A possible role of βA3/A1-crystallin in clathrin-mediated endocytosis and maintenance of microvilli of RPE cells

Abstract

Purpose : We have previously shown that Cryba1 cKO mice, lacking βA3/A1-crystallin specifically in the RPE, present with an age-related macular degeneration (AMD)-like phenotype. This study was undertaken to provide evidence that loss of this protein may trigger abnormal endocytosis and affect the microvillus assembly in RPE cells.

Methods : Cryba1 cKO mice and age-matched Cryba1^fl/fl control mice were used in this study. Western blotting and immunostaining techniques were used to detect the expression of proteins involved in the endocytic pathway and the apical domain. RPE flat-mount culture and total internal reflection fluorescence (TIRF) microscopy were employed for time-lapse live cell imaging to measure the endocytosis activity at the apical side of RPE cells in situ from Cryba1 cKO and Cryba1^fl/fl control mice. A clathrin adenovirus construct (Ad-CMV-FAP-mClta), an Ad-CMV-mCherry-mFNBP1 and a rAV^CMV-LifeAct-TagGFP2 were used to visualize clathrin coated vesicles (CCVs), FNBP1 and actin, respectively, for endocytosis imaging studies.

Results : Immunostaining of F-actin and EBP50 on retina sections and RPE flatmounts showed disorganized microvilli in RPE cells of 1 month old Cryba1 cKO mice. Complete loss of microvilli of some RPE cells was observed in 9 month old cKO mice. Key proteins in the endocytosis pathway were altered in cKO RPE cells compared to control RPE cells. Rab5a expression in cKO RPE cells was reduced at 3 months of age and older. Other endosomal proteins like Appl1 and EEA1 were found to have decreased expression in 9 month old cKO RPE cells. Time-lapse live cell imaging of RPE cells in situ showed insertion failure of CCVs from the apical side to the cytoplasm after EGF stimulation in Cryba1 cKO RPE cells. Inhibited CCV fission was also observed in cKO RPE cells. Phosphorylation levels of Ezrin/Radixin/Moesin and total Rac1, a small GTPase, were substantially reduced in cKO RPE cells even at 3 weeks old, which may be the main reason for F-actin microfilament disassembly and inhibited EGFR endocytosis.

Conclusions : It can be concluded that βA3/A1-crystallin plays a multifaceted role in RPE cells, including involvement in clathrin-dependent endocytosis of EGFR and in maintaining the apical integrity of RPE cells. These findings also indicate that abnormal endocytosis and loss of the apical domain may be a major cause of AMD.

This is a 2020 ARVO Annual Meeting abstract.