Polycystin-2 immunolocalization and function in zebrafish
Polycystin-2 immunolocalization and function in zebrafish
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DOI：
10.1681/asn.2006040412
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发表时间：
2006-10-01
影响因子：
13.6
通讯作者：
Drummond, Iain A.
中科院分区：
文献类型：
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作者：
Obara, Tomoko;Mangos, Steven;Drummond, Iain A.
多囊蛋白-2在肾上皮细胞中作为一种阳离子渗透性瞬时受体电位离子通道发挥作用，当突变时可导致人类常染色体显性多囊肾病。为了进一步探索多囊蛋白-2在体内的功能，本研究对其在斑马鱼胚胎发生过程中的表达和功能进行了研究。pkd2 mRNA是普遍表达的，其在幼体肾脏中的存在可以通过分离肾原的逆转录- pcr证实。抗斑马鱼多囊素-2抗体免疫染色显示，运动肾上皮细胞、纤毛和胞内细胞膜表达蛋白。细胞内定位是片段特异性的；在近端肾段，多囊素-2集中在基底外侧细胞膜上，而在尾端肾原段，多囊素-2集中在根尖下的细胞质囊泡中。polycytin -2也在肌肉细胞和各种与机械传导相关的感觉细胞中表达，包括耳细胞、侧线器官和嗅觉基板。多囊蛋白-2 mRNA表达的破坏导致肾原肾囊肿、体轴弯曲、器官侧边缺陷和脑积水缺陷，这些都可以通过表达人类PKD2 mRNA来挽救。胞外第一环和胞质尾部c端磷酸氟酸簇分选蛋白-1 （PACS-1）结合位点的框内缺失导致多囊蛋白-2错定位到细胞顶端表面。与斑马鱼鞭毛内转运蛋白（IFT）突变体不同，囊肿的形成与纤毛缺陷无关，而是与肾液输出量减少、尾管顶端细胞膜扩张和尾侧肾原肾段闭塞相关。
Polycystin-2 functions as a cation-permeable transient receptor potential ion channel in kidney epithelial cells and when mutated results in human autosomal dominant polycystic kidney disease. For further exploration of the in vivo functions of Polycystin-2, this study examined its expression and function during zebrafish embryogenesis. pkd2 mRNA is ubiquitously expressed, and its presence in the larval kidney could be confirmed by reverse transcription-PCR on isolated pronephroi. Immunostaining with anti-zebrafish Polycystin-2 antibody revealed protein expression in motile kidney epithelial cell cilia and intracellular cell membranes. Intracellular localization was segment specific; in the proximal nephron segment, Polycystin-2 was localized to basolateral cell membranes, whereas in the caudal pronephric segment, Polycystin-2 was concentrated in subapical cytoplasmic vesicles. Polycystin-2 also was expressed in muscle cells and in a variety of sensory cells that are associated with mechanotransduction, including cells of the ear, the lateral line organ, and the olfactory placodes. Disruption of Polycystin-2 mRNA expression resulted in pronephric kidney cysts, body axis curvature, organ laterality defects, and hydrocephalus-defects that could be rescued by expression of a human PKD2 mRNA. In-frame deletions in the first extracellular loop and C-terminal phosphofurin acidic cluster sorting protein-1 (PACS-1) binding sites in the cytoplasmic tail caused Polycystin-2 mislocalization to the apical cell surface. Unlike zebrafish intraflagellar transport protein (IFT) mutants, cyst formation was not associated with cilia defects and instead correlated with reduced kidney fluid output, expansion of caudal duct apical cell membranes, and occlusion of the caudal pronephric nephron segment.