Abstract
Purpose :
We have shown that autophagy plays a crucial function in the formation of the Organelle Free Zone (OFZ), the developmental timing of organelle loss linked to inhibition of JNK signaling.We now investigated whether PI3K signaling pathways, whose inhibition can induce autophagy and apoptosis, regulate the removal of organelles and nuclei from the developing lens.
Methods :
Chick embryo lenses placed in organ culture at E12, prior to OFZ formation, were exposed for 24hrs to the pan-PI3K inhibitor (LY294002), or inhibitors of the two predominant PI3K downstream effectors AKT (MK-2206) and Rac (EHT), with DMSO as control. Distinct regions of differentiation were microdissected for immunoblot analysis or cryosectioned and immunolabeled for confocal imaging to determine the impact on lens organelles and nuclei.
Results :
Both the pan-PI3K inhibitor and the PI3K/Akt-specific inhibitor induced premature loss of lens organelles, mitochondria and endoplasmic reticulum, within 24hrs. While blocking all PI3K signaling pathways also induced premature elimination of nuclei, the inhibition of the PI3K/Akt signaling axis was not alone sufficient to cause loss of nuclei from the center of the lens. In contrast, when Rac, the other primary PI3K effector pathway, is inhibited, nuclei across the fiber cell zone become condensed; however, inhibiting this pathway also was alone insufficient to completely eliminate nuclei within 24hrs. Our studies show that the normal process of nuclear elimination involves a series of changes in nuclear morphology from highly elongated ellipses to a shortened misshapen elliptical form, to a condensed rounded shape prior to fragmentation, accompanied by changes in organization of lamin B and chromatin. We find that the premature elimination of nuclei following exposure to the pan-PI3K inhibitor replicates this same process.
Conclusions :
Autophagy-dependent elimination of lens organelles to form the OFZ is induced by suppression of the PI3K/Akt signaling axis, while nuclear condensation, an essential intermediate in nuclear removal, is induced when Rac, also a downstream effector of PI3K, is inhibited. Although inhibition of either PI3K effector pathway is not alone sufficient for nuclear elimination, blocking all PI3K signaling results in premature loss of both nuclei and organelles, suggesting that nuclear loss involves suppression of multiple PI3K-dependent signaling pathways.
This is a 2020 ARVO Annual Meeting abstract.