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

Phosphorylation of the regulatory myosin light chain (RMLC) regulates the activity of myosin II, which is involved in cell motility in both muscle and non-muscle cells. There are both Ca2+-dependent and independent pathways for the RMLC phosphorylation in smooth muscle cells, and the latter is often involved in abnormal contractility in pathological states. Therefore, pharmacological intervention of the RMLC phosphorylation may have a therapeutic value. In this study, we developed a genetically encoded new biosensor, termed CRCit, to detect the RMLC phosphorylation using fluorescence resonance energy transfer between two variants of the green fluorescent protein fused to both amino and carboxyl termini of RMLC. When expressed in primary cultured vascular smooth muscle cells, CRCit reported the RMLC phosphorylation with a high spatiotemporal resolution. Furthermore, we were able to assay Ca2+-independent phosphorylation of RMLC. CRCit can be used for efficient screening of lead compounds that inhibit abnormal smooth muscle contraction.