Purchase this article with an account.
Na Luo, Emilie Song, Jorge Antonio Alvarado, Yang Sun; Distribution Defects of PI(4,5)P2 in Primary Cilium of Lowe Syndrome Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1734. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Lowe syndrome is a rare X-linked disorder, caused by mutations in gene OCRL1, characterized by bilateral congenital cataracts and glaucoma, mental retardation, and proximal renal tubular dysfunction. Previously, we showed OCRL, an inositol polyphosphate 5-phosphatase which dephosphorylates PI(4,5)P2 to PI(4)P, localizes to the primary cilium. However, the role of phosphoinositides in primary cilium is not defined. The purpose of this study is to determine the distribution of phosphoinositides in the primary cilium of Lowe syndrome cells.
Lentiviral CRISPR/cas9/OCRL1 was constructed and transduced into RPE cells. Two passages of puromycin selection were performed to generate stably mediated cells. All mouse embryonic fibroblasts (MEFs) were isolated from E14.5 embryos. WT or mutated GFP-OCRL were transfected into Lowe patient-derived fibroblasts or Lowe syndrome mouse model-derived MEFs using Lipofectamine LTX. Immunofluorescence was performed to investigate the localization of PI(4,5)P2 or PI(4)P in primary cilium.
Increased level of PI(4,5)P2 (30-45%, compared to 5±3% in control, p<0.001, student t-test) was exhibited in primary cilia of fibroblasts derived from Lowe syndrome patients, and CRISPR/cas9/OCRL1 stably-mediated RPE cells. Ciliary accumulation of PI(4,5)P2 (60±17%, compared to 8±2% in WT MEFs, p<0.001, student t-test) was observed in IOB-/- MEFs derived from Lowe syndrome mouse (Ocrl-/-:Inpp5b-/-:INPP5B+/+ ), whereas the expression of Ocrl (Ocrl+/Y:Inpp5b-/-:INPP5B+/+ ) prevented the PI(4,5)P2 build-up in the cilia. Loss of only Ocrl (Ocrl-/-) in mice resulted in an accumulation of PI(4,5)P2 within the ciliary axoneme (40±9%, p<0.001, student t-test). Furthermore, expression of WT OCRL rescued the elevated PI(4,5)P2 levels in Lowe patient fibroblasts, CRISPR/cas9/OCRL1 mediated RPE cells, and IOB-/- & Ocrl-/- MEFs.
Our findings demonstrate that ciliary phosphoinositide PI(4,5)P2 is regulated by OCRL and support the role of inositol phosphatase in primary cilia signaling.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only