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Rifah Gheyas, Lisa A Brennan, Marc Kantorow, A Sue Menko; A role for the PI3K regulator PIK3IP1 in signaling the autophagy-dependent removal of organelles during lens development. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1712.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies demonstrated a critical role for autophagy in the formation of the lens organelle free zone (OFZ) during lens development. To establish the upstream signal initiating the autophagy-dependent formation of the OFZ we examined inhibition of PI3K signaling by phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). We tested the hypothesis that PIK3IP1 initiates the autophagy-dependent formation of the OFZ through its interaction with the p110 catalytic domain of PI3K.
Chick embryo lenses were separated into epithelial (E) and fiber (F) regions, or microdissected to isolate 4 distinct regions of differentation: central anterior epithelium (EC), equatorial epithelium (EQ), cortical fiber (FC), and nuclear fiber (FP) zones. RNA sequencing followed by functional gene clustering was performed on microdissected lens fractions at E13 and PIK3IP1 expression confirmed by immunoblot. Development-specific molecular linkage of the regulatory PIK3IP1 subunit with PI3K catalytic subunits p110α, p110β, and p110γ was examined by co-immunoprecipitation analysis in E/F regions of D10-15 lenses. PIK3IP1 was overexpressed in ex vivo whole chick embryonic lens cultures and the lenses monitored for loss of organelles.
RNA seq analysis revealed a sharp increase in PIK3IP1 expression in fiber cells of the E13 lens. PIK3IP1 associates with all p110 isoforms examined in lens epithelial and fiber cells from E10-15, suggesting PIK3IP1 is a general common regulator of PI3K signaling throughout lens differentiation. Importantly, association of PIK3IP1 specifically with the p110γ catalytic subunit of PI3K increases significantly in fiber cells at D14, the day before the OFZ forms, consistent with a role in shutting down PI3K signaling to induce the autophagic pathway that removes lens organelles. Overexpression of PIK3IP1 in chick embryo lenses induced premature removal of mitochondria and endoplasmic reticulum, as detected by decreased levels of mitochondrial protein marker TOMM20 and ER protein Grp78/Bip, without affecting expression of α-tubulin or αB-crystallin.
PIK3IP1 associates with PI3K p110γ's catalytic domain and its overexpression results in loss of mitochondria and ER in ex vivo cultured embryonic lenses. These results are consistent with an important role for PIK3IP1 in the initiation of autophagy signaling to form the lens OFZ.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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