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利用RNA干扰技术敲低嗜热四膜虫中的Kin5会阻断Gef1的货物运输。

Kin5 knockdown in Tetrahymena thermophila using RNAi blocks cargo transport of Gef1.

作者信息

Awan Aashir, Bell Aaron J, Satir Peter

机构信息

Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

出版信息

PLoS One. 2009;4(3):e4873. doi: 10.1371/journal.pone.0004873. Epub 2009 Mar 17.

DOI:10.1371/journal.pone.0004873
PMID:19290045
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2653729/
Abstract

A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality.

摘要

构建和维持真核生物纤毛的一个关键过程是鞭毛内运输（IFT）。该过程利用驱动蛋白-2超家族的成员将货物运输到纤毛中（顺行运输），并利用动力蛋白进行逆行运输。我们使用一种新型的RNA干扰敲低方法，分析了同二聚体IFT驱动蛋白-2（Kin5）在四膜虫纤毛运输中的功能。在RNA干扰转化体中，Kin5严重下调并从纤毛中消失，但纤毛并未吸收，尽管顶端结构受到影响。敲低细胞去纤毛后，纤毛再生且细胞能够游动，这表明Kin5并不负责将轴丝前体运输到纤毛中以构建纤毛，但可能运输参与纤毛信号转导的分子，如鸟嘌呤核苷酸交换蛋白（GEF）。Gef1是一种四膜虫纤毛蛋白，目前的免疫共沉淀和免疫荧光研究表明，在缺乏纤毛Kin5的敲低细胞再生纤毛中不存在该蛋白。我们认为Kin5的一个重要货物是Gef1，敲低Kin5会导致细胞死亡。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/58c88995c1ac/pone.0004873.g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/4f64d57254b5/pone.0004873.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/ab44df1c2e9f/pone.0004873.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/26cdf11d07b3/pone.0004873.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/efac65cc4183/pone.0004873.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/5db73a4d8721/pone.0004873.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/703532a39590/pone.0004873.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/19896f9acee2/pone.0004873.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/e681eb248882/pone.0004873.g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/da9ec59541a6/pone.0004873.g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/58c88995c1ac/pone.0004873.g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/4f64d57254b5/pone.0004873.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/ab44df1c2e9f/pone.0004873.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/26cdf11d07b3/pone.0004873.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/efac65cc4183/pone.0004873.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/5db73a4d8721/pone.0004873.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/703532a39590/pone.0004873.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/19896f9acee2/pone.0004873.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/e681eb248882/pone.0004873.g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/da9ec59541a6/pone.0004873.g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0361/2653729/58c88995c1ac/pone.0004873.g010.jpg

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