A ratiometric expressible FRET sensor for phosphoinositides displays a signal change in highly dynamic membrane structures in fibroblasts
A ratiometric expressible FRET sensor for phosphoinositides displays a signal change in highly dynamic membrane structures in fibroblasts
复制标题
DOI：
10.1021/bi035480w
复制
发表时间：
2004-02-24
期刊：
影响因子：
2.9
通讯作者：
Allen, PG
中科院分区：
文献类型：
--
作者：
Cicchetti, G;Biernacki, M;Allen, PG
磷酸肌醇是细胞生长、存活和运动中重要的信号转导中间体。我们已经发明了一种用于多磷酸化磷酸肌醇的荧光传感器，其基于来源于李斯特菌蛋白ActA的肽，所述肽在脂质结合时经历无规卷曲至螺旋转变。该传感器，称为CAY，是一种融合蛋白的青色和黄色荧光蛋白侧翼的肽在其N-和C-末端，分别。CAY显示荧光共振能量转移在体外的磷酸化的磷酸肌醇的情况下，这种能量转移后失去与这些磷脂的相互作用。这些结果表明，经历螺旋到螺旋转变的短肽对于基于FRET的生物传感器的工程化是足够的。CAY主要定位于表达传感器的成纤维细胞的细胞质中，但在活性肌动蛋白动力学区域（如先前已证明含有高水平磷酸肌醇的皱褶）中显示荧光共振能量转移的损失。
Phosphoinositides are important signal transduction intermediates in cell growth, survival, and motility. We have invented a fluorescence sensor for polyphosphorylated phosphoinositides based on a peptide derived from the Listeria protein ActA that undergoes a random coil to helix transition upon lipid binding. The sensor, termed CAY, is a fusion protein of cyan and yellow fluorescent proteins flanking the peptide at its N- and C-termini, respectively. CAY displays fluorescence resonance energy transfer in vitro in the absence of phosphorylated phosphoinositides, and this energy transfer is lost upon interaction with these phospholipids. These results demonstrate that a short peptide undergoing a coil to helix transition can be sufficient for the engineering of a FRET-based biosensor. CAY is predominantly localized to the cytoplasm in fibroblasts expressing the sensor but shows loss of fluorescence resonance energy transfer in regions of active actin dynamics such as ruffles that have previously been demonstrated to contain high levels of phosphoinositides.