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Background Cryo-electron tomography (cryo-ET) enables the 3D visualization of cellular organization in near-native state which plays important roles in the field of structural cell biology. However, due to the low signal-to-noise ratio (SNR), large volume and high content complexity within cells, it remains difficult and time-consuming to localize and identify different components in cellular cryo-ET. To automatically localize and recognize in situ cellular structures of interest captured by cryo-ET, we proposed a simple yet effective automatic image analysis approach based on Faster-RCNN. Results Our experimental results were validated using in situ cyro-ET-imaged mitochondria data. Our experimental results show that our algorithm can accurately localize and identify important cellular structures on both the 2D tilt images and the reconstructed 2D slices of cryo-ET. When ran on the mitochondria cryo-ET dataset, our algorithm achieved Average Precision >0.95. Moreover, our study demonstrated that our customized pre-processing steps can further improve the robustness of our model performance. Conclusions In this paper, we proposed an automatic Cryo-ET image analysis algorithm for localization and identification of different structure of interest in cells, which is the first Faster-RCNN based method for localizing an cellular organelle in Cryo-ET images and demonstrated the high accuracy and robustness of detection and classification tasks of intracellular mitochondria. Furthermore, our approach can be easily applied to detection tasks of other cellular structures as well.

Cryogenic electron microscopy (cryo-EM) can reveal the molecular details of biological processes in their native, cellular environment at atomic resolution. However, few cells are sufficiently thin to permit imaging with cryo-EM. Thinning of frozen cells to <500 nm lamellae by focused-ion-beam (FIB) milling has enabled visualization of cellular structures with cryo-EM. FIB milling represents a significant advance over prior approaches because of its ease of use, scalability, and lack of large-scale sample distortions. However, the amount of damage it causes to a thinned cell section has not yet been determined. We recently described an approach for detecting and identifying single molecules in cryo-EM images of cells using 2D template matching (2DTM). 2DTM is sensitive to small differences between a molecular model (template) and the detected structure (target). Here, we use 2DTM to demonstrate that under the standard conditions used for machining lamellae of biological samples, FIB milling introduces a layer of variable damage that extends to a depth of 60 nm from each lamella surface. This layer of damage limits the recovery of information for in situ structural biology. We find that the mechanism of FIB milling damage is distinct from radiation damage during cryo-EM imaging. By accounting for both electron scattering and FIB milling damage, we estimate that FIB milling damage with current protocols will negate the potential improvements from lamella thinning beyond 90 nm.

Nucleotide-binding and oligomerization domain–like receptor (NLR) proteins oligomerize into multiprotein complexes termed inflammasomes when activated. Their autoinhibition mechanism remains poorly defined. Here, we report the crystal structure of mouse NLRC4 in a closed form. The adenosine diphosphate–mediated interaction between the central nucleotide-binding domain (NBD) and the winged-helix domain (WHD) was critical for stabilizing the closed conformation of NLRC4. The helical domain HD2 repressively contacted a conserved and functionally important α-helix of the NBD. The C-terminal leucine-rich repeat (LRR) domain is positioned to sterically occlude one side of the NBD domain and consequently sequester NLRC4 in a monomeric state. Disruption of ADP-mediated NBD-WHD or NBD-HD2/NBD-LRR interactions resulted in constitutive activation of NLRC4. Together, our data reveal the NBD-organized cooperative autoinhibition mechanism of NLRC4 and provide insight into its activation.

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels 1 . Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle 2 ) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions. Open-access 3D images of whole cells and tissues with combined finer resolution and larger sample size are enabled by advances in focused ion beam-scanning electron microscopy.

Cryo-electron tomography allows the routine visualization of cellular landscapes in three dimensions at nanometer-range resolutions. When combined with single-particle tomography, it is possible to obtain near-atomic resolution structures of frequently occurring macromolecules within their native environment. Two outstanding challenges associated with cryo-electron tomography/single-particle tomography are the automatic identification and localization of proteins, tasks that are hindered by the molecular crowding inside cells, imaging distortions characteristic of cryo-electron tomography tomograms and the sheer size of tomographic datasets. Current methods suffer from low accuracy, demand extensive and time-consuming manual labeling or are limited to the detection of specific types of proteins. Here, we present MiLoPYP, a two-step dataset-specific contrastive learning-based framework that enables fast molecular pattern mining followed by accurate protein localization. MiLoPYP’s ability to effectively detect and localize a wide range of targets including globular and tubular complexes as well as large membrane proteins, will contribute to streamline and broaden the applicability of high-resolution workflows for in situ structure determination. MiLoPYP is a two-step, dataset-specific contrastive learning-based method for fast and accurate detection and localization of a diverse range of target structures in cryo-electron tomography data, enabling improved in situ structural biology.

The cGAS-STING cytoplasmic-DNA-sensing pathway is activated by accumulation of extrachromosomal telomere repeats, and this proliferation-inhibition mechanism is defective in ALT cancer cells. Extrachromosomal telomere repeat (ECTR) DNA is unique to cancer cells that maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, but the role of ECTRs in ALT development remains elusive. We found that induction of ECTRs in normal human fibroblasts activated the cGAS-STING-TBK1-IRF3 signaling axis to trigger IFNβ production and a type I interferon response, resulting in cell-proliferation defects. In contrast, ALT cancer cells are commonly defective in sensing cytosolic DNA. We found that STING expression was inhibited in ALT cancer cell lines and transformed ALT cells. Notably, the ALT suppressors histone H3.3 and the ATRX–Daxx histone chaperone complex were also required to activate the DNA-sensing pathway. Collectively, our data suggest that the loss of the cGAS-STING pathway may be required to evade ECTR-induced anti-proliferation effects and permit ALT development, and this requirement may be exploited for treatments specific to cancers utilizing the ALT pathway.

Fluorescent proteins (FPs) are widely used as optical sensors, whereas other light-absorbing domains have been used for optical control of protein localization or activity. Here, we describe light-dependent dissociation and association in a mutant of the photochromic FP Dronpa, and we used it to control protein activities with light. We created a fluorescent light-inducible protein design in which Dronpa domains are fused to both termini of an enzyme domain. In the dark, the Dronpa domains associate and cage the protein, but light induces Dronpa dissociation and activates the protein. This method enabled optical control over guanine nucleotide exchange factor and protease domains without extensive screening. Our findings extend the applications of FPs from exclusively sensing functions to also encompass optogenetic control.

The surrounding rock of tunnel engineering in an alpine mountainous environment is prone to frequent freeze–thaw action due to fissure water and temperature differential, which leads to crack propagation and even failure in rock. Freezing sandstone CT damage-free scanning studies were conducted. Based on deep learning theory, the U-Net network technique is utilized to naturally merge high-resolution properties of frozen rock CT images in the shrinking path with low-resolution characteristics in the expansion path. Intelligent detection of freezing rock fissures and geometric information parameters at the pixel level has been accomplished. The primary fracture structure and its parameters of the sandstone with natural damage during the freeze–thaw process are obtained, and the pixel-level intelligent identification of the meso-structure and geometric information parameters of the freeze–thaw rock fracture is realized. This justifies the classification of naturally cracked rock under load and freeze–thaw as a discrete time-dimensional evolution system. The dynamic process and mechanical characteristics of meso-damage propagation of naturally fractured rock under freeze–thaw and compression load are investigated using Casrock numerical computation software, which is based on the cellular automata theory. The results reveal that when the number of freeze–thaw cycles rises, the random rate of fracture network structure distribution increases, the uniformity of fracture distribution increases, and the dominating direction decreases. The sandstone's secondary fractures progressively increase as the fracture dominant angle rises, and the rock sample's failure mode eventually shifts from tensile failure to compression-shear mixed failure. When the comprehensive dominant angle of fracture is 60°, the fracture of freeze–thaw rock is more prone to expansion and its mechanical strength deteriorates more. The fractured rock creates narrow strip directional damage along the end of the original fracture when subjected to compressive load, exhibiting typical localization features. The main crack and the secondary crack dominate the crack progression. The number of secondary fractures inside sandstone steadily grows as the fracture's comprehensive dominant angle increases. The direction of the crack penetration development is determined by the comprehensive dominating angle of the fracture.

Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events. We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells.

Diabetes is associated with a dramatic mortality rate due to its vascular complications. Chronic hyperglycemia in diabetes leads to enhanced glycation of erythrocytes and oxidative stress. Even though erythrocytes play a determining role in vascular complications, very little is known about how erythrocyte structure and functionality can be affected by glycation. Our objective was to decipher the impact of glycation on erythrocyte structure, oxidative stress parameters and capacity to interact with cultured human endothelial cells. In vitro glycated erythrocytes were prepared following incubation in the presence of different concentrations of glucose. To get insight into the in vivo relevance of our results, we compared these data to those obtained using red blood cells purified from diabetics or non-diabetics. We measured erythrocyte deformability, susceptibility to hemolysis, reactive oxygen species production and oxidative damage accumulation. Altered structures, redox status and oxidative modifications were increased in glycated erythrocytes. These modifications were associated with reduced antioxidant defence mediated by enzymatic activity. Enhanced erythrocyte phagocytosis by endothelial cells was observed when cultured with glycated erythrocytes, which was associated with increased levels of phosphatidylserine-likely as a result of an eryptosis phenomenon triggered by the hyperglycemic treatment. Most types of oxidative damage identified in in vitro glycated erythrocytes were also observed in red blood cells isolated from diabetics. These results bring new insights into the impact of glycation on erythrocyte structure, oxidative damage and their capacity to interact with endothelial cells, with a possible relevance to diabetes.