Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a ...highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm
tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.
The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates ...the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca2+-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca2+-dependent K+ channels (Kcnn2 and Kcnn3), voltage-gated Ca2+ channels (Cacna1g and Cacna1h), or Ca2+/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca2+ ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca2+-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals.
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•A simple model predicts Ca2+-dependent hyperpolarization regulates sleep duration•Impaired/enhanced Ca2+-dependent hyperpolarization decreases/increases sleep duration•Impaired Ca2+-dependent hyperpolarization increases neural excitability•Impaired Ca2+/calmodulin-dependent kinases (Camk2a/Camk2b) decreases sleep duration
Tatsuki et al. present that a Ca2+-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals. They predicted the hypothesis by a simple averaged-neuron model and verified it by phenotyping 21 KO mice and whole-brain imaging with pharmacological intervention.
SignificanceHuman sleep phenotypes are diversified by genetic and environmental factors, and a quantitative classification of sleep phenotypes would lead to the advancement of biomedical mechanisms ...underlying human sleep diversity. To achieve that, a pipeline of data analysis, including a state-of-the-art sleep/wake classification algorithm, the uniform manifold approximation and projection (UMAP) dimension reduction method, and the density-based spatial clustering of applications with noise (DBSCAN) clustering method, was applied to the 100,000-arm acceleration dataset. This revealed 16 clusters, including seven different insomnia-like phenotypes. This kind of quantitative pipeline of sleep analysis is expected to promote data-based diagnosis of sleep disorders and psychiatric disorders that tend to be complicated by sleep disorders.
Selective autophagy cargos are recruited to autophagosomes primarily by interacting with autophagosomal ATG8 family proteins via the LC3‐interacting region (LIR). The upstream sequence of most LIRs ...contains negatively charged residues such as Asp, Glu, and phosphorylated Ser and Thr. However, the significance of LIR phosphorylation (compared with having acidic amino acids) and the structural basis of phosphorylated LIR–ATG8 binding are not entirely understood. Here, we show that the serine residues upstream of the core LIR of the endoplasmic reticulum (ER)‐phagy receptor TEX264 are phosphorylated by casein kinase 2, which is critical for its interaction with ATG8s, autophagosomal localization, and ER‐phagy. Structural analysis shows that phosphorylation of these serine residues increases binding affinity by producing multiple hydrogen bonds with ATG8s that cannot be mimicked by acidic residues. This binding mode is different from those of other ER‐phagy receptors that utilize a downstream helix, which is absent from TEX264, to increase affinity. These results suggest that phosphorylation of the LIR is critically important for strong LIR–ATG8 interactions, even in the absence of auxiliary interactions.
Synopsis
Casein kinase 2 phosphorylates the serine residues upstream of the core LIR of the endoplasmic reticulum (ER)‐phagy receptor TEX264, which enhances the interaction between TEX264 and ATG8 proteins.
The serine residues upstream of the core LIR of the ER‐phagy receptor TEX264 are phosphorylated by casein kinase 2.
Phosphorylation of serine 271 and 272 increases binding affinity to ATG8 proteins by producing multiple hydrogen bonds with ATG8s.
Casein kinase 2 phosphorylates the serine residues upstream of the core LIR of the endoplasmic reticulum (ER)‐phagy receptor TEX264, which enhances the interaction between TEX264 and ATG8 proteins.
To clarify the significance of quantitative analyses of amyloid proteins in clinical practice and in research relating to systemic amyloidoses, we applied mass spectrometry-based quantification by ...isotope-labeled cell-free products (MS-QBIC) to formalin-fixed, paraffin-embedded (FFPE) tissues. The technique was applied to amyloid tissues collected by laser microdissection of Congo red-stained lesions of FFPE specimens. Twelve of 13 amyloid precursor proteins were successfully quantified, including serum amyloid A (SAA), transthyretin (TTR), immunoglobulin kappa light chain (IGK), immunoglobulin lambda light chain (IGL), beta-2-microglobulin (B2M), apolipoprotein (Apo) A1, Apo A4, Apo E, lysozyme, Apo A2, gelsolin, and fibrinogen alpha chain; leukocyte cell-derived chemotaxin-2 was not detected. The quantification of SAA, TTR, IGK, IGL, and B2M confirmed the responsible proteins, even when the immunohistochemical results were not decisive. Considerable amounts of Apo A1, Apo A4, and Apo E were deposited in parallel amounts with the responsible proteins. Quantification of amyloid protein by MS-QBIC is feasible and useful for the classification of and research on systemic amyloidoses.
The identification of molecular networks at the system level in mammals is accelerated by next-generation mammalian genetics without crossing, which requires both the efficient production of ...whole-body biallelic knockout (KO) mice in a single generation and high-performance phenotype analyses. Here, we show that the triple targeting of a single gene using the CRISPR/Cas9 system achieves almost perfect KO efficiency (96%–100%). In addition, we developed a respiration-based fully automated non-invasive sleep phenotyping system, the Snappy Sleep Stager (SSS), for high-performance (95.3% accuracy) sleep/wake staging. Using the triple-target CRISPR and SSS in tandem, we reliably obtained sleep/wake phenotypes, even in double-KO mice. By using this system to comprehensively analyze all of the N-methyl-D-aspartate (NMDA) receptor family members, we found Nr3a as a short-sleeper gene, which is verified by an independent set of triple-target CRISPR. These results demonstrate the application of mammalian reverse genetics without crossing to organism-level systems biology in sleep research.
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•A triple-target CRISPR method achieved almost perfect knockout efficiency•SSS achieved non-invasive fully automated high-performance sleep phenotyping•Genetics without crossing revealed Nr3a as a short-sleeper gene
Sunagawa et al. present an application of mammalian reverse genetics without crossing by developing two methods. The authors improve KO mice production using a triple-target CRISPR and combine this with a non-invasive respiration-based automated sleep phenotyping system, the Snappy Sleep Stager. Combining these methods, the authors found that the Nr3a KO is a short-sleeper.
Abstract
Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein ...and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis through stabilization of the actin network. MYH9 accumulates in NEK9 LIR mutant cells and mice, and depletion of MYH9 restores ciliogenesis in NEK9 LIR mutant cells. These results suggest that NEK9 regulates ciliogenesis by acting as an autophagy adaptor for MYH9. Given that the LIR in NEK9 is conserved only in land vertebrates, the acquisition of the autophagic regulation of the NEK9–MYH9 axis in ciliogenesis may have possible adaptive implications for terrestrial life.
Sleep is a fundamental property conserved across species. The homeostatic induction of sleep indicates the presence of a mechanism that is progressively activated by the awake state and that induces ...sleep. Several lines of evidence support that such function, namely, sleep need, lies in the neuronal assemblies rather than specific brain regions and circuits. However, the molecular mechanism underlying the dynamics of sleep need is still unclear. This review aims to summarize recent studies mainly in rodents indicating that protein phosphorylation, especially at the synapses, could be the molecular entity associated with sleep need. Genetic studies in rodents have identified a set of kinases that promote sleep. The activity of sleep-promoting kinases appears to be elevated during the awake phase and in sleep deprivation. Furthermore, the proteomic analysis demonstrated that the phosphorylation status of synaptic protein is controlled by the sleep-wake cycle. Therefore, a plausible scenario may be that the awake-dependent activation of kinases modifies the phosphorylation status of synaptic proteins to promote sleep. We also discuss the possible importance of multisite phosphorylation on macromolecular protein complexes to achieve the slow dynamics and physiological functions of sleep in mammals.
Abstract Electroencephalogram (EEG) and electromyogram (EMG) are fundamental tools in sleep research. However, investigations into the statistical properties of rodent EEG/EMG signals in the ...sleep–wake cycle have been limited. The lack of standard criteria in defining sleep stages forces researchers to rely on human expertise to inspect EEG/EMG. The recent increasing demand for analysing large‐scale and long‐term data has been overwhelming the capabilities of human experts. In this study, we explored the statistical features of EEG signals in the sleep–wake cycle. We found that the normalized EEG power density profile changes its lower and higher frequency powers to a comparable degree in the opposite direction, pivoting around 20–30 Hz between the NREM sleep and the active brain state. We also found that REM sleep has a normalized EEG power density profile that overlaps with wakefulness and a characteristic reduction in the EMG signal. Based on these observations, we proposed three simple statistical features that could span a 3D space. Each sleep–wake stage formed a separate cluster close to a normal distribution in the 3D space. Notably, the suggested features are a natural extension of the conventional definition, making it useful for experts to intuitively interpret the EEG/EMG signal alterations caused by genetic mutations or experimental treatments. In addition, we developed an unsupervised automatic staging algorithm based on these features. The developed algorithm is a valuable tool for expediting the quantitative evaluation of EEG/EMG signals so that researchers can utilize the recent high‐throughput genetic or pharmacological methods for sleep research.
The covalent conjugation of ubiquitin family proteins is a widespread post-translational protein modification. In the ubiquitin family, the ATG8 subfamily is exceptional because it is conjugated ...mainly to phospholipids. However, it remains unknown whether other ubiquitin family proteins are also conjugated to phospholipids. Here, we report that ubiquitin is conjugated to phospholipids, mainly phosphatidylethanolamine (PE), in yeast and mammalian cells. Ubiquitinated PE (Ub-PE) accumulates at endosomes and the vacuole (or lysosomes), and its level increases during starvation. Ub-PE is also found in baculoviruses. In yeast, PE ubiquitination is catalyzed by the canonical ubiquitin system enzymes Uba1 (E1), Ubc4/5 (E2), and Tul1 (E3) and is reversed by Doa4. Liposomes containing Ub-PE recruit the ESCRT components Vps27-Hse1 and Vps23 in vitro. Ubiquitin-like NEDD8 and ISG15 are also conjugated to phospholipids. These findings suggest that the conjugation to membrane phospholipids is not specific to ATG8 but is a general feature of the ubiquitin family.
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•PE in endosomes and vacuoles/lysosomes is ubiquitinated•PE is ubiquitinated by Uba1, Ubc4/5, and Tul1 and deubiquitinated by Doa4•Ub-PE can recruit the ESCRT components Vps27-Hse1 and Vps23•The ubiquitin-like proteins NEDD8 and ISG15 are also conjugated to phospholipids
Sakamaki et al. show that the phospholipid phosphatidylethanolamine in endosomes and vacuoles (lysosomes in mammals) is ubiquitinated by the canonical ubiquitin system. The ubiquitin-like proteins NEDD8 and ISG15 are also conjugated to phospholipids, suggesting that the conjugation to phospholipids is a general feature of the ubiquitin family.
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