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Scanning electron microscopy (SEM) has contributed to elucidating the ultrastructure of bio-specimens in three dimensions. SEM imagery detects several kinds of signals, of which secondary electrons (SEs) and backscattered electrons (BSEs) are the main electrons used in biological and biomedical research. SE and BSE signals provide a three-dimensional (3D) surface topography and information on the composition of specimens, respectively. Among the various sample preparation techniques for SE-mode SEM, the osmium maceration method is the only approach for examining the subcellular structure that does not require any reconstruction processes. The 3D ultrastructure of organelles, such as the Golgi apparatus, mitochondria, and endoplasmic reticulum has been uncovered using high-resolution SEM of osmium-macerated tissues. Recent instrumental advances in scanning electron microscopes have broadened the applications of SEM for examining bio-specimens and enabled imaging of resin-embedded tissue blocks and sections using BSE-mode SEM under low-accelerating voltages; such techniques are fundamental to the 3D-SEM methods that are now known as focused ion-beam SEM, serial block-face SEM, and array tomography (i.e., serial section SEM). This technical breakthrough has allowed us to establish an innovative BSE imaging technique called section-face imaging to acquire ultrathin information from resin-embedded tissue sections. In contrast, serial section SEM is a modern 3D imaging technique for creating 3D surface rendering models of cells and organelles from tomographic BSE images of consecutive ultrathin sections embedded in resin. In this article, we introduce our related SEM techniques that use SE and BSE signals, such as the osmium maceration method, semithin section SEM (section-face imaging of resin-embedded semithin sections), section-face imaging for correlative light and SEM, and serial section SEM, to summarize their applications to neural structure and discuss the future possibilities and directions for these methods.

VAMP-associated protein (VAP) is a type IV integral transmembrane protein at the endoplasmic reticulum (ER). Mutations in human VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS). The N-terminal major sperm protein (MSP) domain of VAPB ( Drosophila Vap33) is cleaved, secreted, and acts as a signaling ligand for several cell-surface receptors. Although extracellular functions of VAPB are beginning to be understood, it is unknown how the VAPB/Vap33 MSP domain facing the cytosol is secreted to the extracellular space. Here we show that Vap33 is transported to the plasma membrane, where the MSP domain is exposed extracellularly by topological inversion. The externalized MSP domain is cleaved by Matrix metalloproteinase 1/2 (Mmp1/2). Overexpression of Mmp1 restores decreased levels of extracellular MSP domain derived from ALS8-associated Vap33 mutants. We propose an unprecedented secretion mechanism for an ER-resident membrane protein, which may contribute to ALS8 pathogenesis. VAPB is a type IV transmembrane protein at the ER, and Iiis unknown how the VAPB MSP domain facing the cytosol is secreted. Here, the authors show that secretion of ER the protein VAPB requires topological inversion and Mmp1-mediated cleavage.

Stimulator of interferon genes (STING) is critical for the type I interferon responses to pathogen- or self-derived cytosolic DNA. STING signalling is terminated by ESCRT-driven lysosomal microautophagy. How STING is directly encapsulated by lysosomes has not yet been understood. Here we show that two lysosomal components, a phosphoinositide PI(3,5)P 2 and CHMP4B (a subunit of ESCRT-III subcomplex) are essential for STING encapsulation by lysosomes. Liposome sedimentation assay reveals that CHMP4B binds to PI(3,5)P 2 . The forced recruitment of the catalytic core of Pikfyve (a lipid kinase generating PI(3,5)P 2 ) to early endosomes, recruits a fraction of CHMP4B to early endosomes. CHMP4B mutant, defective in the binding to PI(3,5)P 2 , cannot restore the microautophagic degradation of STING or the resolution of the STING signalling in cells depleted of Chmp4b . Our results reveal a molecular mechanism that terminates innate immune signalling at the lysosomal membrane. Inhibition of Pikfyve, a kinase generating PI(3,5)P2, abolishes CHMP4B (an ESCRT-III subunit) recruitment to lysosomes, and leads to an accumulation of STING vesicles and sustained signaling. Our results reveal a lysosomal PI(3,5)P2/CHMP4B axis that terminates innate immune signaling.

A missense variant from methionine to arginine at codon 232 (M232R) of the prion protein gene accounts for ~ 15% of Japanese patients with genetic prion diseases. However, pathogenic roles of the M232R substitution for the induction of prion disease have remained elusive because family history is usually absent in patients with M232R. In addition, the clinicopathologic phenotypes of patients with M232R are indistinguishable from those of sporadic Creutzfeldt-Jakob disease patients. Furthermore, the M232R substitution is located in the glycosylphosphatidylinositol (GPI)-attachment signal peptide that is cleaved off during the maturation of prion proteins. Therefore, there has been an argument that the M232R substitution might be an uncommon polymorphism rather than a pathogenic mutation. To unveil the role of the M232R substitution in the GPI-attachment signal peptide of prion protein in the pathogenesis of prion disease, here we generated a mouse model expressing human prion proteins with M232R and investigated the susceptibility to prion disease. The M232R substitution accelerates the development of prion disease in a prion strain-dependent manner, without affecting prion strain-specific histopathologic and biochemical features. The M232R substitution did not alter the attachment of GPI nor GPI-attachment site. Instead, the substitution altered endoplasmic reticulum translocation pathway of prion proteins by reducing the hydrophobicity of the GPI-attachment signal peptide, resulting in the reduction of N -linked glycosylation and GPI glycosylation of prion proteins. To the best of our knowledge, this is the first time to show a direct relationship between a point mutation in the GPI-attachment signal peptide and the development of disease.

Background Lymphocyte recruitment into the portal tract is crucial not only for homeostatic immune surveillance but also for many liver diseases. However, the exact route of entry for lymphocytes into portal tract is still obscure. We investigated this question using a rat hepatic allograft rejection model. Methods A migration route was analyzed by immunohistological methods including a recently developed scanning electron microscopy method. Transmigration-associated molecules such as selectins, integrins, and chemokines and their receptors expressed by hepatic vessels and recruited T-cells were analyzed by immunohistochemistry and flow cytometry. Results The immunoelectron microscopic analysis clearly showed CD8β + cells passing through the portal vein (PV) endothelia. Furthermore, the migrating pathway seemed to pass through the endothelial cell body. Local vascular cell adhesion molecule-1 (VCAM-1) expression was induced in PV endothelial cells from day 2 after liver transplantation. Although intercellular adhesion molecule-1 (ICAM-1) expression was also upregulated, it was restricted to sinusoidal endothelia. Recipient T-cells in the graft perfusate were CD25 + CD44 + ICAM-1 + CXCR3 + CCR5 – and upregulated α4β1 or αLβ2 integrins. Immunohistochemistry showed the expression of CXCL10 in donor MHCII high cells in the portal tract as well as endothelial walls of PV. Conclusions We show for the first time direct evidence of T-cell transmigration across PV endothelial cells during hepatic allograft rejection. Interactions between VCAM-1 on endothelia and α4β1 integrin on recipient effector T-cells putatively play critical roles in adhesion and transmigration through endothelia. A chemokine axis of CXCL10 and CXCR3 also may be involved.

The Golgi stress response is an important cytoprotective system that enhances Golgi function in response to cellular demand, while cells damaged by prolonged Golgi stress undergo cell death to ensure the survival of organisms. OSW-1, a natural compound with anticancer activity, acts as a potent inhibitor of OSBP that transports cholesterol and phosphatidylinositol-4-phosphate (PI4P) at contact sites between the endoplasmic reticulum and the Golgi apparatus. Previously, we reported that OSW-1 induces the Golgi stress response, resulting in Golgi stress-induced transcription and cell death. However, the underlying molecular mechanism has been unknown. To reveal the mechanism of a novel pathway of the Golgi stress response regulating transcriptional induction and cell death (the cholesterol pathway), we performed a genome-wide knockout screen and found that transcriptional induction as well as cell death induced by OSW-1 was repressed in HeLa cells deficient in factors involved in the PI4P metabolism, such as PITPNB and PI4KB genes. Our data indicate that OSW-1 induces Golgi stress-dependent transcriptional induction and cell death through dysregulation of the PI4P metabolism in the Golgi apparatus.

Stimulator of interferon genes (STING) is critical for the type I interferon responses to pathogen- or self-derived cytosolic DNA. STING signalling is terminated by ESCRT-driven lysosomal microautophagy. How STING is directly encapsulated by lysosomes has not yet been understood. Here we show that two lysosomal components, a phosphoinositide PI(3,5)P and CHMP4B (a subunit of ESCRT-III subcomplex) are essential for STING encapsulation by lysosomes. Liposome sedimentation assay reveals that CHMP4B binds to PI(3,5)P . The forced recruitment of the catalytic core of Pikfyve (a lipid kinase generating PI(3,5)P ) to early endosomes, recruits a fraction of CHMP4B to early endosomes. CHMP4B mutant, defective in the binding to PI(3,5)P , cannot restore the microautophagic degradation of STING or the resolution of the STING signalling in cells depleted of Chmp4b. Our results reveal a molecular mechanism that terminates innate immune signalling at the lysosomal membrane.

Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived cytosolic DNA. STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven lysosomal microautophagy (LMA), the impairment of which leads to sustained inflammatory responses. It has been unknown how ESCRT targets STING directly to lysosomes. Here, through kinase inhibitor screening and knockdown experiments of all the individual components of ESCRT, we show that STING degradation requires PIKfyve (a lipid kinase that generates PI(3,5)P2) and CHMP4B/C (components of ESCRT-III subcomplex). Knockdown of Pikfyve or Chmp4b/c results in the accumulation of STING vesicles of a recycling endosomal origin in the cytosol, leading to sustained type I interferon response. CHMP4B/C localize at lysosomes and their lysosomal localization is abolished by interference with PIKfyve activity. Our results identify lysosomal ESCRT-III as a PI(3,5)P2 effector, reveal a role of the less characterized phosphoinositide PI(3,5)P2 in lysosomal biology, and provide insights into the molecular framework that distinguishes LMA from other cellular processes engaged with ESCRT.

Social connections are essential for human health. While curiosity and empathy are crucial psychological factors for a fulfilling life connected with others, it is unclear if acquired environmental factors influence them. In this cross-sectional observational study, 1,311 men and women aged 20–79 years living in Japan were observed to explore how lifestyle factors such as diet, sleep, and leisure activities (such as exercise and hobbies) impact curiosity and empathy. A hierarchical multiple regression analysis revealed that diet and leisure activities impacted curiosity, whereas hobbies influenced cognitive empathy but not affective empathy. Structural equation modeling indicated that men’s curiosity was influenced by diet, leisure activities, and work, whereas women’s curiosity was influenced by leisure activities and work. These findings suggest that diet and leisure activities can enhance curiosity and cognitive empathy, leading to improved well-being.

Social anxiety disorder (SAD) develops in the early teens and is a common disorder among university students. Understanding the predictive factors of SAD comorbid with major depressive episode (MDE) is important for student mental health care. The aim of this study was to identify the personality traits that predict SAD with MDE by analyzing longitudinal data of Japanese university students. In this retrospective study, Japanese university students who visited the health care center of Hokkaido University for the first time were divided into the following four groups: \"Control\" (n = 43), \"MDE\" (n = 16), \"SAD\" (n = 28), and \"SAD with MDE\" (n = 61) based on the Patient Health Questionnaire-9 (PHQ-9), the Liebowitz Social Anxiety Scale, and core anxiety symptoms for SAD in the Mini International Neuropsychiatric Interview during screening. Predictors for SAD with MDE were identified by a four-group comparison of the Temperament and Character Inventory and PHQ-9 data previously obtained at the enrollment using analysis of variance and post-hoc tests. Upon comparing the four groups using analysis of variance, there were significant differences in the baseline PHQ-9 summary score, Harm-Avoidance (HA), and Self-Directedness (SD). According to results of the post-hoc test, all three showed a significant difference between the \"Control\" and \"SAD with MDE.\" Furthermore, there was a significant difference in HA scores between \"SAD\" and \"Control.\" In all the groups, the mean time from enrollment to the first visit to the center was >2 years. A higher HA score at baseline is a predictor of SAD with or without MDE. Higher PHQ-9 summary and lower SD scores at baseline are predictive factors of SAD with MDE.