The preparation of histological specimens from animals and humans is a multi-step process comprising tissue collection, fixation, and dehydration, followed by paraffin embedding. Each process can be ...achieved using different methods and substances. For example, dehydration may not be required depending on the substance used for embedding. The freezing technique described in the present study can be used for tissue collection and fixation. Tissues obtained using “in vivo cryotechnique (IVCT)” reflect blood flow and protein localization in body fluids at the time of tissue collection, making it an indispensable method in histological analyses of the future. This study utilized the IVCT to capture histological images of dynamic objects from multiple viewpoints and elucidate the mechanism underlying their movement control at the molecular level.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Pancreatic cancer is one of the most fatal cancers without druggable molecular targets. Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcriptional factor that promotes malignancy in ...various cancers including pancreatic cancer. Herein, we found that HIF-1 is accumulated in normoxic or moderate hypoxic areas of pancreatic cancer xenografts in vivo and is active even during normoxia in pancreatic cancer cells in vitro. This prompted us to analyze whether the HIF-1 activator Mint3 contributes to malignant features of pancreatic cancer. Mint3 depletion by shRNAs attenuated HIF-1 activity during normoxia and cell proliferation concomitantly with accumulated p21 and p27 protein in pancreatic cancer cells. Further analyses revealed that Mint3 increased transcription of the oncogenic ubiquitin ligase SKP2 in pancreatic cancer cells via HIF-1. This Mint3-HIF-1-SKP2 axis also promoted partial epithelial-mesenchymal transition, stemness features, and chemoresistance in pancreatic cancer cells. Even in vivo, Mint3 depletion attenuated tumor growth of orthotopically inoculated human pancreatic cancer AsPC-1 cells. Database and tissue microarray analyses showed that Mint3 expression is correlated with SKP2 expression in human pancreatic cancer specimens and high Mint3 expression is correlated with poor prognosis of pancreatic cancer patients. Thus, targeting Mint3 may be useful for attenuating the malignant features of pancreatic cancer.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large ...volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cancer cells adapt to various stress conditions by optimizing gene expression profiles via transcriptional and translational regulation. However, whether and how EXOSC9, a component of the RNA ...exosome complex, regulates adaptation to stress conditions and tumorigenicity in cancer cells remain unclear. Here, we examined the effects of EXOSC9 depletion on cancer cell growth under various stress conditions. EXOSC9 depletion attenuated growth and survival under various stress conditions in cancer cells. Interestingly, this also decreased the number of P-bodies, which are messenger ribonucleoprotein particles (mRNPs) required for stress adaptation. Meanwhile, EXOSC2/EXOSC4 depletion also attenuated P-body formation and stress resistance with decreased EXOSC9 protein. EXOSC9-mediated stress resistance and P-body formation were found to depend on the intact RNA-binding motif of this protein. Further, RNA-seq analyses identified 343 EXOSC9-target genes, among which, APOBEC3G contributed to defects in stress resistance and P-body formation in MDA-MB-231 cells. Finally, EXOSC9 also promoted xenografted tumor growth of MDA-MB-231 cells in an intact RNA-binding motif-dependent manner. Database analyses further showed that higher EXOSC9 activity, estimated based on the expression of 343 target genes, was correlated with poorer prognosis in some cancer patients. Thus, drugs targeting activity of the RNA exosome complex or EXOSC9 might be useful for cancer treatment.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Microglia are the resident macrophages of the central nervous system and play complex roles in the milieu of diseases including the primary diseases of myelin. Although mitochondria are critical for ...cellular functions and survival in the nervous system, alterations in and the roles of mitochondrial dynamics and associated signaling in microglia are still poorly understood. In the present study, by combining immunohistochemistry and 3D ultrastructural analyses, we show that mitochondrial fission/fusion in reactive microglia is differentially regulated from that in monocyte-derived macrophages and the ramified microglia of normal white matter in myelin disease models. Mouse cerebral microglia in vitro demonstrated that stimulation of TLR4 with lipopolysaccharide, widely used to examine microglial reactions, caused the activation of the mitochondrial fission protein, dynamin-related protein 1 (Drp1) and enhanced production of reactive oxygen species (ROS). The increase in the ROS level activated 5' adenosine monophosphate-activated protein kinase (AMPK), and facilitated elongation of mitochondria along the microtubule tracks. These results suggest that the polymorphic regulation of mitochondrial fission and fusion in reactive microglia is mediated by distinct signaling under inflammatory conditions, and modulates microglial phenotypes through the production of ROS.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
A membrane skeletal molecular complex, protein 4.1G–membrane palmitoylated protein 6 (MPP6)–Lin7–cell adhesion molecule 4 (CADM4), is incorporated in Schwann cells, especially in Schmidt–Lanterman ...incisures (SLIs), in the mouse peripheral nervous system (PNS). MPP6, Lin7, and CADM4 are transported to SLIs by 4.1G. In this study, we created MPP6-deficient mice and evaluated myelin structure and MPP6 protein complexes. In SLIs in MPP6-deficient nerves, Lin7 was rarely detected by immunohistochemistry and western blotting, but the localization and amount of CADM4 and 4.1G were not altered. Motor activity was not significantly impaired in a tail-suspension test, but the sciatic nerves of MPP6-deficient mice had thicker myelin in internodes by electron microscopy compared to that of wild-type mice. These results indicate that the MPP6–Lin7 complex regulates myelin formation.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, VSZLJ, ZAGLJ
We previously demonstrated that a membrane skeletal molecular complex, 4.1G–membrane palmitoylated protein 6 (MPP6)–cell adhesion molecule 4, is incorporated in Schwann cells in the peripheral ...nervous system (PNS). In this study, we evaluated motor activity and myelin ultrastructures in 4.1G-deficient (−/−) mice. When suspended by the tail, aged 4.1G
−/−
mice displayed spastic leg extension, especially after overwork. Motor-conduction velocity in 4.1G
−/−
mice was slower than that in wild-type mice. Using electron microscopy, 4.1G
−/−
mice exhibited myelin abnormalities: myelin was thicker in internodes, and attachment of myelin tips was distorted in some paranodes. In addition, we found a novel function of 4.1G for sorting a scaffold protein, Lin7, due to disappearance of the immunolocalization and reduction of the production of Lin7c and Lin7a in 4.1G
−/−
sciatic nerves, as well as the interaction of MPP6 and Lin7 with immunoprecipitation. Thus, we herein propose 4.1G functions as a signal for proper formation of myelin in PNS.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, VSZLJ, ZAGLJ
The primary cilium is a hair-like immotile organelle with specific membrane receptors, including the receptor of Hedgehog signaling, smoothened. The cilium organized in preosteoblasts promotes ...differentiation of the cells into osteoblasts (osteoblast differentiation) by mediating Hedgehog signaling to achieve bone formation. Notably, 4.1G is a plasma membrane-associated cytoskeletal protein that plays essential roles in various tissues, including the peripheral nervous system, testis, and retina. However, its function in the bone remains unexplored. In this study, we identified 4.1G expression in the bone. We found that, in the 4.1G-knockout mice, calcium deposits and primary cilium formation were suppressed in the trabecular bone, which is preosteoblast-rich region of the newborn tibia, indicating that 4.1G is a prerequisite for osteoblast differentiation by organizing the primary cilia in preosteoblasts. Next, we found that the primary cilium was elongated in the differentiating mouse preosteoblast cell line MC3T3-E1, whereas the knockdown of 4.1G suppressed its elongation. Moreover, 4.1G-knockdown suppressed the induction of the cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation. These results demonstrate a new regulatory mechanism of 4.1G in bone formation that promotes the primary ciliogenesis in the differentiating preosteoblasts and induction of cilia-mediated osteoblast differentiation, resulting in bone formation at the newborn stage.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Schmidt-Lanterman incisure (SLI) is a circular-truncated cone shape in the myelin internode that is a specific feature of myelinated nerve fibers formed in Schwann cells in the peripheral nervous ...system (PNS). The SLI circular-truncated cones elongate like spring at the narrow sites of beaded appearance nerve fibers under the stretched condition. In this chapter, we demonstrate various molecular complexes in SLI, and especially focus on membrane skeleton, protein 4.1G-membrane protein palmitoylated 6 (MPP6)-cell adhesion molecule 4 (CADM4). 4.1G was essential for the molecular targeting of MPP6 and CADM4 in SLI. Motor activity and myelin ultrastructures were abnormal in 4.1G-deficient mice, indicating the 4.1G function as a signal for proper formation of myelin in PNS. Thus, SLI probably has potential roles in the regulation of adhesion and signal transduction as well as in structural stability in Schwann cell myelin formation.
The protein 4.1 and membrane palmitoylated protein (MPP) families were originally found as components in the erythrocyte membrane skeletal protein complex, which helps maintain the stability of ...erythrocyte membranes by linking intramembranous proteins and meshwork structures composed of actin and spectrin under the membranes. Recently, it has been recognized that cells and tissues ubiquitously use this membrane skeletal system. Various intramembranous proteins, including adhesion molecules, ion channels, and receptors, have been shown to interact with the 4.1 and MPP families, regulating cellular and tissue dynamics by binding to intracellular signal transduction proteins. In this review, we focus on our previous studies regarding genetically modified animal models, especially on 4.1G, MPP6, and MPP2, to describe their functional roles in the peripheral nervous system, the central nervous system, the testis, and bone formation. As the membrane skeletal proteins are located at sites that receive signals from outside the cell and transduce signals inside the cell, it is necessary to elucidate their molecular interrelationships, which may broaden the understanding of cell and tissue functions.
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