Real-time bioimaging of the cellular function of an actin-regulatory protein, gelsolin

明胶蛋白是一种肌动蛋白调节蛋白，至少有三种不同的活性，即成核、切断和封顶。这些活性受到钙离子(Ca^&lt；2+&gt；)、pH和多磷酸肌醇的严格调控。明胶蛋白通过与丝状肌动蛋白(F-)和单体肌动蛋白(G-)相互作用，并通过各种机制控制肌动蛋白聚合物的长度来调节细胞内肌动蛋白的动态。在本研究中，我们试图通过荧光共振能量转移(FRET)获得活细胞内明胶蛋白激活的时空图像。为了在细胞中进行明胶蛋白活性的生物成像，将小鼠明胶蛋白基因插入绿色荧光蛋白的衍生物ECFP和EYFP之间，创建FRET探针(Cgy)，然后构建Cgy的哺乳动物表达载体。将Cgy表达载体导入明胶蛋白基因敲除小鼠成纤维细胞中，Western印迹分析证实Cgy在成纤维细胞中有表达。Cgy在G细胞中的表达增加了细胞爬行的速度，表明Cgy嵌合明胶蛋白在细胞中具有功能。EYFP在细胞外周的荧光强度降低，而在胞核附近的荧光强度较高，提示FRET发生在细胞的中央区，而明胶蛋白在细胞边缘被激活，这是因为FRET的丢失。

Gelsolin, an actin-regulatory protein has at least three different activities, nucleating, severing and capping. These activities are tightly regulated by calcium ions (Ca^<2+>), pH, and polyphosphoinositides. Gelsolin regulates actin dynamics in cells by interaction with both filamentous (F-) and monomeric (G-) actins and controlling the length of actin polymers through a variety of mechanisms. In this study, we sought to obtain an image of spatio-temporal activation of gelsolin in the living cells by means of FRET (Fluorescence Resonance Energy Transfer).For bioimaging of gelsolin activities in cells, mouse gelsolin cDNA were inserted between ECFP and EYFP, derivatives of green fluorescence protein, to create a probe (CGY) for FRET, and then the mammalian expression vector for CGY was constructed. The CGY expression vector was transfected in fibroblast derived from gelsolin knock-out mice and expression of CGY was confirmed by western blot analysis. The expression of CGY in G- cells resulted in increased rate of cell crawling, indicating CGY, chimeric gelsolin protein was functional in cells. Fluorescence intensity of EYFP was decreased at cell peripheral, whereas it was higher around nulei, suggesting FRET was occurred at the central region of cells, and gelsolin was activated at the edge of cells, because FRET was lost.