一种用于定量活细胞和生物体内非肌肉肌球蛋白 II 磷酸化动力学的基因编码生物传感器策略。

A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms.

机构信息

Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, US NIH, Bethesda, MD 20814, USA.

出版信息

Cell Rep. 2018 Jul 24;24(4):1060-1070.e4. doi: 10.1016/j.celrep.2018.06.088.

Complex cell behaviors require dynamic control over non-muscle myosin II (NMMII) regulatory light chain (RLC) phosphorylation. Here, we report that RLC phosphorylation can be tracked in living cells and organisms using a homotransfer fluorescence resonance energy transfer (FRET) approach. Fluorescent protein-tagged RLCs exhibit FRET in the dephosphorylated conformation, permitting identification and quantification of RLC phosphorylation in living cells. This approach is versatile and can accommodate several different fluorescent protein colors, thus enabling multiplexed imaging with complementary biosensors. In fibroblasts, dynamic myosin phosphorylation was observed at the leading edge of migrating cells and retracting structures where it persistently colocalized with activated myosin light chain kinase. Changes in myosin phosphorylation during C. elegans embryonic development were tracked using polarization inverted selective-plane illumination microscopy (piSPIM), revealing a shift in phosphorylated myosin localization to a longitudinal orientation following the onset of twitching. Quantitative analyses further suggested that RLC phosphorylation dynamics occur independently from changes in protein expression.

复杂的细胞行为需要对非肌肉肌球蛋白 II（NMMII）调节轻链（RLC）磷酸化进行动态控制。在这里，我们报告了一种同源转移荧光共振能量转移（FRET）方法，可用于在活细胞和生物体中跟踪 RLC 磷酸化。荧光蛋白标记的 RLC 在去磷酸化构象中表现出 FRET，允许在活细胞中鉴定和定量 RLC 磷酸化。这种方法具有多功能性，可以适应几种不同的荧光蛋白颜色，从而能够与互补的生物传感器进行多路复用成像。在成纤维细胞中，在迁移细胞的前沿和回缩结构中观察到动态肌球蛋白磷酸化，并且它与激活的肌球蛋白轻链激酶持续共定位。使用偏振倒置选择性平面照明显微镜（piSPIM）跟踪 C. elegans 胚胎发育过程中的肌球蛋白磷酸化变化，发现在抽搐开始后，磷酸化肌球蛋白的定位发生了向纵向的转变。定量分析进一步表明，RLC 磷酸化动力学的发生与蛋白质表达的变化无关。