Extensive structural rearrangement of intraflagellar transport trains underpins bidirectional cargo transport.

Bidirectional transport in cilia is carried out by polymers of the IFTA and IFTB protein complexes, called anterograde and retrograde intraflagellar transport (IFT) trains. Anterograde trains deliver cargoes from the cell to the cilium tip, then convert into retrograde trains for cargo export. We set out to understand how the IFT complexes can perform these two directly opposing roles before and after conversion. We use cryoelectron tomography and in situ cross-linking mass spectrometry to determine the structure of retrograde IFT trains and compare it with the known structure of anterograde trains. The retrograde train is a 2-fold symmetric polymer organized around a central thread of IFTA complexes. We conclude that anterograde-to-retrograde remodeling involves global rearrangements of the IFTA/B complexes and requires complete disassembly of the anterograde train. Finally, we describe how conformational changes to cargo-binding sites facilitate unidirectional cargo transport in a bidirectional system.

The molecular architecture of the nuclear basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

Mammalian oocytes store proteins for the early embryo on cytoplasmic lattices.

Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development.

Molecular mechanisms of stress-induced reactivation in mumps virus condensates.

Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.

Better, Faster, Cheaper: Recent Advances in Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.

Metabolic analysis as a driver for discovery, diagnosis, and therapy.

Metabolic anomalies contribute to tissue dysfunction. Current metabolism research spans from organelles to populations, and new technologies can accommodate investigation across these scales. Here, we review recent advancements in metabolic analysis, including small-scale metabolomics techniques amenable to organelles and rare cell types, functional screening to explore how cells respond to metabolic stress, and imaging approaches to non-invasively assess metabolic perturbations in diseases. We discuss how metabolomics provides an informative phenotypic dimension that complements genomic analysis in Mendelian and non-Mendelian disorders. We also outline pressing challenges and how addressing them may further clarify the biochemical basis of human disease.

Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice.

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

Cone-shaped HIV-1 capsids are transported through intact nuclear pores.

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

The molecular basis for sarcomere organization in vertebrate skeletal muscle.

Sarcomeres are force-generating and load-bearing devices of muscles. A precise molecular picture of how sarcomeres are built underpins understanding their role in health and disease. Here, we determine the molecular architecture of native vertebrate skeletal sarcomeres by electron cryo-tomography. Our reconstruction reveals molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band, and Z-disc and demonstrates that α-actinin cross-links antiparallel actin filaments by forming doublets with 6-nm spacing. Structures of myosin, tropomyosin, and actin at ~10 Å further reveal two conformations of the "double-head" myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases.

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.

3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway.

Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.

Molecular Architecture of the SARS-CoV-2 Virus.

SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, the detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryoelectron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in pre- and postfusion conformations were determined to average resolutions of 8.7-11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed overall processing states of the native glycans highly similar to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNPs) and their higher-order assemblies were revealed. Overall, these characterizations revealed the architecture of the SARS-CoV-2 virus in exceptional detail and shed light on how the virus packs its ∼30-kb-long single-segmented RNA in the ∼80-nm-diameter lumen.

In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.

Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.

Clinically Applicable AI System for Accurate Diagnosis, Quantitative Measurements, and Prognosis of COVID-19 Pneumonia Using Computed Tomography.

Many COVID-19 patients infected by SARS-CoV-2 virus develop pneumonia (called novel coronavirus pneumonia, NCP) and rapidly progress to respiratory failure. However, rapid diagnosis and identification of high-risk patients for early intervention are challenging. Using a large computed tomography (CT) database from 3,777 patients, we developed an AI system that can diagnose NCP and differentiate it from other common pneumonia and normal controls. The AI system can assist radiologists and physicians in performing a quick diagnosis especially when the health system is overloaded. Significantly, our AI system identified important clinical markers that correlated with the NCP lesion properties. Together with the clinical data, our AI system was able to provide accurate clinical prognosis that can aid clinicians to consider appropriate early clinical management and allocate resources appropriately. We have made this AI system available globally to assist the clinicians to combat COVID-19.

ß-Arrestin-Biased Allosteric Modulator of NTSR1 Selectively Attenuates Addictive Behaviors.

Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages ß-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a ß-arrestin-biased agonist but also extends profound ß-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and ß-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.

The In Situ Structure of Parkinson's Disease-Linked LRRK2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.

mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding.

Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.

Identifying Medical Diagnoses and Treatable Diseases by Image-Based Deep Learning.

The implementation of clinical-decision support algorithms for medical imaging faces challenges with reliability and interpretability. Here, we establish a diagnostic tool based on a deep-learning framework for the screening of patients with common treatable blinding retinal diseases. Our framework utilizes transfer learning, which trains a neural network with a fraction of the data of conventional approaches. Applying this approach to a dataset of optical coherence tomography images, we demonstrate performance comparable to that of human experts in classifying age-related macular degeneration and diabetic macular edema. We also provide a more transparent and interpretable diagnosis by highlighting the regions recognized by the neural network. We further demonstrate the general applicability of our AI system for diagnosis of pediatric pneumonia using chest X-ray images. This tool may ultimately aid in expediting the diagnosis and referral of these treatable conditions, thereby facilitating earlier treatment, resulting in improved clinical outcomes. VIDEO ABSTRACT.

Chromosome Translocation Inflates Bacillus Forespores and Impacts Cellular Morphology.

The means by which the physicochemical properties of different cellular components together determine bacterial cell shape remain poorly understood. Here, we investigate a programmed cell-shape change during Bacillus subtilis sporulation, when a rod-shaped vegetative cell is transformed to an ovoid spore. Asymmetric cell division generates a bigger mother cell and a smaller, hemispherical forespore. The septum traps the forespore chromosome, which is translocated to the forespore by SpoIIIE. Simultaneously, forespore size increases as it is reshaped into an ovoid. Using genetics, timelapse microscopy, cryo-electron tomography, and mathematical modeling, we demonstrate that forespore growth relies on membrane synthesis and SpoIIIE-mediated chromosome translocation, but not on peptidoglycan or protein synthesis. Our data suggest that the hydrated nucleoid swells and inflates the forespore, displacing ribosomes to the cell periphery, stretching septal peptidoglycan, and reshaping the forespore. Our results illustrate how simple biophysical interactions between core cellular components contribute to cellular morphology.