of 3D cardiomyocyte ultrastructures such as the T-tubular system, sarcoplasmic reticulum, mitochondria and microtubules. (C) 2016 The Authors. Published by Elsevier Ltd.

Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications.

Contemporary models for neuronal migration are grounded in the view that virtually all functionally relevant microtubules (MTs) in migrating neurons are attached to the centrosome, which occupies a position between the nucleus and a short leading process. It is assumed that MTs do not undergo independent movements but rather transduce forces that enable movements of the centrosome and nucleus. The present results demonstrate that although this is mostly true, a small fraction of the MTs are centrosome-unattached, and this permits limited sliding of MTs. When this sliding is pharmacologically inhibited, the leading process becomes shorter, migration of the neuron deviates from its normal path, and the MTs within the leading process become buckled. Partial depletion of ninein, a protein that attaches MTs to the centrosome, leads to greater numbers of centrosome-unattached MTs as well as greater sliding of MTs. Concomitantly, the soma becomes less mobile and the leading process acquires an elongated morphology akin to an axon.

A benign, clonable tag for the localization of proteins by electron microscopy of cells would be valuable, especially if it provided labelling with high signal-to-noise ratio and good spatial resolution. Here we explore the use of metallothionein as such a localization marker. We have achieved good success with desmin labelled in vitro and with a component of the yeast spindle pole body labelled in cells. Heavy metals added after fixation and embedding or during the process of freeze-substitution fixation provide readily visible signals with no concern that the heavy atoms are affecting the behaviour of the protein in its physiological environment. However, our methods did not work with protein components of the nuclear pore complex, suggesting that this approach is not yet universally applicable. We provide a full description of our optimal labelling conditions and other conditions tried, hoping that our work will allow others to label their own proteins of interest and/or improve on the methods we have defined.

Many cells and organisms react to depletion of nutrients with a low-energy program. Yeast cells response to nutrient starvation by entering a quiescent state. This state is defined by a substantial increase in the viscosity of the cell cytoplasm, a phenomenon we term "cell-freezing", or cytoplasmic freezing. Cell-freezing is a phenomenon that has been discovered recently through measurements of the viscoelastic properties of the cytoplasm of fission yeast cells. It occurs as a response to carbon starvation and leads to a dramatic immobilization of all visible sub-cellular structures, which suggests that the preservation of the internal organization of the cell in this quiescent state is its main biological function. Recently we found evidence that the transition of viscosity of the cell cytoplasm could be reliably reproduced by starving cells of glucose. Also, there is evidence that septins might be involved. Septins were known to form fibers in vitro, and during cell division. Hence, we proposed that a dense and homogeneous network will be formed by septins when the cell enters the starvation state and that this network is sufficiently sturdy to immobilize all major cell components. We used conventional and Cryo-Electron Microscopy (cryo-EM) combined with tomographic 3-D analysis, that allowed us visualizing polymeric structures resembling that of septin filaments, and their interaction with subcellular structures. In particular, high pressure freezing followed by vitrified sectioning (Cryo-microtome) technology preserved the original condition of cell to molecular detail. Here we display for the first time the proposed filamentous network in starved fission yeast cells. Apart from filament bundles we also found some other interesting novel sub-cellular structures.

Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ±60 and ±70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1–2° or less and a tilt range of ±60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.

Triglyceride-rich lipoproteins (TRLs) undergo lipolysis by lipoprotein lipase (LPL), an enzyme that is transported to the capillary lumen by an endothelial cell protein, GPIHBP1. For LPL-mediated lipolysis to occur, TRLs must bind to the lumen of capillaries. This process is often assumed to involve heparan sulfate proteoglycans (HSPGs), but we suspected that TRL margination might instead require GPIHBP1. Indeed, TRLs marginate along the heart capillaries of wild-type but not Gpihbp1(-/-) mice, as judged by fluorescence microscopy, quantitative assays with infrared-dye-labeled lipoproteins, and EM tomography. Both cell-culture and in vivo studies showed that TRL margination depends on LPL bound to GPIHBP1. Notably, the expression of LPL by endothelial cells in Gpihbp1(-/-) mice did not restore defective TRL margination, implying that the binding of LPL to HSPGs is ineffective in promoting TRL margination. Our studies show that GPIHBP1-bound LPL is the main determinant of TRL margination.

Defects in flagella growth are related to a number of human diseases. Central to flagellar growth is the organization of microtubules that polymerize from basal bodies to form the axoneme, which consists of hundreds of proteins. Flagella exist in all eukaryotic phyla, but neither the mechanism by which flagella grow nor the conservation of this process in evolution are known. Here, we study how protein complexes assemble onto the growing axoneme tip using (cryo) electron tomography. In Chlamydomonas reinhardtii microtubules and associated proteins are added simultaneously. However, in Trypanosoma brucei, disorganized arrays of microtubules are arranged into the axoneme structure by the later addition of preformed protein complexes. Post assembly, the T. brucei transition zone alters structure and its association with the central pair loosens. We conclude that there are multiple ways to form a flagellum and that species-specific structural knowledge is critical before evaluating flagellar defects.