INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells

Abstract

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-2xP4M^SidM, PH^PLCδ1-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.