For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 μm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.

OBJECTIVE: A recent genome-wide association study (GWAS) reported a significant genetic association between rs34330 of cyclin-dependent kinase inhibitor 1B (CDKN1B) and risk of systemic lupus erythematosus (SLE) in Han Chinese. This study aims to validate the reported association and elucidate the biochemical mechanisms underlying the variant's effect.

METHODS: We performed allelic association with SLE followed by meta-analysis across 11 independent cohorts (n=28,872). We applied in silico bioinformatics and experimental validation in SLE-relevant cell lines to determine the functional consequences of rs34330.

RESULTS: We replicated genetic association between SLE and rs34330 (P =5.29x10 , OR (95% CI)=0.84 (0.81-0.87)). Follow-up bioinformatics and eQTL analysis suggest that rs34330 is located in active chromatin and potentially regulates several target genes. Using luciferase and ChIP-qPCR, we demonstrated substantial allele-specific promoter and enhancer activity, and allele-specific binding of three histone marks (H3K27ac, H3K4me3, H3K4me1), RNA pol II, CTCF, and a critical immune transcription factor (IRF-1). Chromosome conformation capture (3C) detected long-range chromatin interactions between rs34330 and the promoters of neighboring genes APOLD1 and DDX47, and effects on CDKN1B and the other target genes were directly validated by CRISPR-based genome editing. Finally, CRISPR-dCas9-based epigenetic activation/silencing confirmed these results. Gene-edited cell lines also showed higher levels of proliferation and apoptosis.

CONCLUSION: Collectively, these findings suggest a mechanism whereby the rs34330 risk allele (C) influences the presence of histone marks, RNA pol II, and the IRF-1 transcription factor to regulate expression of several target genes linked to proliferation and apoptosis, which potentially underlie the association of rs34330 with SLE.

Regulated vesicle exocytosis is a key response to extracellular stimuli in diverse physiological processes; including hormone regulated short-term urine concentration. In the renal collecting duct, the water channel aquaporin-2 localizes to the apical plasma membrane as well as small, sub-apical vesicles. In response to stimulation with the antidiuretic hormone, arginine vasopressin, aquaporin-2 containing vesicles fuse with the plasma membrane, which increases collecting duct water reabsorption and thus, urine concentration. The nano-scale size of these vesicles has limited analysis of their 3D organization. Using a cell system combined with 3D super resolution microscopy, we provide the first direct analysis of the 3D network of aquaporin-2 containing exocytic vesicles in a cell culture system. We show that aquaporin-2 vesicles are 43 ± 3nm in diameter, a size similar to synaptic vesicles, and that one fraction of AQP2 vesicles localized with the sub-cortical F-actin layer and the other localized in between the F-actin layer and the plasma membrane. Aquaporin-2 vesicles associated with F-actin and this association was enhanced in a serine 256 phospho-mimic of aquaporin-2, whose phosphorylation is a key event in antidiuretic hormone-mediated aquaporin-2 vesicle exocytosis.

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. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

The lack of efficient tools to label multiple endogenous targets in cell lines without staining or fixation has limited our ability to track physiological and pathological changes in cells over time via live-cell studies. Here, we outline the FAST-HDR vector system to be used in combination with CRISPR-Cas9 to allow visual live-cell studies of up to three endogenous proteins within the same cell line. Our approach utilizes a novel set of advanced donor plasmids for homology-directed repair and a streamlined workflow optimized for microscopy-based cell screening to create genetically modified cell lines that do not require staining or fixation to accommodate microscopy-based studies. We validated this new methodology by developing two advanced cell lines with three fluorescent-labeled endogenous proteins that support high-content imaging without using antibodies or exogenous staining. We applied this technology to study seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) viral proteins to understand better their effects on autophagy, mitochondrial dynamics, and cell growth. Using these two cell lines, we were able to identify the protein ORF3a successfully as a potent inhibitor of autophagy, inducer of mitochondrial relocalization, and a growth inhibitor, which highlights the effectiveness of live-cell studies using this technology.

Internal models are nowadays customarily used in different domains of science and engineering to describe how living organisms or artificial computational units embed their acquired knowledge about recurring events taking place in the surrounding environment. This article reviews the internal model principle in control theory, bioengineering, and neuroscience, illustrating the fundamental concepts and theoretical developments of the few last decades of research.

To accurately track self-location, animals need to integrate their movements through space. In amniotes, representations of self-location have been found in regions such as the hippocampus. It is unknown whether more ancient brain regions contain such representations and by which pathways they may drive locomotion. Fish displaced by water currents must prevent uncontrolled drift to potentially dangerous areas. We found that larval zebrafish track such movements and can later swim back to their earlier location. Whole-brain functional imaging revealed the circuit enabling this process of positional homeostasis. Position-encoding brainstem neurons integrate optic flow, then bias future swimming to correct for past displacements by modulating inferior olive and cerebellar activity. Manipulation of position-encoding or olivary neurons abolished positional homeostasis or evoked behavior as if animals had experienced positional shifts. These results reveal a multiregional hindbrain circuit in vertebrates for optic flow integration, memory of self-location, and its neural pathway to behavior.Competing Interest StatementThe authors have declared no competing interest.