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Tia Kowal, BIAO WANG, Philipp Prosseda, Jorge Antonio Alvarado, Wei He, ke ning, Yang Hu, Yang Sun; Role of inositol phosphatase OCRL in microtubule nucleation: Implications for Oculocerebrorenal Syndrome of Lowe. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4934. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Lowe syndrome results from mutation in OCRL, an inositol phosphatase which dephosphorylates PI(4,5)P2 to PI4P. This X-linked recessive disorder presents with congenital cataracts, glaucoma, renal dysfunction and brain abnormalities. The mechanism of OCRL causing cataracts and glaucoma is not well understood. Impairment in structural support, including modified cytoskeletal proteins and microtubule-organization elements could be initiating factors in congenital cataracts and glaucoma. Here we investigate the role of OCRL mediated microtubule nucleation in the pathogenesis of ocular phenotypes of Lowe syndrome.
Immunostaining with antibodies targeting γ-tubulin and OCRL, and transfection of OCRL-GFP were used to observe OCRL localization in the phases of the cell cycle in hTERT-RPE cells. OCRL’s localization within the centrosomal structure was analyzed by structure illumination microscopy. Fibroblasts from unrelated Lowe syndrome patients (Lowe1 and Lowe2) and NHF cells were immunostained for α- and γ-tubulin to determine microtubule organization. To confirm microtubule disorganization observed in Lowe1 and Lowe2 cells was a result of OCRL deficiency, immunostaining for tubulins was performed on OCRL knockout MEFs and on RPE cells in which OCRL was knocked down by siRNA. Microtubule dynamics were analyzed by live cell imaging of cells transfected with EB3-mCherry, a microtubule plus end protein.
OCRL was found to localize to centrosomes during all phases of the cell cycle. Localization of OCRL was determined to be at the proximal end of both mother and daughter centrioles, based on its position to CEP164 and C-NAP (n=30 cells in each group, p <0.05 ANOVA). OCRL deficiency resulted in disrupted microtubule nucleation from centrosomes as observed in Lowe1, Lowe2, OCRL knock out MEFs and OCRL knock down by siRNA. This was confirmed by live cell imaging of OCRL knock down and Lowe patient cells (n=50 cells in each group, p < 0.05 pair t-test). Lowe patient cells were observed to have a loss of SSX2IP, a microtubule anchoring factor, at the centrosome. As a consequence of microtubule disorganization, lysosome localization was disrupted.
OCRL localizes to the centrosome and regulates centrosomal microtubule nucleation, which may play critical roles in microtubule-based organelle positioning and may contribute to cataract or glaucoma development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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