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. 2024 Apr;38(7):e25031.
doi: 10.1002/jcla.25031. Epub 2024 Mar 21.

INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells

Denghui Zhai  1 , Lamei Li  1 , Cheng Chen  1 , Xue Wang  1 , Ruming Liu  1 , Ying Shan  1
Affiliations

INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells

Denghui Zhai et al. J Clin Lab Anal. 2024 Apr.

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-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.

Keywords: INPP5E; PI(4,5)P2; PI4P; ciliary base; primary cilia.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Localization of PI4P and PI(4,5)P2 on cilia in RPE1 cells. (A) Immunofluorescence detected the localization of PI4P in RPE1 cells cultured in serum‐free medium for 48 h. (B) The fluorescence intensity of PI4P and Ace‐tubulin in Panel A was assessed using ImageJ. (C) Immunofluorescence detected the localization of PI(4,5)P2 in RPE1 cells cultured in serum‐free medium for 48 h. (D) The fluorescence intensity of PI(4,5)P2 and γ‐tubulin in Panel B was assessed using ImageJ. Scale bar, 10 μm.
FIGURE 2
FIGURE 2
Localization of INPP5E on cilia in RPE1 cells. (A) Immunofluorescence detected the localization of INPP5E in RPE1 cells with siControl (up) or siINPP5E (down) treated and cultured in serum‐free medium for 48 h. (B, C) The fluorescence intensity of INPP5E and Ace‐tubulin in Panel A was assessed using ImageJ. (D) Comparison of the gene sequences after INPP5E KO displayed by SnapGene. (E) Synthesized amino acids after INPP5E KO displayed by SnapGene. (F) Immunofluorescence detected the localization of INPP5E in RPE1 cells (up) or INPP5E KO cells (down) cultured in serum‐free medium for 48 h. (G, H) The fluorescence intensity of INPP5E and Ace‐tubulin in Panel F was assessed using ImageJ. Scale bar, 10 μm.
FIGURE 3
FIGURE 3
Localization of PI4P on cilia in siRNA‐treated cells or INPP5E KO cells. (A) Immunofluorescence detected the localization of PI4P in RPE1 cells treated with siRNA and cultured in serum‐free medium for 48 h. (B, C) The fluorescence intensity of PI4P and Ace‐tubulin in Panel A was assessed using ImageJ. (D) Immunofluorescence detected the localization of PI4P in INPP5E KO RPE1 cells cultured in serum‐free medium for 48 h. (E, F) The fluorescence intensity of PI4P and Ace‐tubulin in Panel D was assessed using ImageJ. Scale bar, 10 μm.
FIGURE 4
FIGURE 4
Localization of PI(4,5)P2 on cilia in siRNA‐treated cells or INPP5E KO cells. (A) Immunofluorescence detected the localization of PI(4,5)P2 in RPE1 cells treated with siRNA and cultured in serum‐free medium for 48 h. (B, C) The fluorescence intensity of PI(4,5)P2 and Ace‐tubulin in Panel A was assessed using ImageJ. (D) Immunofluorescence detected the localization of PI(4,5)P2 in INPP5E KO RPE1 cells cultured in serum‐free medium for 48 h. (E, F) The fluorescence intensity of PI(4,5)P2 and Ace‐tubulin in Panel D was assessed using ImageJ. Scale bar, 10 μm.
FIGURE 5
FIGURE 5
Localization of PI4P and PI(4,5)P2 on cilia marked with Smoothened‐tRFP (SMO‐tRFP). (A) Immunofluorescence detected the localization of PI(4,5)P2 in SMO‐tRFP RPE1 cells cultured in serum‐free medium for 48 h. (B) The fluorescence intensity of PI(4,5)P2 and Ace‐tubulin in Panel A was assessed using ImageJ. (C) Immunofluorescence detected the localization of PI4P in SMO‐tRFP RPE1 cells cultured in serum‐free medium for 48 h. (D) The fluorescence intensity of PI4P and Ace‐tubulin in Panel A was assessed using ImageJ. Scale bar, 10 μm.
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