A targeted RNAi screen identifies factors affecting diverse stages of receptor-mediated transcytosis.
A targeted RNAi screen identifies factors affecting diverse stages of receptor-mediated transcytosis.
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DOI：
10.1083/jcb.201609035
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发表时间：
2017-02
期刊：
影响因子：
--
通讯作者：
Lencer W
中科院分区：
文献类型：
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作者：
Nelms B;Dalomba NF;Lencer W
转胞吞作用在建立细胞极性和介导转运大货物穿过上皮屏障中起重要作用，但其分子基础尚不清楚。Nelms等人提出了一个新的受体介导的转胞吞作用基因数据集，并表明顶端和基底侧再循环和转胞吞途径在遗传上是可分离的。通过胞吞转运的内体转运是蛋白质和其他大货物穿过正常组织中的上皮屏障的主要机制。转胞吞作用对于上皮细胞和其他极化细胞中建立和维持膜极性也是必不可少的。为了鉴定该途径的新组分，我们对Fcγ受体FcRn双向转胞吞IgG所必需的因子进行了高通量RNA干扰筛选。该筛选鉴定了23个基因，其抑制导致FcRn介导的转胞吞作用的可再现减少。对四个顶级基因（EXOC 2、EXOC 7、PARD 6B和LEPROT）的脉冲追踪动力学转运测定揭示了对顶侧和基底侧回收和胞吞途径的不同影响，表明这些途径在遗传上是可分离的。我们还发现了一个强大的依赖PARD6B顶端，而不是基底侧，回收，牵连这个细胞极性基因的组装或维护顶端内体系统。该数据集深入了解囊泡运输如何适应分化细胞类型的专门功能，并为上皮细胞运输开辟了新的研究途径。
Transcytosis plays an important role in establishing cell polarity and in mediating transport of large cargo across epithelial barriers, but its molecular basis is unclear. Nelms et al. present a new dataset of genes involved in receptor-mediated transcytosis and show that the apical and basolateral recycling and transcytotic pathways are genetically separable. Endosome transport by transcytosis is the primary mechanism by which proteins and other large cargo traverse epithelial barriers in normal tissue. Transcytosis is also essential for establishing and maintaining membrane polarity in epithelia and other polarized cells. To identify novel components of this pathway, we conducted a high-throughput RNA interference screen for factors necessary for the bidirectional transcytosis of IgG by the Fcγ receptor FcRn. This screen identified 23 genes whose suppression resulted in a reproducible decrease in FcRn-mediated transcytosis. Pulse-chase kinetic transport assays on four of the top-ranking genes (EXOC2, EXOC7, PARD6B, and LEPROT) revealed distinct effects on the apical and basolateral recycling and transcytotic pathways, demonstrating that these pathways are genetically separable. We also found a strong dependence on PARD6B for apical, but not basolateral, recycling, implicating this cell polarity gene in assembly or maintenance of the apical endosomal system. This dataset yields insights into how vesicular transport is adapted to the specialized functions of differentiated cell types and opens new research avenues into epithelial trafficking.