in vivo imaging analysis of cGMP dynamics in the smooth muscle of circulatory system

2014 Fiscal Year Final Research Report

• Project/Area Number: 25840050
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: The University of Tokushima
• Principal Investigator: HORIKAWA Kazuki 徳島大学, ヘルスバイオサイエンス研究部, 教授 (70420247)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: イメージング / cGMP / 血管平滑筋

Outline of Final Research Achievements

cGMP, one of the most important second messenger, is responsible for a variety of physiological events. To better understand the regulating mechanisms of cGMP signaling, quantitative analysis of cGMP dynamics with the help of indicator has been waited. Here, we engineered a genetically encoded biosensor that reports the concentration change of cGMP. It consists cGMP sensing motif whose N- and C-terminus were tagged with GFP variants, in which cGMP dynamics was reported in the form of FRET signal changes. By optimizing FRET parameters and the property of cGMP sensing motifs, we developed a series of improved indicators whose dynamic range was increased to 5-fold including high- to low-affinity variants. Our cell-based assay revealed that these indicators nicely report spatio-temporal dymnamics of cGMP associated with the spontaneous activity in the chemotacting cells which had not been detected with previously reported indicators.

Free Research Field

生物学