High-confidence 3D template matching for cryo-electron tomography.

Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.

Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography.

The use of protoplasts in plant biology has become a convenient tool for the application of transient gene expression. This model system has allowed the study of plant responses to biotic and abiotic stresses, protein location and trafficking, cell wall dynamics, and single-cell transcriptomics, among others. Although well-established protocols for isolating protoplasts from different plant tissues are available, they have never been used for studying plant cells using cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET). Here we describe a workflow to prepare root protoplasts from Arabidopsis thaliana plants for cryo-ET. The process includes protoplast isolation and vitrification on EM grids, and cryo-focused ion beam milling (cryo-FIB), with the aim of tilt series acquisition. The whole workflow, from growing the plants to the acquisition of the tilt series, may take a few months. Our protocol provides a novel application to use plant protoplasts as a tool for cryo-ET.

In situ architecture of the ER-mitochondria encounter structure.

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.

The architecture of Cidec-mediated interfaces between lipid droplets.

Lipid droplets (LDs) are intracellular organelles responsible for storing surplus energy as neutral lipids. Their size and number vary enormously. In white adipocytes, LDs can reach 100 µm in diameter, occupying >90% of the cell. Cidec, which is strictly required for the formation of large LDs, is concentrated at interfaces between adjacent LDs and facilitates directional flux of neutral lipids from the smaller to the larger LD. The mechanism of lipid transfer is unclear, in part because the architecture of interfaces between LDs remains elusive. Here we visualize interfaces between LDs by electron cryo-tomography and analyze the kinetics of lipid transfer by quantitative live fluorescence microscopy. We show that transfer occurs through closely apposed monolayers, is slowed down by increasing the distance between the monolayers, and follows exponential kinetics. Our data corroborate the notion that Cidec facilitates pressure-driven transfer of neutral lipids through two "leaky" monolayers between LDs.

Structures of the eukaryotic ribosome and its translational states in situ.

Ribosomes translate genetic information into primary structure. During translation, various cofactors transiently bind to the ribosome that undergoes prominent conformational and structural changes. Different translational states of ribosomes have been well characterized in vitro. However, to which extent the known translational states are representative of the native situation inside cells has thus far only been addressed in prokaryotes. Here, we apply cryo-electron tomography to cryo-FIB milled Dictyostelium discoideum cells combined with subtomogram averaging and classification. We obtain an in situ structure that is locally resolved up to 3 Angstrom, the distribution of eukaryotic ribosome translational states, and unique arrangement of rRNA expansion segments. Our work demonstrates the use of in situ structural biology techniques for identifying distinct ribosome states within the cellular environment.

Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids.

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments.

Architecture of cell-cell junctions in situ reveals a mechanism for bacterial biofilm inhibition.

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam-milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.

Tricalbins Contribute to Cellular Lipid Flux and Form Curved ER-PM Contacts that Are Bridged by Rod-Shaped Structures.

Lipid flow between cellular organelles occurs via membrane contact sites. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). How these proteins regulate membrane architecture to transport lipids across the aqueous space between bilayers remains unknown. Using correlative microscopy, electron cryo-tomography, and high-throughput genetics, we address the interplay of architecture and function in budding yeast. We find that ER-PM contacts differ in protein composition and membrane morphology, not in intermembrane distance. In situ electron cryo-tomography reveals the molecular organization of tricalbin-mediated contacts, suggesting a structural framework for putative lipid transfer. Genetic analysis uncovers functional overlap with cellular lipid routes, such as maintenance of PM asymmetry. Further redundancies are suggested for individual tricalbin protein domains. We propose a modularity of molecular and structural functions of tricalbins and of their roles within the cellular network of lipid distribution pathways.

Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis.

During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

The RNA-Binding Protein Scp160p Facilitates Aggregation of Many Endogenous Q/N-Rich Proteins.

The RNA-binding protein Scp160p is the yeast homolog of the conserved vigilin protein family. These proteins influence a variety of nuclear and cytoplasmic functions. One of Scp160p's reported roles is to increase translation elongation efficiency in a manner related to codon usage. Thus, it can affect translation speed and co-translational folding of nascent peptides. We used polyglutamine (polyQ) reporters to assess Scp160p's effect on protein synthesis and observed that, in the absence of Scp160p, aggregation of polyQ is reduced and toxicity is abolished. We additionally took a proteomic approach and analyzed the impact of Scp160p on the aggregation of endogenous proteins under normal growth conditions. In the absence of Scp160p, aggregation of many Q/N-rich proteins was reduced. Because aggregation mediated by these regions can be important for the proteins' functions, Scp160p may affect many processes via aggregation of Q/N-rich proteins.

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ.

Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation.

Perspective on architecture and assembly of membrane contact sites.

Membrane contact sites (MCS) are platforms of physical contact between different organelles. They are formed through interactions involving lipids and proteins, and function in processes such as calcium and lipid exchange, metabolism and organelle biogenesis. In this article, we discuss emerging questions regarding the architecture, organisation and assembly of MCS, such as: What is the contribution of different components to the interaction between organelles? How is the specific composition of different types of membrane contacts sites established and maintained? How are proteins and lipids spatially organised at MCS and how does that influence their function? How dynamic are MCS on the molecular and ultrastructural level? We highlight current state of research and point out experimental approaches that promise to contribute to a spatiomechanistic understanding of MCS functions.

Reducing Lissencephaly-1 levels augments mitochondrial transport and has a protective effect in adult Drosophila neurons.

Defective transport of mitochondria in axons is implicated in the pathogenesis of several age-associated neurodegenerative diseases. However, the regulation and function of axonal mitochondrial motility during normal ageing is poorly understood. Here, we use novel imaging procedures to characterise axonal transport of these organelles in the adult Drosophila wing nerve. During early adult life there is a boost and progressive decline in the proportion of mitochondria that are motile, which is not due to general changes in cargo transport. Experimental inhibition of the mitochondrial transport machinery specifically in adulthood accelerates the appearance of focal protein accumulations in ageing axons, which is suggestive of defects in protein homeostasis. Unexpectedly, lowering levels of Lissencephaly-1 (Lis1), a dynein motor co-factor, augments axonal mitochondrial transport in ageing wing neurons. Lis1 mutations suppress focal protein accumulations in ageing neurons, including those caused by interfering with the mitochondrial transport machinery. Our data provide new insights into the dynamics of mitochondrial motility in adult neurons in vivo, identify Lis1 as a negative regulator of transport of these organelles, and provide evidence of a link between mitochondrial movement and neuronal protein homeostasis.

Optimal guideRNAs for re-directing deaminase activity of hADAR1 and hADAR2 in trans.

Adenosine deaminases that act on RNA (ADAR) are a class of enzymes that catalyze the conversion of adenosine to inosine in RNA. Since inosine is read as guanosine ADAR activity formally introduces A-to-G point mutations. Re-addressing ADAR activity toward new targets in an RNA-dependent manner is a highly rational, programmable approach for the manipulation of RNA and protein function. However, the strategy encounters limitations with respect to sequence and codon contexts. Selectivity is difficult to achieve in adenosine-rich sequences and some codons, like 5'-GAG, seem virtually inert. To overcome such restrictions, we systematically studied the possibilities of activating difficult codons by optimizing the guideRNA that is applied in trans. We find that all 5'-XAG codons with X = U, A, C, G are editable in vitro to a substantial amount of at least 50% once the guideRNA/mRNA duplex is optimized. Notably, some codons, including CAG and GAG, accept or even require the presence of 5'-mismatched neighboring base pairs. This was unexpected from the reported analysis of global editing preferences on large double-stranded RNA substrates. Furthermore, we report the usage of guanosine mismatching as a means to suppress unwanted off-site editing in proximity to targeted adenosine bases. Together, our findings are very important to achieve selective and efficient editing in difficult codon and sequence contexts.