摘要
目的探究肌萎缩侧索硬化症蛋白2(ALS2)对初级纤毛发生的调控作用及其可能机制。方法通过免疫荧光染色检测ALS2蛋白的亚细胞定位。采用siRNA干涉特异性敲低RPE⁃1细胞中ALS2 mRNA含量,检测ALS2蛋白含量减少对初级纤毛的影响。利用Western印迹检测初级纤毛组装与去组装过程中ALS2蛋白的动态变化。建立初级纤毛去组装实验体系,检测ALS2蛋白缺失对初级纤毛去组装过程的影响,Time⁃laps活细胞实时成像技术检测ALS2对初级纤毛去组装过程中纤毛末端剪切的影响。结果免疫荧光显示,ALS2定位于初级纤毛发生的基体——中心体周围。在RPE⁃1细胞中敲低ALS2会显著提高初级纤毛异常发生的比例。蛋白水平动态检测结果显示,在初级纤毛去组装过程中ALS2蛋白表达水平逐渐增加;敲低ALS可显著抑制纤毛去组装过程。活细胞实时成像显示,ALS2蛋白缺失可抑制初级纤毛去组装过程的末端剪切。结论ALS2作为中心体定位蛋白,通过促进初级纤毛末端剪切的正常进行,调控初级纤毛组装与去组装之间的动态平衡,防止纤毛异常生长引起的纤毛类疾病的发生。
Objective To investigate the regulatory effect of amyotrophic lateral sclerosis protein 2(ALS2)on primary cilia and the possible mechanism.Methods The subcellular localization of ALS2 was detected with immunofluorescence.Subsequently,siRNA interference was used to knock down the content of ALS2 mRNA in RPE-1 cells specifically and the effect of reduced ALS2 protein content on primary cilia was studied.The protein level of ALS2 during ciliary assembly and disassembly was detected by Western blotting.A primary experimental system for ciliary disassembly was established to determine the effect of ALS2 protein deletion on the disassembly process of primary cilia.Finally,time-laps live-cell real time imaging was used to detect the effect of ALS2 on ciliary decapitation during disassembly of primary cilia.Results Immunofluorescence results showed that ALS2 was localized around the centrosome,which was the matrix of primary ciliogenesis.Knockdown of ALS2 in RPE-1 cells significantly increased the proportion of aberrant ciliogenesis.The results of dynamic detection of protein levels showed that the protein expression level of ALS2 gradually increased during the disassembly of primary cilia.Knocking down ALS significantly inhibited the cilia disassembly process.Live cell real time imaging showed that deletion of ALS2 protein inhibited ciliary decapitation during disassembly of primary cilia.Conclusion As a centrosome localization protein,ALS2 regulates the dynamic balance between primary ciliary assembly and disassembly by promoting the ciliary decapitation,and prevents the occurrence of ciliary diseases caused by abnormal growth of cilia.
作者
何春雨
张必雨
简肖肖
王雯
张宇程
李慧艳
胡怀斌
HE Chunyu;ZHANG Biyu;JIAN Xiaoxiao;WANG Wen;ZHANG Yucheng;LI Huiyan;HU Huaibin(National Center of Biomedical Analysis,Beijing 100850,China)
出处
《军事医学》
CAS
CSCD
2023年第8期571-575,共5页
Military Medical Sciences
基金
国家自然科学基金青年科学基金项目(8200113021)。
作者简介
何春雨,男,硕士研究生,研究方向:初级纤毛调控,E⁃mail:cyhe@xmail.ncba.ac.cn;通信作者:胡怀斌,Tel:010⁃66930344,E⁃mail:hbhu@xmail.ncba.ac.cn。
