The binding of A3/A1-crystallin and PITP indicates its location in the Golgi and endoplasmic reticulum (ER). (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that A3/A1-crystallin binds to phosphatidylinositol transfer protein (PITP) and CZC24832 that A3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that A3/A1-crystallin acquired its RPE function before evolving CZC24832 as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITP/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity. cKO mice (conditional knockout of specifically in the RPE) exhibit an age-related macular LAG3 degeneration (AMD)-like phenotype23,24,26,27. We have also found that A3/A1-crystallin may play an important role in maintaining RPE polarity. This protein is enriched at the apical region of polarized RPE cells and is not expressed in non-polarized RPE cells, such as CZC24832 non-polarized cultured RPE cell lines27. Furthermore, RPE cells lacking mRNA by leaky ribosomal scanning28. The production of two polypeptides may contribute to the complexity of the intriguingly diverse functions of this moonlighting protein. By utilizing our well-characterized cKO mouse model and RPE/choroid/sclera flatmounts, we herein suggest another important function of A3/A1-crystallin: modulating the PI(4,5)P2 pool in RPE cells via the PITP/PLC signaling axis, with subsequent effects on cell polarity and EGFR signaling. Results cKO RPE show age-related microvilli defects We investigated the RPE microvilli on retinal sections and RPE flatmounts from cKO and age-matched floxed control mice by immunostaining for F-actin and EBP50. At 1 month of age, the apical microvilli of RPE cells in floxed mice are strong and well interdigitated with photoreceptor outer segments, while in cKO mice they are disorganized. This disorganization became more severe with age (Fig.?1a, b). By 4 months of age, microvilli in RPE cells from cKO mice had collapsed, progressing to near complete loss of microvilli in some RPE cells by 9 months. These abnormalities can be visualized by the orthogonal projection of z-stack confocal images on RPE flatmounts where the XY panels are taken from the same apical plane (Fig.?1b). The lower magnification images of retina cryosections and RPE flatmounts showed a patchy pattern of microvilli abnormalities in cKO RPE cells (Supplementary Fig.?1a, b). Ultrastructurally, the RPE exhibited disorganized interdigitation of microvilli with photoreceptor outer segments in 2-month-old cKO retinas, and complete loss of microvilli in some RPE cells by 20 months (Fig.?1c). It is worth noting that this control RPE cells also showed moderate age-related microvilli disorganization (Fig.?1aCc). Our data are in agreement with another group that showed progressive microvilli atrophy (shortening) in aging rats5. Open in a separate windows Fig. 1 cKO RPE show age-related microvilli defects.a Immunostaining for EBP50 (red) and CZC24832 F-actin (phalloidin, green) on retina sections showed disorganized microvilli in 1-month-old cKO RPE cells, and microvilli loss (arrows) in RPE cells of 9-month-old cKO mice compared to age-matched control. Mag: magnified area layed out in merged image. DAPI (blue). Scale bar: 20?m. Graph shows the average microvilli height in control and cKO retina sections measured by the length measurement tool in ZEN software based on the staining results. For each biological repeat, three representative values from different RPE locations (center, middle, peripheral).
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