Dendritic epidermal T cells (DETCs) found in mouse skin are NKG2D-positive γδ T cells involved in immune surveillance and wound repair. It is assumed that the interaction of an NKG2D receptor on DETCs and an MHC class I-like NKG2D ligand on keratinocytes activates DETCs, which then secrete cytokines promoting wound repair. However, direct evidence that DETC activation through NKG2D signaling promotes wound repair is not available. In the present study, we generated mAbs for an NKG2D ligand H60c previously suggested to be expressed specifically on skin keratinocytes. Local administration of H60c-specific mAb inhibited activation of DETCs and significantly delayed wound repair. Likewise, administration of NKG2D-specific mAb impaired wound repair to a similar extent. The delay in wound closure resulting from the blockade of the NKG2D pathway was comparable to that observed in γδ T cell-deficient mice. These results indicate that H60c/NKG2D interactions play a critical role in wound repair. Reassessment of binding affinities showed that H60c monomers bind to NKG2D with affinity (Kd = 26 ± 3.2 nM) comparable to those of other high-affinity NKG2D ligands. H60c is transcribed not only in skin but also in tissues such as tongue and female reproductive tract known to contain epithelium-resident γδ T cells expressing invariant TCRs, suggesting a more general role for H60c in the maintenance of epithelial integrity.

No abstract

Fascin, an actin‐bundling protein, is present in the filopodia and lamellipodia of growth cones. However, few studies have examined lamellipodial fascin because it is difficult to observe. In this study, we evaluated lamellipodial fascin. We visualized the actin meshwork of lamellipodia in live growth cones by super‐resolution microscopy. Fascin was colocalized with the actin meshwork in lamellipodia. Ser39 of fascin is a well‐known phosphorylation site that controls the binding of fascin to actin filaments. Fluorescence recovery after photobleaching experiments with confocal microscopy showed that binding of fascin was controlled by phosphorylation of Ser39 in lamellipodia. Moreover, TPA, an agonist of protein kinase C, induced phosphorylation of fascin and dissociation from actin filaments in lamellipodia. Time series images showed that dissociation of fascin from the actin meshwork was induced by TPA. As fascin dissociated from actin filaments, the orientation of the actin filaments became parallel to the leading edge. The angle of actin filaments against the leading edge was changed from 73° to 15°. This decreased the elasticity of the lamellipodia by 40%, as measured by atomic force microscopy. These data suggest that actin bundles made by fascin contribute to elasticity of the growth cone.

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 simultaneously 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 spectroscopyThe 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 i...

No abstract