Suppression of PI3K signaling is linked to autophagy activation and the spatiotemporal induction of the lens organelle free zone.

The terminal steps of lens cell differentiation require elimination of all organelles to create a central Organelle Free Zone (OFZ) that is required for lens function of focusing images on the retina. Previous studies show that the spatiotemporal elimination of these organelles during development is autophagy-dependent. We now show that the inhibition of PI3K signaling in lens organ culture results in the premature induction of autophagy within 24 h, including a significant increase in LAMP1+ lysosomes, and the removal of lens organelles from the center of the lens. Specific inhibition of just the PI3K/Akt signaling axis was directly linked to the elimination of mitochondria and ER, while pan-PI3K inhibitors that block all PI3K downstream signaling removed all organelles, including nuclei. Therefore, blocking the PI3K/Akt pathway was alone insufficient to remove nuclei. RNAseq analysis revealed increased mRNA levels of the endogenous inhibitor of PI3K activation, PIK3IP1, in differentiating lens fiber cells preceding the induction of OFZ formation. Co-immunoprecipitation confirmed that PIK3IP1 associates with multiple PI3K p110 isoforms just prior to formation of the OFZ, providing a likely endogenous mechanism for blocking all PI3K signaling and activating the autophagy pathway required to form the OFZ during lens development.