September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Functional Assessment of FYCO1 Underlines the Association with Autophagy and Confirms the Indispensable Role in Lens Development
Author Affiliations & Notes
  • Firoz Kabir
    The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Shahid Yar Khan
    The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Sheikh Riazuddin
    National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
    National Centre for Genetic Diseases, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan
  • Javed Akram
    Allama Iqbal Medical College, University of Health Sciences, Lahore, Pakistan
    National Centre for Genetic Diseases, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan
  • J. Fielding Hejtmancik
    Ophthalmic Genetics and Visual Function Branch, National Eye Institute, Bethesda, Maryland, United States
  • S Amer Riazuddin
    The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Firoz Kabir, None; Shahid Khan, None; Sheikh Riazuddin, None; Javed Akram, None; J. Fielding Hejtmancik, None; S Amer Riazuddin, None
  • Footnotes
    Support  R01EY022714
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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