3P177 Comparison of the delivery effects of two antibody carrier systems(Cell biology,Poster,The 52th Annual Meeting of the Biophysical Society of Japan(BSJ2014))
3P177 二つの抗体送達システムにおける送達作用の比較(細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

Kuwahara Kana, Harada Kazuki, Yamamoto Takenori, Shinohara Yasuo
2014 Seibutsu Butsuri  
The kappa opioid receptor (kOR), a G-protein-coupled receptor, was expressed in the plasma membrane (PM) of cultured cells and observed at the single-molecule level. The kOR molecules underwent thermal diffusion, but dynamically concentrated in many submicron areas: many molecules enter and exit from the area continually, but the average molecular density remained high in the area (hotspot). kOR's scaffolding protein EBP50, which could link kOR to the actin filament via ERM proteins, is
more » ... roteins, is dynamically present in the hotspots, but at variance with the general view of the kOR-EBP50 interaction, their interactions are transient, with a colocalization lifetime of ~0.3 s. These results indicate a possibility that kOR signaling occurs in transient kOR oligomers including EBP50. 3P176 Noonan 症候群における SOS を介した Ras positive feedback 異常の生細胞一分子解析 Dysregulations of SOS-mediated positive feedback on RAS activation in Noonan syndrome observed using single molecule imaging Noonan syndrome (NS) is a congenital disorder with cardiac disease. The 10 % of NS patients has mutation on SOS, an activator (guanine-nucleotide exchange factor, GEF) of a small GTPase RAS, a crucial factor of cell proliferation. In this study, we observed fluorescently labelled SOS molecules on the plasma membrane using single-molecule imaging and analyzed the dissociation kinetics reflecting GEF activity. It is known that GEF activity of SOS is stimulated by interaction with the active form of RAS (positive feedback), which is crucial for RAS activation in living cells. Our observation indicated that roles of the positive feedback were different among types of SOS mutants in NS. Mutants that become gain of function might exist independent of the positive-feedback. 3P177 二つの抗体送達システムにおける送達作用の比較 Comparison of the delivery effects of two antibody carrier systems Experimental systems, which can deliver antibodies into living cells, are useful to develop antibody therapies. In these systems, the delivery strategy using cell-penetrating peptide has some advantages such as low toxicity. We here compared two kinds of the peptide-based antibody delivery system: the system using protein A module and that using a peptide with hydrophobic tryptophan-rich motif. We delivered the fluorescent labeled antibodies to HeLa cells using each delivery system. As a result, both systems enabled to deliver the antibodies into the cells. However, the system using a hydrophobic tryptophan-rich motif caused the red-shift of the fluorescent spectrum. The possible reasons for the shift will be discussed. 3P178 Amoeba proteus 細胞膜の 3 次元曲率に関する研究 Amoeba proteus migrates actively and during the locomotion the shape of cell surface is dynamically deformed. In this study, we observed the surface structure of A. proteus in three dimensional spaces using a modified digital scanned light-sheet microscope. Interestingly, pseudopods were actively elongated to various directions in three dimensional spaces, which have never seen in two dimensional observations. To characterize the surface structure of the cell, we calculated three dimensional curvatures (Gaussian curvature and Mean curvature) of the structure of cell surface and compared the curvatures with direction of cell locomotion. As a result, cell surface curvatures were unevenly distributed in the cell depending on polarity of cell migration. 3P179 電子顕微鏡法によるヒト毛乳頭細胞の一次繊毛の構造解析 Structural analysis of primary cilia in human follicle dermal papilla cells by electron microscopy Most cells of multicellular organisms have primary cilia, which are single, non-motile, and sensory cilia. They have been reported to detect and transform mechanical stimulation to intracellular, but the mechanisms are not still well. Dermal papilla cells (DPCs) with primary cilia (Matsushima, 2013) in the skin can induce hair growth. Here we observed the structure of DPCs (2D and clumps) by scanning electron microscopy (SEM) for microstructural analysis. In SEM we prepared the specimens under nearliving conditions with ionic liquid and so. Moreover we challenged CLEM, which can observe the cilia by SEM after identifying them by fluorescence microscopy. Here we report the recent progress to get the ultrastructure of cells and primary cilia under near-living conditions. 3P180 Intranulear particles include RPS or RPL subunits Apodized phase contrast microscope (APC) reduces halo artifacts with spacial filtering.and reveals fine structures without any staining. To identify structures in the APC images with fluorescent marker, our external APC system have simultaneous imaging system of APC (546nm) and fluorescence image (594nm-700nm). Here, we re-examined and identified intracellular structures observed with APC in simultaneous image records of APC and fluorescence. APC directly revealed 1) ER 2) mitochondria, 3) PML body, and 4) intranuclear granules. The granules included, ribosomal protein of small (RPS) or large (RPL) subunit, but RPS and RPL were not co-localized in the granules. Farther image data will be discussed in the presentation.
doi:10.2142/biophys.54.s278_3 fatcat:p3vhkmzbpnaxrnmzjgwkg4bs5m