Cryo-electron tomography: A long journey to the inner space of cells.

Cryogenic electron tomography (cryo-ET) is the application of tomographic principles of data acquisition and reconstruction to frozen-hydrated biological specimens. It combines a close-to-life preservation of cellular structures with the power of high-resolution three-dimensional imaging, which allows us to study the molecular architecture of cells, or their molecular sociology, in unprecedented detail.

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment.

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.

In Situ Architecture and Cellular Interactions of PolyQ Inclusions.

Expression of many disease-related aggregation-prone proteins results in cytotoxicity and the formation of large intracellular inclusion bodies. To gain insight into the role of inclusions in pathology and the in situ structure of protein aggregates inside cells, we employ advanced cryo-electron tomography methods to analyze the structure of inclusions formed by polyglutamine (polyQ)-expanded huntingtin exon 1 within their intact cellular context. In primary mouse neurons and immortalized human cells, polyQ inclusions consist of amyloid-like fibrils that interact with cellular endomembranes, particularly of the endoplasmic reticulum (ER). Interactions with these fibrils lead to membrane deformation, the local impairment of ER organization, and profound alterations in ER membrane dynamics at the inclusion periphery. These results suggest that aberrant interactions between fibrils and endomembranes contribute to the deleterious cellular effects of protein aggregation. VIDEO ABSTRACT.

Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

Structure and topography of the synaptic V-ATPase-synaptophysin complex.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here, we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the protein gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogs synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

A Selective Autophagy Pathway for Phase-Separated Endocytic Protein Deposits.

Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.

Stress- and ubiquitylation-dependent phase separation of the proteasome.

The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins1,2. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes3-8, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation.

Vimentin regulates nuclear segmentation in neutrophils.

Granulocytes are indispensable for various immune responses. Unlike other cell types in the body, the nuclei of granulocytes, particularly neutrophils, are heavily segmented into multiple lobes. Although this distinct morphological feature has long been observed, the underlying mechanism remains incompletely characterized. In this study, we utilize cryo-electron tomography to examine the nuclei of mouse neutrophils, revealing the cytoplasmic enrichment of intermediate filaments on the concave regions of the nuclear envelope. Aided by expression profiling and immuno-electron microscopy, we then elucidate that the intermediate-filament protein vimentin is responsible for such perinuclear structures. Of importance, exogenously expressed vimentin in nonimmune cells is sufficient to form cytoplasmic filaments wrapping on the concave nuclear surface. Moreover, genetic deletion of the protein causes a significant reduction of the number of nuclear lobes in neutrophils and eosinophils, mimicking the hematological condition of the Pelger-Huët anomaly. These results have uncovered a new component establishing the nuclear segmentation of granulocytes.

In situ snapshots along a mammalian selective autophagy pathway.

Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are carriers responsible for delivering their content to a lytic compartment for destruction. The cargo can be of diverse origin, ranging from macromolecular complexes to protein aggregates, organelles, and even invading pathogens. Each cargo is unique in composition and size, presenting different challenges to autophagosome biogenesis. Among the largest cargoes targeted by the autophagy machinery are intracellular bacteria, which can, in the case of Salmonella, range from 2 to 5 µm in length and 0.5 to 1.5 µm in width. How phagophores form and expand on such a large cargo remains mechanistically unclear. Here, we used HeLa cells infected with an auxotrophic Salmonella to study the process of phagophore biogenesis using in situ correlative cryo-ET. We show that host cells generate multiple phagophores at the site of damaged Salmonella-containing vacuoles (SCVs). The observed double membrane structures range from disk-shaped to expanded cup-shaped phagophores, which have a thin intermembrane lumen with a dilating rim region and expand using the SCV, the outer membrane of Salmonella, or existing phagophores as templates. Phagophore rims establish different forms of contact with the endoplasmic reticulum (ER) via structurally distinct molecular entities for membrane formation and expansion. Early omegasomes correlated with the marker Double-FYVE domain-Containing Protein 1 (DFCP1) are observed in close association with the ER without apparent membrane continuity. Our study provides insights into the formation of phagophores around one of the largest selective cargoes.

In situ structural analysis reveals membrane shape transitions during autophagosome formation.

Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.

Deep learning improves macromolecule identification in 3D cellular cryo-electron tomograms.

Cryogenic electron tomography (cryo-ET) visualizes the 3D spatial distribution of macromolecules at nanometer resolution inside native cells. However, automated identification of macromolecules inside cellular tomograms is challenged by noise and reconstruction artifacts, as well as the presence of many molecular species in the crowded volumes. Here, we present DeepFinder, a computational procedure that uses artificial neural networks to simultaneously localize multiple classes of macromolecules. Once trained, the inference stage of DeepFinder is faster than template matching and performs better than other competitive deep learning methods at identifying macromolecules of various sizes in both synthetic and experimental datasets. On cellular cryo-ET data, DeepFinder localized membrane-bound and cytosolic ribosomes (roughly 3.2 MDa), ribulose 1,5-bisphosphate carboxylase-oxygenase (roughly 560 kDa soluble complex) and photosystem II (roughly 550 kDa membrane complex) with an accuracy comparable to expert-supervised ground truth annotations. DeepFinder is therefore a promising algorithm for the semiautomated analysis of a wide range of molecular targets in cellular tomograms.

Gel-like inclusions of C-terminal fragments of TDP-43 sequester stalled proteasomes in neurons.

Aggregation of the multifunctional RNA-binding protein TDP-43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C-terminal fragments of ~25 kDa ("TDP-25") accumulate in cytoplasmic inclusions. Here, we analyze gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP-25 inclusions are amorphous, and photobleaching experiments reveal gel-like biophysical properties that are less dynamic than nuclear TDP-43. Compared with full-length TDP-43, the TDP-25 interactome is depleted of low-complexity domain proteins. TDP-25 inclusions are enriched in 26S proteasomes adopting exclusively substrate-processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP-25 impairs proteostasis, and this inhibitory function is enhanced by ALS-causing TDP-43 mutations. These findings support a patho-physiological relevance of proteasome dysfunction in ALS/FTD.

The native 3D organization of bacterial polysomes.

Recent advances have led to insights into the structure of the bacterial ribosome, but little is known about the 3D organization of ribosomes in the context of translating polysomes. We employed cryoelectron tomography and a template-matching approach to map 70S ribosomes in vitrified bacterial translation extracts and in lysates of active E. coli spheroplasts. In these preparations, polysomal arrangements were observed in which neighboring ribosomes are densely packed and exhibit preferred orientations. Analysis of characteristic examples of polysomes reveals a staggered or pseudohelical organization of ribosomes along the mRNA trace, with the transcript being sequestered on the inside, the tRNA entrance sites being accessible, and the polypeptide exit sites facing the cytosol. Modeling of elongating nascent polypeptide chains suggests that this arrangement maximizes the distance between nascent chains on adjacent ribosomes, thereby reducing the probability of intermolecular interactions that would give rise to aggregation and limit productive folding.

The cryo-electron microscopy structure of huntingtin.

Huntingtin (HTT) is a large (348 kDa) protein that is essential for embryonic development and is involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and the regulation of transcription. Although an integrative understanding of the biological functions of HTT is lacking, the large number of identified HTT interactors suggests that it serves as a protein-protein interaction hub. Furthermore, Huntington's disease is caused by a mutation in the HTT gene, resulting in a pathogenic expansion of a polyglutamine repeat at the amino terminus of HTT. However, only limited structural information regarding HTT is currently available. Here we use cryo-electron microscopy to determine the structure of full-length human HTT in a complex with HTT-associated protein 40 (HAP40; encoded by three F8A genes in humans) to an overall resolution of 4 Å. HTT is largely α-helical and consists of three major domains. The amino- and carboxy-terminal domains contain multiple HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat-like organization. HAP40 binds in a cleft and contacts the three HTT domains by hydrophobic and electrostatic interactions, thereby stabilizing the conformation of HTT. These data rationalize previous biochemical results and pave the way for improved understanding of the diverse cellular functions of HTT.

Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor.

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the Gs-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.

Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex.

The class B glucagon-like peptide-1 (GLP-1) G protein-coupled receptor is a major target for the treatment of type 2 diabetes and obesity. Endogenous and mimetic GLP-1 peptides exhibit biased agonism-a difference in functional selectivity-that may provide improved therapeutic outcomes. Here we describe the structure of the human GLP-1 receptor in complex with the G protein-biased peptide exendin-P5 and a Gαs heterotrimer, determined at a global resolution of 3.3 Å. At the extracellular surface, the organization of extracellular loop 3 and proximal transmembrane segments differs between our exendin-P5-bound structure and previous GLP-1-bound GLP-1 receptor structure. At the intracellular face, there was a six-degree difference in the angle of the Gαs-α5 helix engagement between structures, which was propagated across the G protein heterotrimer. In addition, the structures differed in the rate and extent of conformational reorganization of the Gαs protein. Our structure provides insights into the molecular basis of biased agonism.

Structure of the adenosine-bound human adenosine A1 receptor-Gi complex.

The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound ß2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides molecular insights into receptor and G-protein selectivity.

A transformation clustering algorithm and its application in polyribosomes structural profiling.

Improvements in cryo-electron tomography sample preparation, electron-microscopy instrumentations, and image processing algorithms have advanced the structural analysis of macromolecules in situ. Beyond such analyses of individual macromolecules, the study of their interactions with functionally related neighbors in crowded cellular habitats, i.e. 'molecular sociology', is of fundamental importance in biology. Here we present a NEighboring Molecule TOpology Clustering (NEMO-TOC) algorithm. We optimized this algorithm for the detection and profiling of polyribosomes, which play both constitutive and regulatory roles in gene expression. Our results suggest a model where polysomes are formed by connecting multiple nonstochastic blocks, in which translation is likely synchronized.

Improvements in cryo-electron tomography sample preparation, electron-microscopy instrumentations, and image processing algorithms have advanced the structural analysis of macromolecules in situ. Beyond such analyses of individual macromolecules, the study of their interactions with functionally related neighbors in crowded cellular habitats, i.e. "molecular sociology", is of fundamental importance in biology. Here we present a NEighboring Molecule TOpology Clustering (NEMO-TOC) algorithm. We optimized this algorithm for the detection and profiling of polyribosomes, which play both constitutive and regulatory roles in gene expression. Our results suggest a model where polysomes are formed by connecting multiple nonstochastic blocks, in which translation is likely synchronized.

Author Correction: Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.