Open lecture on “Cellular System”

Open lecture on “Cellular System”

Professor Takao Hamakubo, Assistant Professor Kazuyuki Masuda, Professor Shigeo Ihara and others in the field of econometric biomedicine published papers to visualize and quantitate signals from trimeric G protein. Visualization of ligand-induced Gi protein activation in chemotaxing cells. Kazuyuki Masuda, Jun-ichi Kitakami, Tohru Kozasa, Tatsuhiko Kodama, Sigeo Ihara and Takao Hamakubo FASEB J. Published online before print November 23, 2016, doi: 10.1096 / fj.201601102R Cell chemotaxis is a phenomenon related to many pathological conditions such as physiological response of cells, inflammatory response and invasion of cancer. Chemotaxis via G protein-coupled receptor (GPCR) is caused by extracellular chemotactic factors specifically binding to GPCR and activating intracellular trimeric G protein . Furthermore, it has been clarified that the trimeric G protein sequentially activates molecules downstream of intracellular signal transduction. Up to now, the mechanism of chemotaxis has been positively studied by fluorescence imaging etc. regarding the activation control of the molecule downstream of the signal. On the other hand, it was difficult to directly observe changes in the activity of receptors and trimeric G proteins upstream of the chemotactic signal in dynamically changing cells, so there were many unclear points . In order to clarify how cells are perceived by a slight concentration difference of extracellular chemotactic factors, in particular by visualization of the local activity change of trimeric G protein during cell chemotaxis Has been expected. To detect the activity of trimeric G protein, we have developed a novel molecular probe (R10-Gi FRET probe) using fluorescence resonance energy transfer (FRET). The developed R10-Gi FRET probe conjugated with Gi-coupled GPCR and showed ligand-stimulated G protein activation in a ligand-stimulated manner. It also showed an increase in FRET signal associated with G protein activation. Human leukemia-derived HL-60 cells expressed a chemotactic factor leukotriene B4 (LTB4) -specific receptor (BLT1) when differentiated into neutrophils and showed chemotaxis according to the concentration gradient of LTB4 I will. We have found that it is possible to measure trimeric G protein activity change upon chemotaxis of neutrophil-like HL-60 cells to LTB4 by using R10-Gi FRET probe. In addition, the measurement of neutrophil-like HL-60 cells revealed that trimeric G protein activation in the chemotaxis direction of cells occurs in a local region rather than what has been thought up to now. The R10 – Gi FRET probe developed this time is thought to be superior to signal measurement in cells that dynamically change its shape than conventional molecular probes, and is expected to be applied to various research in the future.