{"title":"INPP5E 调节 RPE1 细胞纤毛上磷脂的分布。","authors":"Denghui Zhai, Lamei Li, Cheng Chen, Xue Wang, Ruming Liu, Ying Shan","doi":"10.1002/jcla.25031","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Background</h3>\n \n <p>Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>The EGFP-2xP4M<sup>SidM</sup>, PH<sup>PLCδ1</sup>-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The <i>INPP5E</i> KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P<sub>2</sub> and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P<sub>2</sub> is localized at the base of cilia. Knocking down or knocking out <i>INPP5E</i> alters this distribution, resulting in the distribution of PI(4,5)P<sub>2</sub> along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P<sub>2</sub> is located in the ciliary membrane labeled by SMO-tRFP.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P<sub>2</sub> localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P<sub>2</sub>, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.</p>\n </section>\n </div>","PeriodicalId":15509,"journal":{"name":"Journal of Clinical Laboratory Analysis","volume":"38 7","pages":""},"PeriodicalIF":2.6000,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jcla.25031","citationCount":"0","resultStr":"{\"title\":\"INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells\",\"authors\":\"Denghui Zhai, Lamei Li, Cheng Chen, Xue Wang, Ruming Liu, Ying Shan\",\"doi\":\"10.1002/jcla.25031\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> Background</h3>\\n \\n <p>Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Methods</h3>\\n \\n <p>The EGFP-2xP4M<sup>SidM</sup>, PH<sup>PLCδ1</sup>-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The <i>INPP5E</i> KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P<sub>2</sub> and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Results</h3>\\n \\n <p>In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P<sub>2</sub> is localized at the base of cilia. Knocking down or knocking out <i>INPP5E</i> alters this distribution, resulting in the distribution of PI(4,5)P<sub>2</sub> along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P<sub>2</sub> is located in the ciliary membrane labeled by SMO-tRFP.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Conclusions</h3>\\n \\n <p>INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P<sub>2</sub> localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P<sub>2</sub>, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.</p>\\n </section>\\n </div>\",\"PeriodicalId\":15509,\"journal\":{\"name\":\"Journal of Clinical Laboratory Analysis\",\"volume\":\"38 7\",\"pages\":\"\"},\"PeriodicalIF\":2.6000,\"publicationDate\":\"2024-03-21\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jcla.25031\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Clinical Laboratory Analysis\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/jcla.25031\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MEDICAL LABORATORY TECHNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Clinical Laboratory Analysis","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/jcla.25031","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MEDICAL LABORATORY TECHNOLOGY","Score":null,"Total":0}
INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells
Background
Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function.
Methods
The EGFP-2xP4MSidM, PHPLCδ1-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ.
Results
In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P2 is located in the ciliary membrane labeled by SMO-tRFP.
Conclusions
INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P2, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.
期刊介绍：
Journal of Clinical Laboratory Analysis publishes original articles on newly developing modes of technology and laboratory assays, with emphasis on their application in current and future clinical laboratory testing. This includes reports from the following fields: immunochemistry and toxicology, hematology and hematopathology, immunopathology, molecular diagnostics, microbiology, genetic testing, immunohematology, and clinical chemistry.
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