Simulating the Cellular Context in Synthetic Datasets for Cryo-Electron Tomography.

Cryo-electron tomography (cryo-ET) allows to visualize the cellular context at macromolecular level. To date, the impossibility of obtaining a reliable ground truth is limiting the application of deep learning-based image processing algorithms in this field. As a consequence, there is a growing demand of realistic synthetic datasets for training deep learning algorithms.

Atg18 oligomer organization in assembled tubes and on lipid membrane scaffolds.

Autophagy-related protein 18 (Atg18) participates in the elongation of early autophagosomal structures in concert with Atg2 and Atg9 complexes. How Atg18 contributes to the structural coordination of Atg2 and Atg9 at the isolation membrane remains to be understood. Here, we determined the cryo-EM structures of Atg18 organized in helical tubes, Atg18 oligomers in solution as well as on lipid membrane scaffolds.

MemBrain: A deep learning-aided pipeline for detection of membrane proteins in Cryo-electron tomograms.

Background And Objective: Cryo-electron tomography (cryo-ET) is an imaging technique that enables 3D visualization of the native cellular environment at sub-nanometer resolution, providing unpreceded insights into the molecular organization of cells. However, cryo-electron tomograms suffer from low signal-to-noise ratios and anisotropic resolution, which makes subsequent image analysis challenging. In particular, the efficient detection of membrane-embedded proteins is a problem still lacking satisfactory solutions.

Statistical spatial analysis for cryo-electron tomography.

Cryo-electron tomography (cryo-ET) is uniquely suited to precisely localize macromolecular complexes in situ, that is in a close-to-native state within their cellular compartments, in three-dimensions at high resolution. Point pattern analysis (PPA) allows quantitative characterization of the spatial organization of particles. However, current implementations of PPA functions are not suitable for applications to cryo-ET data because they do not consider the real, typically irregular 3D shape of cellular compartments and molecular complexes.

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.

In situ architecture of neuronal α-Synuclein inclusions.

The molecular architecture of α-Synuclein (α-Syn) inclusions, pathognomonic of various neurodegenerative disorders, remains unclear. α-Syn inclusions were long thought to consist mainly of α-Syn fibrils, but recent reports pointed to intracellular membranes as the major inclusion component. Here, we use cryo-electron tomography (cryo-ET) to image neuronal α-Syn inclusions in situ at molecular resolution.

Trans-synaptic assemblies link synaptic vesicles and neuroreceptors.

Synaptic transmission is characterized by fast, tightly coupled processes and complex signaling pathways that require a precise protein organization, such as the previously reported nanodomain colocalization of pre- and postsynaptic proteins. Here, we used cryo-electron tomography to visualize synaptic complexes together with their native environment comprising interacting proteins and lipids on a 2- to 4-nm scale. Using template-free detection and classification, we showed that tripartite trans-synaptic assemblies (subcolumns) link synaptic vesicles to postsynaptic receptors and established that a particular displacement between directly interacting complexes characterizes subcolumns.

The structural basis of Rubisco phase separation in the pyrenoid.

Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii.

Reliable estimation of membrane curvature for cryo-electron tomography.

Curvature is a fundamental morphological descriptor of cellular membranes. Cryo-electron tomography (cryo-ET) is particularly well-suited to visualize and analyze membrane morphology in a close-to-native state and molecular resolution. However, current curvature estimation methods cannot be applied directly to membrane segmentations in cryo-ET, as these methods cannot cope with some of the artifacts introduced during image acquisition and membrane segmentation, such as quantization noise and open borders.

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.

Dynamic instability of clathrin assembly provides proofreading control for endocytosis.

Clathrin-mediated endocytosis depends on the formation of functional clathrin-coated pits that recruit cargos and mediate the uptake of those cargos into the cell. However, it remains unclear whether the cargos in the growing clathrin-coated pits are actively monitored by the coat assembly machinery. Using a cell-free reconstitution system, we report that clathrin coat formation and cargo sorting can be uncoupled, indicating that a checkpoint is required for functional cargo incorporation.

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.

The Eukaryotic CO-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization.

Approximately 30%-40% of global CO fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division.

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).

A novel method to increase LinLog CMOS sensors' performance in high dynamic range scenarios.

Images from high dynamic range (HDR) scenes must be obtained with minimum loss of information. For this purpose it is necessary to take full advantage of the quantification levels provided by the CCD/CMOS image sensor. LinLog CMOS sensors satisfy the above demand by offering an adjustable response curve that combines linear and logarithmic responses.