Conduit Sarah E, Davies Elizabeth M, Fulcher Alex J, Oorschot Viola, Mitchell Christina A
Cancer Program, Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
Monash Micro Imaging, Monash University, Clayton, VIC, Australia.
Front Cell Dev Biol. 2021 Apr 30;9:634649. doi: 10.3389/fcell.2021.634649. eCollection 2021.
Primary cilia are evolutionary conserved microtubule-based organelles that protrude from the surface of most mammalian cells. Phosphoinositides (PI) are membrane-associated signaling lipids that regulate numerous cellular events via the recruitment of lipid-binding effectors. The temporal and spatial membrane distribution of phosphoinositides is regulated by phosphoinositide kinases and phosphatases. Recently phosphoinositide signaling and turnover has been observed at primary cilia. However, the precise localization of the phosphoinositides to specific ciliary subdomains remains undefined. Here we use superresolution microscopy (2D stimulated emission depletion microscopy) to map phosphoinositide distribution at the cilia transition zone. PI(3,4,5)P and PI(4,5)P localized to distinct subregions of the transition zone in a ring-shape at the inner transition zone membrane. Interestingly, the PI(3,4,5)P subdomain was more distal within the transition zone relative to PtdIns(4,5)P. The phosphoinositide effector kinase pAKT(S473) localized in close proximity to these phosphoinositides. The inositol polyphosphate 5-phosphatase, INPP5E, degrades transition zone phosphoinositides, however, studies of fixed cells have reported recombinant INPP5E localizes to the ciliary axoneme, distant from its substrates. Notably, here using live cell imaging and optimized fixation/permeabilization protocols INPP5E was found concentrated at the cilia base, in a distribution characteristic of the transition zone in a ring-shaped domain of similar dimensions to the phosphoinositides. Collectively, this superresolution map places the phosphoinositides in situ with the transition zone proteins and reveals that INPP5E also likely localizes to a subdomain of the transition zone membrane, where it is optimally situated to control local phosphoinositide metabolism.
初级纤毛是进化保守的基于微管的细胞器，从大多数哺乳动物细胞表面突出。磷酸肌醇（PI）是与膜相关的信号脂质，通过募集脂质结合效应器来调节众多细胞事件。磷酸肌醇的时空膜分布由磷酸肌醇激酶和磷酸酶调节。最近在初级纤毛处观察到了磷酸肌醇信号传导和周转。然而，磷酸肌醇在特定纤毛亚结构域的精确定位仍不明确。在这里，我们使用超分辨率显微镜（二维受激发射损耗显微镜）来绘制纤毛过渡区的磷酸肌醇分布。PI(3,4,5)P和PI(4,5)P在内过渡区膜处以环形定位于过渡区的不同子区域。有趣的是，相对于PtdIns(4,5)P，PI(3,4,5)P子结构域在过渡区内更靠远端。磷酸肌醇效应激酶pAKT(S473)定位于这些磷酸肌醇附近。肌醇多磷酸5-磷酸酶INPP5E可降解过渡区的磷酸肌醇，然而，对固定细胞的研究报告称重组INPP5E定位于纤毛轴丝，远离其底物。值得注意的是，在这里使用活细胞成像和优化的固定/通透方案发现INPP5E集中在纤毛基部，其分布特征与过渡区相似，呈与磷酸肌醇尺寸相似的环形结构域。总的来说，这张超分辨率图谱将磷酸肌醇与过渡区蛋白定位在一起，并揭示INPP5E也可能定位于过渡区膜的一个子结构域，在那里它处于最佳位置来控制局部磷酸肌醇代谢。
