3P174 Molecule-selective lateral-diffusion barrier in the neuronal axon membrane(Cell biology,Poster,The 52th Annual Meeting of the Biophysical Society of Japan(BSJ2014))
3P174 神経細胞膜の分子選択的な並進拡散障壁(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))

Miyahara Manami, Nakada Chieko, Kalay Ziya, Matsui Toshiki, Iwata Hiroo, Fujiwara Takahiro, Kusumi Akihiro
2014 Seibutsu Butsuri  
Measurement of cell mechanical property is crucial for both understanding various cell functions and diagnosing cell disease. Previous atomic force microscopy (AFM), in which a stepwise change in frequency was employed, revealed that the cell complex shear modulus, G* exhibited a large cell-cell variation depending on frequency [1]. However the AFM method required a measurement time longer than the conventional force curve measurements. Here, we proposed a multi-frequency AFM for obtaining
more » ... taneously G* of cell in a wide frequency range and demonstrated that this AFM technique allowed us to measure the powerlaw frequency dependence of G* [2] within several seconds. [1] Cai et al. Biophys. Using chemotaxis signal processing system, Escherichia coli (E. coli) regulates rotational direction of flagellar motor. When E. coli cell recognizes chemoattractant by transmembrane receptor, rotational direction of the flagellar motor changes to counterclockwise direction. In order to investigate kinetic property of the signal processing induced by chemoattractant, we statistically measured time course of directional biases of rotationing tethered cells. By using caged chemoattractants and hightemporal resolution measurement system (250 Hz), we traced precisely the directional biases triggered by photolysis of caged attractants. We would like to discuss about detail of the kinetic property in the signal processing induced by chemoattractant in this annual meeting. 3P171 蛍光相関分光法を用いた単一細胞由来のグルココルチコイド レセプター二量体形成と転写活性の定量 Quantification of glucocorticoid receptor homo-dimer and transcriptional activity in single cell by fluorescence correlation spectroscopy The relationship between the amounts of homo-dimeric glucocorticoid receptor (GR) and transcriptional activity is studied using single-cell method combined with Fluorescence Correlation Spectroscopy (FCS) and microwell chip (FCS-microwell system). It is well-known that GR translocates to nucleus after ligand binding and then, homo-dimeric GR binds to genome and activates its functions as transcriptional factor. However, the relationship between the amounts of homo-dimeric GR and its transcriptional activity remains unclear, yet. In this study, FCSmicrowell system was established, namely the amounts of homo-dimeric GR and transcriptional activity were determined at single-cell lysate in microwell simultaneously by FCS and fluorescent reporter assay. 3P172 超解像顕微鏡法による上皮成長因子受容体クラスタリングの 定量解析 Activation of epidermal growth factor receptor (EGFR), which evokes various cellular signaling, has been suggested to be regulated through diffusion-driven receptor clustering. For a precise analysis of the EGFR clustering, photo-activation localization microscopy (PALM) was employed to distinguish adjacent oligomers with a distance of shorter than the conventional optical resolution. The distribution of oligomer size quantified by a hierarchical clustering algorithm on the PALM data was distinct from that obtained by the previous methods. The localization accuracy has been improved for enabling investigation of the spatial receptor arrangement in oligomers. The clustering dependent mechanism of signaling regulation unveiled by the novel method will be discussed. 3P173 FRAP による、成長円錐のアクチンおよびアクチン関連タン パク質の動態解析 FRAP experiments on actin and actin associate proteins in growth cones Growth cones are enlargement of cytoplasm which appear at the tip of growing dendrites or axons. The growth cone movement plays an important roles in path finding and neuronal navigation. The movement is controlled by the balance between rate of polymerization of g-actin and of retrograde flow of f-actin. Growth cone advances when rate of polymerization is faster than that of retrograde flow. The actin dynamics is regulated by actin associate proteins. We, therefore, performed FRAP experiments on actin, arp2/3 and other actin associate proteins. We observed not only retrograde flow of actin but also anterograde movement. Mechanism of anterograde movement of actin and future experiments will also be discussed. 3P174 神経細胞膜の分子選択的な並進拡散障壁 Molecule-selective lateral-diffusion barrier in the neuronal axon membrane The presence of a diffusion barrier in the plasma membrane (PM) in the neuronal initial segment (IS) region has been established by us and others. Here, we found that GPI-anchored proteins (GPI-APs) diffuse rapidly in the IS-PM (only ~2x slower than in other PM regions), but not phospholipids, showing that the diffusion barrier is a molecule-selective filter. Ultrahigh-speed single-molecule tracking revealed that the IS-PM is partitioned into ~60-nm compartments, and that GPI-APs hop to an adjacent compartment an average of once every ~2.0 ms, but phospholipids do so ~300x less frequently. With an increase in hydrophilicity in the headgroup moiety, the hop frequency was increased, suggesting that the molecules' vertical location in the PM affects the hop frequency. -S277 -Poster, Day 3
doi:10.2142/biophys.54.s277_6 fatcat:dwco3nwnwnfwzdq2gtc2lc6lme