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GFP associates with microfilaments in fixed cells.
Histochemistry and Cell Biology ( IF 2.3 ) Pub Date : 2001-08-02 , DOI: 10.1007/s004180100293
H D Schmitz 1 , J Bereiter-Hahn
Affiliation  

The discovery of the green fluorescent protein (GFP) and its use as a marker for proteins in cells revolutionised cell biology. Among its applications are the intracellular localisation of proteins and the investigation of the organisation, regulation and dynamics of the cytoskeleton. GFP itself is considered to be an inert protein, homogeneously distributed within the cytoplasm. Here we investigated the intracellular distribution of GFP in an amphibian and in various mammalian cell lines (XTH2, CHO-K1, HaCaT, MDCK, NIH-3T3) by confocal laser scanning microscopy. After paraformaldehyde fixation GFP became associated with microfilaments in all the cell lines investigated. This interaction was not impaired by detergent treatment (1% Brij 58 for 10 min). In contrast to the F-actin binding of GFP in fixed cells, association of GFP with stress fibres was not detectable in living cells. The actin-binding property of GFP might contribute also to the interaction of fusion proteins with microfilaments. Thus, careful controls are unavoidable in investigating (weak) actin-binding proteins in fixed cells. Because no association of GFP with microfilaments was detectable in living cells, it is recommended to monitor the intracellular distribution of GFP-tagged proteins in vivo.

中文翻译:

GFP与固定细胞中的微丝结合。

绿色荧光蛋白(GFP)的发现及其作为细胞蛋白质标记的用途彻底改变了细胞生物学。它的应用包括蛋白质的细胞内定位以及细胞骨架的组织,调节和动力学研究。GFP本身被认为是一种惰性蛋白,均匀地分布在细胞质内。在这里,我们通过共聚焦激光扫描显微镜研究了两栖动物和各种哺乳动物细胞系(XTH2,CHO-K1,HaCaT,MDCK,NIH-3T3)中GFP的细胞内分布。在多聚甲醛固定后,GFP在所有研究的细胞系中都与微丝结合。洗涤剂处理(1%Brij 58 10分钟)不会削弱这种相互作用。与固定细胞中GFP的F-肌动蛋白结合不同,在活细胞中无法检测到GFP与应激纤维的相关性。GFP的肌动蛋白结合特性也可能有助于融合蛋白与微丝的相互作用。因此,在研究(弱)固定细胞中的肌动蛋白结合蛋白时不可避免地要进行仔细的控制。由于在活细胞中未检测到GFP与微丝的缔合,因此建议在体内监测GFP标记蛋白的细胞内分布。
更新日期:2019-11-01
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