Purchase this article with an account.
Myoungsup Sim, April Nettesheim, Angela Dixon, Paloma Borrajo Liton; Nuclear LC3 triggers ribophagy upon cyclic mechanical stretch in human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5125.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We have previously reported the activation of autophagy in human trabecular meshwork (TM) primary cells in response to mechanical stress. Here, we investigate the role of stretch-induced autophagy in TM cell homeostasis.
Human TM cells were subjected to cyclic mechanical stretch (CMS, 8 or 20% elongation,1 cycle/sec) for up to 24 hours. Nuclear localization of LC3 was assessed in nuclear fraction by western blot, and via time-lapse live cell imaging and confocal microscopy using GFP-LC3, tandem fluorescent-tagged LC3 (tfLC3) or immunocytochemistry. Autophagosomal formation was evaluated by electron microscopy (EM) analysis. Blockage of nuclear export and import was achieved using leptomycin B (lepB), an inhibitor for CRM-1 (exportin-1) dependent nuclear export and wheat germ agglutinin, a nuclear import inhibitor. Localization of LC3 in subnuclear organelles and nuclear LC3-interacting proteins were examined by co-staining of various marker proteins including fibrillarin (nucleolus), promyelocytic leukemia (PML nuclear body) and co-immunoprecipitation analysis, respectively.
Western blot and confocal microscopy showed that CMS increased the steady-state protein levels of LC3-II in both cytosolic (1.68 ± 0.42 folds) and nuclear fraction (2.05 ± 0.83 folds) in TM cells compared to non-stretched controls (p<0.05). We also observed higher number of endogenous (CNT vs CMS; 11.00 ± 9.16 vs. 25.93 ± 15.81, p <0.001, n=59) and exogenous (CNT vs CMS; 23.15 ± 13.80 vs. 75.79 ± 32.93, p <0.001, n=27) LC3 aggregates per nucleus in the stretched cells. EM and immunocytochemical analyses revealed that the nuclear LC3 aggregates do not lead to autophagosome formation in the nucleus, but interact with the nucleolus where ribosomal biogenesis primarily takes place. Most interestingly, nuclear LC3 co-immunoprecipitated with NUFIP1, a ribosome receptor for ribophagy. The complex NUFIP1/LC3 was found to shuttle from the nucleus into the cytoplasm upon CMS
Our results strongly indicate that nuclear LC3 plays a key role in the surveillance of nucleolus function and triggers ribophagy to maintain TM cellular homeostasis in response to CMS. We further hypothesize that dysregulation of this response contributes to the loss of TM homeostasis in the glaucomatous outflow pathway.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only