Phosphoinositide metabolism during membrane ruffling and macropinosome formation in EGF-stimulated A431 cells
Phosphoinositide metabolism during membrane ruffling and macropinosome formation in EGF-stimulated A431 cells
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DOI：
10.1016/j.yexcr.2007.02.012
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发表时间：
2007-04-15
影响因子：
3.7
通讯作者：
Hatae, Tanenori
中科院分区：
文献类型：
--
作者：
Araki, Nobukazu;Egami, Youhei;Hatae, Tanenori
在表皮生长因子刺激的A431细胞中，发现磷酸肌醇3-激酶（PI 3 K）抑制剂干扰巨胞饮体的形成，而不影响膜皱褶和肌动蛋白聚合。YFP标记的磷脂酶C δ 1-普列克底物蛋白同源结构域（YFP-PLC-PH）相对于膜靶向CFP（CFP-Mem）的荧光强度比的活细胞成像和定量图像分析表明，形成巨胞饮杯的膜皱褶中PI（4，5）P-2的浓度增加到平面质膜中的两倍以上。PI（4，5）P-2在巨胞饮体关闭前达到最大值，并随着巨胞饮杯的关闭而迅速下降。相反，基于YFP-Akt-PH或YFP-Bruton酪氨酸激酶（Btk）-PH/CFP-Mem比率可视化的PI（3，4，5）P-3浓度在巨胞饮体形成位点局部增加，并在巨胞饮体闭合时达到峰值。PI（4，5）P-2和PI（3，4，5）P-3的动力学似乎与巨胞饮期间的肌动蛋白重塑机制相关。从使用抑制剂和合成的磷酸肌醇的药理学数据和其他数据，可以得出结论，PI（4，5）P-2消除和PI（3，4，5）P-3通过PI 3 K的生产可能是从膜皱褶形成巨胞饮体的关键。这项研究强调，局部控制水平的磷酸肌醇是重要的调节肌动蛋白结合蛋白的功能，影响膜结构的变化。(c)2007爱思唯尔公司All rights reserved.
Inhibitors of phosphoinositide 3-kinase (PI3K) were found to perturb macropinosome formation without affecting the membrane ruffling and actin polymerization in epidermal growth factor-stimulated A431 cells. Live-cell imaging and quantitative image analysis of the fluorescence intensity ratio of the YFP-tagged phospholipase C delta 1-pleckstrin homology domain (YFP-PLC-PH) relative to membrane-targeted CFP (CFP-Mem) demonstrated that the concentration of PI(4,5)P-2 in the membrane ruffles forming macropinocytic cups increased to more than double that in planar plasma membranes. The PI(4,5)P-2 level in the membrane reached its maximum just before macropinosome closure and rapidly fell as the macropinocytic cups closed. In contrast, the PI(3,4,5)P-3 concentrations visualized based on the YFP-Akt-PH or YFP-Bruton's tyrosine kinase (Btk)-PH/CFP-Mem ratio increased locally at the site of macropinosome formation and peaked at the time of macropinosome closure. The kinetics of PI(4,5)P-2 and PI(3,4,5)P-3 appeared to be mechanistically linked to actin remodeling during macropinocytosis. From the pharmacological data using inhibitors and synthetic phosphoinositides and other data, it could be concluded that both PI(4,5)P-2 elimination and PI(3,4,5)P-3 production by PI3K might be crucial for macropinosome formation from membrane ruffles. This study emphasizes that locally controlled levels of phosphoinositides are important for regulating the function of actin-binding proteins which effect changes in the membrane architecture. (c) 2007 Elsevier Inc. All rights reserved.