Actin binding of ezrin is activated by specific recognition of PIP2-functionalized lipid bilayers
Actin binding of ezrin is activated by specific recognition of PIP2-functionalized lipid bilayers
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DOI：
10.1021/bi702542s
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发表时间：
2008-03-25
期刊：
影响因子：
2.9
通讯作者：
Janshoff, Andreas
中科院分区：
文献类型：
--
作者：
Janke, Matthias;Herrig, Alexander;Janshoff, Andreas
我们通过PIP2的识别，定量地研究了Ezrin从休眠状态转换到活性的F-肌动蛋白结合状态的机制。为此，我们建立了一种新的模拟Ezrin介导的膜-细胞骨架附着的体外模型，并比较了通过His标记连接到显示Ni-NTA的脂质双层或结合到含有PIP2的支撑膜上的Ezrin的F-肌动蛋白结合能力。用荧光和胶体探针显微镜(CPM)证实了Ezrin的构象转换为能够结合F-肌动蛋白的活性构象。荧光图像显示荧光标记的F-肌动蛋白仅附着在PIP2结合的Ezrin上。首次利用带有F-肌动蛋白纤维人工骨架的胶体小球定量测量了Ezrin-F-肌动蛋白界面的最大粘附力和粘附功。我们发现，PIP2结合的Ezrin和F-肌动蛋白之间的粘附功明显大于通过His标签结合到膜上的F-肌动蛋白和Ezrin之间的粘附功。总的来说，这些数据表明Ezrin的激活可以作为PIP2结合的结果而发生，并且不需要额外的辅因子。
In a quantitative manner, we investigated the mechanism of switching ezrin from the dormant to the active, F-actin binding state by recognition of PIP2. For this purpose, we established a novel in vitro model mimicking ezrin-mediated membrane-cytoskeleton attachment and compared the F-actin binding capability of ezrin that either had been coupled via a His tag to a lipid bilayer displaying Ni-NTA or had been bound to supported membranes containing PIP2. Epifluorescence and colloidal probe microscopy (CPM) were employed to demonstrate ezrin's conformational switch into an active conformation capable of binding F-actin. Epifluorescence images revealed attachment of fluorescently labeled F-actin solely to PIP2-bound ezrin. For the first time, colloidal spheres equipped with an artificial cytoskeleton composed of firmly attached F-actin filaments were used to measure quantitatively the maximal adhesion forces and the work of adhesion of the ezrin-F-actin interface. We found that the work of adhesion between PIP2-bound ezrin and F-actin is substantially larger than that measured between F-actin and ezrin bound to the membrane via the His tag. Collectively, these data indicate that activation of ezrin can occur as a consequence of PIP2 binding and does not require additional cofactors.