Real-time imaging of myosin II regulatory light-chain phosphorylation using a new protein biosensor.

@article{citeulike:2570103, abstract = {Phosphorylation of the RMLC (regulatory myosin light chain) regulates the activity of myosin II, which is critically involved in the motility of both muscle and non-muscle cells. There are both Ca2+-dependent and -independent pathways for RMLC phosphorylation in smooth-muscle cells, and the latter pathway is often involved in an abnormal contractility in pathological states such as asthma and hypertension. Therefore pharmacological interventions of RMLC phosphorylation may have a therapeutic value. In the present study, we developed a new genetically encoded biosensor, termed CRCit (ECFP-RMLC-Citrine, where ECFP is enhanced cyan fluorescent protein), that detects RMLC phosphorylation using fluorescence resonance energy transfer between two variants of the green fluorescent protein fused to both the N- and C-termini of RMLC. When expressed in primary cultured vascular smooth-muscle cells, CRCit detected the Ca2+-dependent RMLC phosphorylation with a high spatiotemporal resolution. Furthermore, we could specifically assay the agonist-induced Ca2+-independent phosphorylation of RMLC when Ca2+ signalling in cells expressing CRCit was suppressed. Thus CRCit may also be used for the high throughput screening of compounds that inhibit abnormal smooth-muscle contraction.}, address = {Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan.}, author = {Yamada, A. and Hirose, K. and Hashimoto, A. and Iino, M. }, citeulike-article-id = {2570103}, doi = {10.1042/BJ20040778}, issn = {1470-8728}, journal = {Biochem J}, keywords = {live\_cell\_imaging, microscopy, phosphorylation, technique}, month = {January}, number = {Pt 2}, pages = {589--594}, posted-at = {2008-03-21 14:45:12}, priority = {0}, title = {Real-time imaging of myosin II regulatory light-chain phosphorylation using a new protein biosensor.}, url = {http://dx.doi.org/10.1042/BJ20040778}, volume = {385}, year = {2005} }

TY - JOUR ID - citeulike:2570103 L3 - citeulike-article-id:2570103 TI - Real-time imaging of myosin II regulatory light-chain phosphorylation using a new protein biosensor. JF - Biochem J VL - 385 IS - Pt 2 SP - 589 EP - 594 AD - Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan. SN - 1470-8728 N2 - Phosphorylation of the RMLC (regulatory myosin light chain) regulates the activity of myosin II, which is critically involved in the motility of both muscle and non-muscle cells. There are both Ca2+-dependent and -independent pathways for RMLC phosphorylation in smooth-muscle cells, and the latter pathway is often involved in an abnormal contractility in pathological states such as asthma and hypertension. Therefore pharmacological interventions of RMLC phosphorylation may have a therapeutic value. In the present study, we developed a new genetically encoded biosensor, termed CRCit (ECFP-RMLC-Citrine, where ECFP is enhanced cyan fluorescent protein), that detects RMLC phosphorylation using fluorescence resonance energy transfer between two variants of the green fluorescent protein fused to both the N- and C-termini of RMLC. When expressed in primary cultured vascular smooth-muscle cells, CRCit detected the Ca2+-dependent RMLC phosphorylation with a high spatiotemporal resolution. Furthermore, we could specifically assay the agonist-induced Ca2+-independent phosphorylation of RMLC when Ca2+ signalling in cells expressing CRCit was suppressed. Thus CRCit may also be used for the high throughput screening of compounds that inhibit abnormal smooth-muscle contraction. KW - live_cell_imaging KW - microscopy KW - phosphorylation KW - technique AU - Yamada, A AU - Hirose, K AU - Hashimoto, A AU - Iino, M PY - 2005/01/15/ UR - http://dx.doi.org/10.1042/BJ20040778 ER -