title: Clathrin-dependent endocytosis plays a predominant role in cellular uptake of double-stranded RNA in the red flour beetle.
abstract: RNA interference (RNAi) is a highly conserved gene regulatory mechanism in eukaryotic organisms; however, an understanding of mechanisms of cellular uptake of double-stranded RNA (dsRNA) in different organisms remains elusive. By using pharmacological inhibitors of different endocytic pathways in conjunction with RNAi of a marker gene (lethal giant larvae, TcLgl) in the red flour beetle (Tribolium castaneum), we demonstrated that two inhibitors (chlorpromazine and bafilomycin-A1) of clathrin-dependent endocytosis can nearly abolish or significantly diminish RNAi of TcLgl, whereas methyl-β-cyclodextrin and cytochalasin-D, known to inhibit other endocytic pathways, showed no effect on RNAi of TcLgl. By using Cy3-labeled TcLgl dsRNA, we observed significantly reduced cellular uptake of TcLgl dsRNA in midgut cells after larvae were injected with each of the two clathrin-dependent endocytosis inhibitors. By using an "RNAi of RNAi" strategy, we further demonstrated that suppression of each transcript of the four key genes encoding clathrin heavy chain (TcChc), clathrin coat assembly protein AP50 (TcAP50), vacuolar (H(+))-ATPase subunit H (TcVhaSFD) and a ras-related protein (TcRab7) in clathrin-dependent endocytosis by RNAi can significantly impair RNAi of TcLgl. These results support our conclusion that clathrin-dependent endocytosis is a major mechanism in cellular uptake of dsRNA in T. castaneum. Our study also provides new insights into improving RNAi efficiency by enhancing dsRNA endosomal release.
authors: Department of Entomology, Kansas State University, Manhattan, KS 66506, USA; Department of Entomology, China Agricultural University, Beijing 100094, China.
authors: Da
authors: D
authors: Xiao
authors: Department of Entomology, China Agricultural University, Beijing 100094, China.
authors: Xiwu
authors: X
authors: Gao
authors: Department of Entomology, Kansas State University, Manhattan, KS 66506, USA; School of Life Sciences, Anhui Agricultural University, Hefei, Anhui 230036, China.
authors: Jiaping
authors: J
authors: Xu
authors: Department of Entomology, Kansas State University, Manhattan, KS 66506, USA.
authors: Xiao
authors: X
authors: Liang
authors: Department of Entomology, Kansas State University, Manhattan, KS 66506, USA; College of Life Sciences, Yunnan Normal University, Kunming, Yunnan 650092, China.
authors: Qingqing
authors: Q
authors: Li
authors: Department of Entomology, Kansas State University, Manhattan, KS 66506, USA.
authors: Jianxiu
authors: J
authors: Yao
authors: Department of Entomology, Kansas State University, Manhattan, KS 66506, USA. Electronic address: kzhu@ksu.edu.
authors: Kun Yan
authors: KY
authors: Zhu
mesh_terms: D000818:Animals; D022163:Clathrin-Coated Vesicles / Q000502:physiology*; D004705:Endocytosis; D007814:Larva / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism*; D014231:Tribolium / Q000378:metabolism*
chemical_list: D012330:RNA, Double-Stranded
keywords: Cellular uptake; Clathrin-dependent endocytosis; RNA interference; Tribolium castaneum