The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro
The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro
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DOI：
10.1091/mbc.e08-07-0772
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发表时间：
2009-05-15
影响因子：
3.3
通讯作者：
Lipschutz, Joshua H.
中科院分区：
文献类型：
--
作者：
Zuo, Xiaofeng;Guo, Wei;Lipschutz, Joshua H.
初级纤毛存在于许多上皮细胞类型上，包括肾小管上皮细胞，其中它们被认为参与流量传感，并且与囊性肾病如常染色体显性遗传性多囊肾病的发病机制有关。我们以前本地化的exocyst，一个八蛋白质复合物参与膜贩运，到初级纤毛的Madin-Darby犬肾细胞，并表明它参与了cystogenesis。在这里，使用短发夹RNA（shRNA）敲低外囊表达和稳定转染诱导外囊过表达，我们表明，外囊蛋白Sec 10调节初级纤毛发生。使用免疫荧光，扫描和透射电子显微镜，初级纤毛只含有基体中看到Sec 10敲低细胞，并增加纤毛发生中看到Sec 10过表达细胞。这些表型似乎并不是因为细胞极性的总体变化，因为顶端，基底外侧和紧密连接蛋白保持适当的本地化。当细胞在胶原基质中生长时，Sec 10敲低阻止正常的囊肿形态发生，而Sec 10过表达导致囊肿发生增加。用抗犬shRNA的人Sec 10转染拯救了表型，证明了特异性。最后，Par 3最近被证明可以调节初级纤毛的生物发生。通过免疫荧光和免疫共沉淀共定位了Par 3和外囊，这与外囊在靶向和对接携带初级纤毛发生所需蛋白质的囊泡中的作用一致。
Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, in which they are felt to participate in flow sensing and have been linked to the pathogenesis of cystic renal disorders such as autosomal dominant polycystic kidney disease. We previously localized the exocyst, an eight-protein complex involved in membrane trafficking, to the primary cilium of Madin-Darby canine kidney cells and showed that it was involved in cystogenesis. Here, using short hairpin RNA (shRNA) to knockdown exocyst expression and stable transfection to induce exocyst overexpression, we show that the exocyst protein Sec10 regulates primary ciliogenesis. Using immunofluorescence, scanning, and transmission electron microscopy, primary cilia containing only basal bodies are seen in the Sec10 knockdown cells, and increased ciliogenesis is seen in Sec10-overexpressing cells. These phenotypes do not seem to be because of gross changes in cell polarity, as apical, basolateral, and tight junction proteins remain properly localized. Sec10 knockdown prevents normal cyst morphogenesis when the cells are grown in a collagen matrix, whereas Sec10 overexpression results in increased cystogenesis. Transfection with human Sec10 resistant to the canine shRNA rescues the phenotype, demonstrating specificity. Finally, Par3 was recently shown to regulate primary cilia biogenesis. Par3 and the exocyst colocalized by immunofluorescence and coimmunoprecipitation, consistent with a role for the exocyst in targeting and docking vesicles carrying proteins necessary for primary ciliogenesis.