Wang Weishu, Shan Ying, Liu Ruming, Li Dengwen, Zhou Jun, Lu Quanlong, Zhao Huijie
Department of Genetics and Cell Biology, State Key Laboratory of Medicinal Chemical Biology, Haihe Laboratory of Cell Ecosystem, College of Life Sciences, Nankai University, Tianjin, China.
Center for Cell Structure and Function, Shandong Provincial Key Laboratory of Animal Resistance Biology, College of Life Sciences, Shandong Normal University, Jinan, China.
J Clin Lab Anal. 2025 May;39(9):e70000. doi: 10.1002/jcla.70000. Epub 2025 Apr 7.
Primary cilia are organelles formed on the cell surface. They can act as cellular antennae to sense signals and play important roles in various biological processes. Abnormalities in primary cilia lead to a variety of diseases collectively known as ciliopathies. Intraflagellar transport protein 20 (IFT20) has been implicated in ciliogenesis.
IFT20 knockout cell lines were established using the CRISPR-Cas9 gene editing technology. The GFP-IFT20 plasmid was constructed with the Gateway cloning system. Protein levels were detected via immunoblotting, and the localization of IFT20, acetylated α-tubulin, ARL13B, CP110, MKS3, IFT88, and IFT140 in wild-type and IFT20 knockout cells was examined by immunofluorescence microscopy. The fluorescence intensities were analyzed using ImageJ. Data quantifications and mass spectrometry results were analyzed using GraphPad Prism and Metascape.
The IFT20 deficiency impaired ciliogenesis and reduced cilium length. IFT20 depletion did not affect the removal of centriolar coiled-coil protein 110 (CP110) from the mother centriole or the recruitment of Meckel-Gruber syndrome type 3 (MKS3) to the transition zone. Mass spectrometry analysis revealed that proteins interacting with IFT20 were mainly IFT components. IFT20 knockout decreased the levels of both IFT88 and IFT140, and abrogated IFT88 localization at the basal body and ciliary axoneme. IFT20 knockout also impaired IFT140 localization at the ciliary axoneme but did not affect its localization at the basal body.
IFT20 is involved in ciliogenesis by regulating the level and localization of other IFT proteins and may have important implications in ciliopathies and related diseases.
初级纤毛是在细胞表面形成的细胞器。它们可作为细胞的天线来感知信号，并在各种生物学过程中发挥重要作用。初级纤毛异常会导致多种疾病，统称为纤毛病。鞭毛内运输蛋白20（IFT20）与纤毛发生有关。
使用CRISPR-Cas9基因编辑技术建立IFT20基因敲除细胞系。利用Gateway克隆系统构建GFP-IFT20质粒。通过免疫印迹检测蛋白水平，并通过免疫荧光显微镜检查野生型和IFT20基因敲除细胞中IFT20、乙酰化α-微管蛋白、ARL13B、CP110、MKS3、IFT88和IFT140的定位。使用ImageJ分析荧光强度。使用GraphPad Prism和Metascape分析数据定量和质谱结果。
IFT20缺陷损害了纤毛发生并缩短了纤毛长度。IFT20缺失不影响中心粒卷曲螺旋蛋白110（CP110）从母中心粒的去除或梅克尔-格鲁伯综合征3型（MKS3）向过渡区的募集。质谱分析显示，与IFT20相互作用的蛋白质主要是IFT成分。IFT20基因敲除降低了IFT88和IFT140的水平，并消除了IFT88在基体和纤毛轴丝的定位。IFT20基因敲除也损害了IFT140在纤毛轴丝的定位，但不影响其在基体的定位。
IFT20通过调节其他IFT蛋白的水平和定位参与纤毛发生，可能对纤毛病和相关疾病具有重要意义。
