シンポジウム

シンポジウム 53 脳の細胞内外の構造を超解像可視化技術で明らかにする 53 Fantastic voyage into the intra- and inter-cellular structures in the brain 座長：野住素広（新潟大院・医歯学・神経生化学）・本多敦子（新潟大・医学部・医学科研究推進センター）

2022年7月2日　14:00～14:24　ラグナガーデンホテル羽衣：中　第9会場 3S09a-01 STED顕微鏡でみる生きたニューロン・グリア相互作用 Dissecting neuron-glia interaction using live STED microscopy 有薗美沙(1)、Valentin Nägerl(2) 1. 京都大学大学院医学研究科、2. ボルドー大学 Misa Arizono(1), Valentin Nägerl(2) 1. Kyoto University Graduate School of Medicine, 2. Bordeaux University Keyword: neuron-glia interaction, extracellular space, STED microscopy, lattice light sheet microscopy Astrocytic Ca²⁺ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the structure of tripartite synapses with regular light microscopy. Using 3D-STED microscopy in living organotypic brain slices, we observed that the spongiform domain of astrocytes was composed of round enlarged nodes and thin shafts, which often formed ring-like structures. FRAP experiments and Ca²⁺ imaging showed that nodes were biochemical compartments and Ca²⁺ microdomains. Mapping astrocytic Ca²⁺ signals onto STED images of tripartite synapses showed that node Ca²⁺ signals were associated with individual synapses, identifying nodes as the likely functional astrocytic component of tripartite synapses. Applying the SUper-resolution SHadow Imaging technique (SUSHI) to astrocyte-labeled organotypic hippocampal brain slices, we were also able to concurrently image the complex entwinement of astrocytes and the extracelluar space in detail, providing a new angle to understanding the physiology of the tripartite synapse. To further characterize the properties of nodes, we recently established ultra-fast Ca²⁺ imaging in 2D and 3D using lattice light-sheet microscopy, which reconciles high temporal and spatial resolution with low phototoxicity. We show that node Ca²⁺signals can be as transient as < 100 ms and can be highly restricted in space. Finally, we show that glutamate uncaging elicits Ca²⁺responses in nodes, indicating that nodes are equipped with the signaling machinery to respond to synaptic activity. Our combined super-resolution and light-sheet imaging approaches open up the possibility to dissect the bidirectional crosstalk between the neuronal and glial elements of tripartite synapses, and evaluate its impact on neural circuit function with unprecedented spatial and temporal resolution and sensitivity.		2022年7月2日　14:24～14:48　ラグナガーデンホテル羽衣：中　第9会場 3S09a-02 3D-SEM法によるオルガネラの立体構造解明 Elucidation of three-dimensional organelle architecture using three-dimensional scanning electron microscopy techniques 甲賀大輔(1) 1. 旭川医科大学 Daisuke Koga(1) 1. Asahikawa Medical University Keyword: SEM, 3D, organelle Scanning electron microscopy (SEM) has been widely used to analyze the three-dimensional (3D) topography of biological specimens. Most studies using SEM are concerned with the 3D morphology of tissues and cell surface architecture. Among numerous SEM techniques, the osmium maceration method is the only approach that allows the 3D visualization of membranous organelles, such as the Golgi apparatus, mitochondria and endoplasmic reticulum, without reconstruction. In the maceration method, soluble cytoplasmic proteins are removed from the freeze-fractured surface of cells using a diluted osmium tetroxide solution, which enables the direct 3D visualization of organelle architecture. We have investigated the 3D ultrastructure of the Golgi apparatus in different cell types by high-resolution SEM of osmium-macerated tissues and revealed the morphological diversity of this organelle. However, it is difficult to visualize the entire shape of the Golgi apparatus because structures located deep below the freeze-cracked surface of osmium-macerated tissues are invisible to SEM. To overcome this limitation, we developed a 3D imaging technique, serial section SEM, which is based on the systematic collection of images from serial ultrathin sections on a solid substrate, such as a glass microscope slide. This imaging technique enables the 3D reconstruction of subcellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum and Golgi apparatus. We have demonstrated the full shape of the Golgi apparatus in various cell types and demonstrated that its configuration varies depending on the cell type; the Golgi apparatus in cerebellar Purkinje cells forms a basket-like structure, while it is spherical in shape in pituitary gonadotropes. We have also confirmed that the Golgi apparatus forms a continuous structure, which is located in a large area of the cytoplasm. Here, we present beautiful organelle images obtained by the osmium maceration method and precise 3D-reconstructed models of the Golgi apparatus in different cell types obtained using the serial section SEM technique. In addition, the potential of the 3D-SEM techniques for organelle research will be discussed.

2022年7月2日　14:48～15:12　ラグナガーデンホテル羽衣：中　第9会場 3S09a-03 神経成長円錐における膜交通ダイナミクスの超解像ライブイメージング解析 Dynamics of membrane traffic in the neuronal growth cone revealed by super-resolution live imaging 戸島拓郎(1) 1. 理化学研究所光量子工学研究センター Takuro Tojima(1) 1. RIKEN Center for Advanced Photonics Keyword: growth cone, membrane traffic, super-resolution live imaging To create precise neuronal networks during embryonic brain development, the axonal growth cone migrates long distance along the correct path. On the way to the final target, the growth cone changes its migration direction in response to the graded distributions of extracellular axon guidance cues. We previously demonstrated that localized exocytosis and endocytosis play essential roles in growth cone responses to the cues, i.e., vesicle-associated membrane protein 2 (VAMP2)-mediated exocytosis upregulated on one side of the growth cone drives attractive turning, whereas localized upregulation of clathrin-mediated endocytosis drives repulsive turning (Tojima et al, Nat Neurosci 10:58-66. 2007; Tojima et al, J Neurosci 29:7886-7897. 2009; Tojima et al, Neuron 66:370-377. 2010; Tojima et al, J Neurosci 34:7165-7178. 2014; Tojima et al, Nat Rev Neurosci 12:191-203. 2011). However, it remains unclear what cargo proteins are exocytosed and endocytosed in the growth cone, and how the intracellular membrane traffic system orchestrates cargo delivery to, and removal from, the growth cone plasma membrane in a spatiotemporally-regulated manner. As a first step to address these questions, we visualize intracellular membranous organelles, e.g., the endoplasmic reticulum and Golgi apparatus, in living growth cones by super-resolution confocal live imaging microscopy (SCLIM) we developed. We also visualize, using the retention using selective hooks (RUSH) system (Boncompain et al, Nat Methods 9: 493-498. 2012), intracellular traffic of a variety of newly-synthesized neuronal proteins from the endoplasmic reticulum to the plasma membrane in growth cones. Our data will provide new information to explore the essential roles of local membrane traffic systems in growth cone motility.		2022年7月2日　15:12～15:36　ラグナガーデンホテル羽衣：中　第9会場 3S09a-04 生きた神経細胞における膜マイクロドメイン動態の超解像イメージング解析 Visualizing dynamics of membrane microdomains in living neurons by super-resolution imaging 本多敦子(1,2) 1. 新潟大学医学部医学科、2. 新潟大学大学院医歯学総合研究科 ATSUKO HONDA(1,2) 1. Sch of Med, Fac of Med, Niigata Univ, Niigata, JAPAN, 2. Grad Sch of Med and Dent Sciences, Niigata Univ, Niigata, Japan Keyword: GROWTH CONE, LIPID RAFT, AXON GROWTH, Structured Illumination Microscopy Membrane microdomains (lipid rafts) are functional subdomains of cell membrane enriched cholesterol and sphingolipids, and they serve as platforms for accumulating specific membrane proteins. Previous biochemical- and pharmacological experimental evidence have indicated that lipid raft microdomains are crucial for various biological functions including signal transduction, however it has been difficult to visualize the structure and dynamics of the microdomains in living cell by conventional microscopy. Recently, super-resolution imaging techniques with specific probes for lipid rafts have begun to provide information of microdomain dynamics and the spatial signaling transductions at microdomains in living cells. In this study, we focused on the membrane microdomains in the axonal growth cone, a unique structure at the tip of the growing axon. During brain development, neurons extent their axon to be guided by the growth cone, and in where many receptors and signaling molecules are specifically concentrated. Using structured illumination microscopy (SIM), we have reported the Laminin-dependent accumulation of the signaling molecules on the cholesterol-enriched microdomains through glycoprotein M6a in the growth cones. We further examined structural and functional characters of the microdomains using the lipid probes based on the toxins or the chemical probes sensing lipid-ordered phase of the membrane. In addition, clustering of the fluorescence-labeled signaling molecules in the lipid rafts were observed in living neuronal cells. We found that lipids components of the membrane microdomains were spatially enriched in the axonal growth cones and they were frequently clustered with signaling molecules at the tip of filopodia in the growth cone. Here, we will discuss about the dynamics- and functions of the lipid rafts in the axonal growth cone characterized by super-resolution imaging.

2022年7月2日　15:36～16:00　ラグナガーデンホテル羽衣：中　第9会場 3S09a-05 超解像陰影法による複数オルガネラの同時可視化 Visualization of various organelle dynamics by super-resolution shadow imaging of intracellular space 野住素広(1)、五十嵐道弘(1)、U. Valentin Nägerl(2) 1. 新潟大学大学院医歯学総合研究科 Motohiro Nozumi(1), Michihiro Igarashi(1), U. Valentin Nägerl(2) 1. Grad Sch Med Dent, Niigata Univ, Niigata, Japan, 2. IINS, Univ of Bordeaux/ CNRS, Bordeaux, France Keyword: SUPER-RESOLUTION MICROSCOPY, ORGANELLES During axonal growth, vesicle and organelle movements are observed together with cytoskeletal reorganization in the growth cone, which leads the axon. The process can be visualized by fluorescently labeling the cytoskeleton and organelle markers. But it is necessary to select the organelles to be visualized before observation, and there are limits to the types of organelles that can be detected simultaneously. To break through these limitations, we attempted to visualize various intracellular structures simultaneously using a single fluorescent dye. We have adopted the recent super-resolution shadow imaging technique (SUSHI), which was originally developed to image the extracellular space of living brain tissue using 3D-STED microscopy. As in the case of SUSHI (Tønnesen J et al. Cell 172: 1108[‘18]), membrane-delimited subcellular structures like organelles will cast tell-tale “shadows” if surrounded by a bright sea of fluorescence and imaged with sufficiently high spatial resolution. Using cultured cells that cytosolically expressed the fluorescent protein Citrine, we could readily discriminate negative images of various organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus, endosome, lysosome and also cytoskeleton. In addition, this method enabled us to distinguish them without specific labeling and to monitor their movements and morphological changes over time. This new intracellular live imaging method, combined with the original SUSHI may reveal various phenomena in the brain at the organelle level.