The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in mammalian cells
The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in mammalian cells
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DOI：
10.1016/s0171-9335(98)80075-7
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发表时间：
1998-10-01
影响因子：
6.6
通讯作者：
Marriott, G
中科院分区：
文献类型：
--
作者：
Choidas, A;Jungbluth, A;Marriott, G
gfp -肌动蛋白基因融合的产物，在多种哺乳动物细胞系中永久或短暂转染，被证明是肌动蛋白细胞骨架组织和动力学的合适的、内在的探针。在活的Swiss 3T3和MH 3T3细胞中，发现融合蛋白在板足、丝状足、局灶接触和应力纤维中积累。此外，通过比较通透化细胞中GFP-actin和Cy3.5-phalloidin （F-actin的独立标记物）的荧光图像，我们发现这两种荧光信号完全重叠。在被单核增生李斯特菌感染的转染了gfp -肌动蛋白的Hela细胞中，融合蛋白的荧光显示在一个运动细菌后面形成的富含f -肌动蛋白的彗尾中动态关联。在稳定转染的PC12细胞中，gfp -肌动蛋白平均占总肌动蛋白的5%，这些细胞表现出正常的生长行为，并对神经生长因子的处理作出反应，延长神经突样延伸，其丝状足状尖端密集排列着丝状gfp -肌动蛋白。最后，GFP-actin在活细胞中的光漂白衰变时间为63秒，远远超过荧光素标记的肌动蛋白偶联物，并且GFP-actin在光漂白过程中对细胞骨架几乎没有损伤。在证明了GFP-actin作为细胞骨架探针的适用性后，其荧光被用于长期成像研究，旨在记录大鼠膀胱NET-II癌细胞在生长因子介导的24小时上皮向间质转化过程中细胞骨架的变化。本征荧光探针还用于研究肌动蛋白细胞骨架的组织和单个间充质NET-II细胞在上皮细胞群中缓慢迁移的行为。
The product of a GFP-actin gene fusion, permanently or transiently transfected in diverse mammalian cell lines, was shown to be a suitable, intrinsic probe of both the organization and dynamics of the actin cytoskeleton. In live Swiss 3T3 and MH 3T3 cells, the fusion protein was found to accumulate in lamellipodia, filopodia, focal contacts and stress fibers. Furthermore, comparisons of fluorescence images of GFP-actin and Cy3.5-phalloidin, an independent marker of F-actin, in permeabilized cells shelved a complete overlap of the two fluorescence signals. In GFP-actin-transfected Hela cells that had been infected with Listeria monocytogenes, the fluorescence of the fusion protein was shown to dynamically associate in the F-actin rich comet tail that formed behind a motile bacterium. In stable transfectants of PC12 cells, GFP-actin constituted on the average 5% of the total actin - these cells exhibited normal growth behavior and responded to treatment with nerve growth factor by extending neurite-like extensions, the filopodia-like tips of which were densely packed with filamentous GFP-actin. Finally, the photobleaching decay time of GFP-actin in live cells of 63 seconds was much longer than that of fluorescein-labeled actin conjugates and little or no damage to the cytoskeleton was found during the photobleaching of GFP-actin. Having shown the suitability of GFP-actin as a probe of the cytoskeleton, its fluorescence was used in long-term imaging studies aimed at documenting changes in the cytoskeleton of rat bladder NET-II carcinoma cells during the 24-hour growth factor-mediated epithelia to mesenchyme transformation. The intrinsic fluorescent probe was also used to investigate the organization of the actin cytoskeleton and behavior of individual mesenchyme NET-II cells slowly migrating through a colony of epithelia cells.