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Firoz Kabir, Shahid Yar Khan, Sheikh Riazuddin, Javed Akram, J. Fielding Hejtmancik, S Amer Riazuddin; Functional Assessment of FYCO1 Underlines the Association with Autophagy and Confirms the Indispensable Role in Lens Development. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
FYVE and coiled-coil domain containing 1 (FYCO1) an LC3, Rab7, and PI3P interacting protein that plays a critical role in microtubule plus-end-directed transport of autophagic vesicles and has been implicated in autophagy. Mutations in FYCO1 have been associated with autosomal recessive congenital cataracts (arCC). Here, we investigate the role of FYCO1 in autophagy, in general, and lens development and maintenance, in particular.
We developed a Fyco1-/- conditional knockout (KO) mouse model and confirmed the knockdown of Fyco1 expression through quantitative real-time PCR (qRT-PCR). In parallel, we developed a human lens epithelial (HLE) knock-in (KI) cell line through CRISPR/Cas9 strategy harboring the nonsense mutation (c.2206C>T, p.Q736*) that has been associated with congenital cataracts. The off-target effects of CRISPER/Cas9 editing were investigated by high-throughput sequencing. The role of FYCO1 in autophagy was examined by both estimating the levels of lipidated LC3 and measuring autophagic flux through flow cytometry.
Real-time PCR analyses illustrated no expression of Fyco1 at different developmental stages in Fyco1-/- KO mice lens. The KO mice develop bilateral cataracts as early as two weeks of age that progressed to mature cataracts by 12 weeks. Histological analysis of Fyco1-/- mice exhibits severely disorganized lens with large vacuoles, swollen lens fiber cells, and liquefaction. The CRISPER/Cas9 engineered HLE KI cells show only residual expression of FYCO1. We observed a two-hour increase in the population doubling time while the cell cycle analysis illustrates a 6% increase in G1 population (p<0.005) for KI cell line compared with wild-type cells. Flow cytometer based quantification revealed a lower (69%) LC3 lipidation in KI cell line compared to wild-type HLE cells. Likewise, measurement of the autophagic flux in live cells through Cyto-ID staining of autophagic compartments illustrate a reduced autophagic flux (22.2%) in KI cell line compared with wild-type cells.
In summary, our data confirms the association of FYCO1 with autophagy in human lens epithelial cells and its indispensable role in ocular lens development and maintenance of its transparency.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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