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

肌动蛋白调节蛋白凝溶胶蛋白细胞功能的实时生物成像

基本信息

批准号：
13670001
负责人：
FUJITA Hisakazu
金额：
$ 1.98万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670001/
关键词：
Gelsolin GFP-fusion protein Bioimaging Fluorescence Resonance Energy Transfer : FRET

项目摘要

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.

凝溶胶蛋白是一种肌动蛋白调节蛋白，具有至少三种不同的活性：成核、切割和加帽。这些活性受到钙离子（Ca^<2+>）、pH和多磷酸肌醇的严格调节。凝溶胶蛋白通过与丝状（F-）和单体（G-）肌动蛋白相互作用并通过多种机制控制肌动蛋白聚合物的长度来调节细胞中的肌动蛋白动力学。本研究将小鼠凝溶胶蛋白cDNA插入到绿色荧光蛋白衍生物ECFP和EYFP之间，构建了荧光共振能量转移（FRET）探针CGY，并构建了CGY的哺乳动物表达载体。将CGY表达载体转染来自凝溶胶蛋白基因敲除小鼠的成纤维细胞，并通过蛋白质印迹分析证实CGY的表达。CGY在G-细胞中的表达导致细胞爬行速率增加，表明CGY嵌合凝溶胶蛋白在细胞中是功能性的。EYFP的荧光强度在细胞周边减弱，而在细胞核周围增强，说明FRET发生在细胞的中心区域，而凝溶胶蛋白在细胞边缘被激活，因为FRET丢失。