βA3/A1-crystallin regulates apical polarity and EGFR endocytosis in retinal pigmented epithelial cells.
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ABSTRACT:
SUBMITTER: Shang P
PROVIDER: S-EPMC8266859 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.
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Project description:We have previously reported that in the Nuc1 rat, which has a spontaneous mutation in Cryba1 (the gene encoding βA3/A1-crystallin), astrocytes exhibit decreased Notch signaling, leading to reduced promoter activity for glial fibrillary acidic protein (GFAP). Interestingly, in both Nuc1 astrocytes and in wild type astrocytes following knockdown of Cryba1, vascular endothelial growth factor (VEGF) secretion is decreased. This has led us to explore signaling mediators that could be regulated by βA3/A1-crystallin to modulate both GFAP and VEGF. Several studies have shown that the signal transducer and activator of transcription 3 (STAT3) is involved in the co-regulation of GFAP and VEGF. We show that STAT3 and βA3/A1-crystallin may co-regulate each other in astrocytes. Such co-regulation would create a positive feedback circuit; i.e., in the cytosol of astrocytes, βA3/A1-crystallin is necessary for the phosphorylation of STAT3, which then dimerizes and translocates to the nucleus to form DNA-binding complexes, activating transcription of Cryba1. This stoichiometric co-regulation of STAT3 and Cryba1 could potentiate expression of GFAP and secretion of VEGF, both of which are essential for maintaining astrocyte and blood vessel homeostasis in the retina. Consistent with this idea, Cryba1 knockout mice exhibit an abnormal astrocyte pattern and defective remodeling of retinal vessels.
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Project description:Phagocytosis of the shed outer segment discs of photoreceptors is a major function of the retinal pigmented epithelium (RPE). We demonstrate for the first time that ?A3/A1-crystallin, a major structural protein of the ocular lens, is expressed in RPE cells. Further, by utilizing the Nuc1 rat, in which the ?A3/A1-crystallin gene is mutated, we show that this protein is required by RPE cells for proper degradation of outer segment discs that have been internalized in phagosomes. We also demonstrate that in wild-type RPE, ?A3/A1-crystallin is localized to the lysosomes. However, in the Nuc1 RPE, ?A3/A1-crystallin fails to translocate to the lysosomes, perhaps because misfolding of the mutant protein masks sorting signals required for proper trafficking. The digestion of phagocytized outer segments requires a high level of lysosomal enzyme activity, and cathepsin D, the major enzyme responsible for proteolysis of the outer segments, is decreased in mutant RPE cells. Interestingly, our results also indicate a defect in the autophagy process in the Nuc1 RPE, which is probably also linked to impaired lysosomal function, because phagocytosis and autophagy might share common mechanisms in degradation of their targets. ?A3/A1-crystallin is a novel lysosomal protein in RPE, essential for degradation of phagocytosed material.
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Project description:PurposeThe RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that βA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells.MethodsRPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of βA3/A1-crystallin.ResultsβA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of βA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and vimentin, downregulation of E-cadherin, and increased cell migration. βA3/A1-crystallin binds to cortactin, and loss of βA3/A1-crystallin resulted in increased P-cortactinY421. The RPE from AMD samples had increased Snail and vimentin, and decreased E-cadherin, compared to age-matched controls.ConclusionsWe introduced a novel concept of dry AMD initiation induced by lysosomal clearance defects in the RPE and subsequent attempts by RPE cells to avoid the resulting stress by undergoing EMT. We demonstrate that βA3/A1-crystallin is a potential therapeutic target for AMD through rejuvenation of lysosomal dysfunction and potentially, reversal of EMT.
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Project description:Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process. Nuc1 is a spontaneous mutation in the Sprague-Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the betaA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate betaA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina.
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Project description:The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers.
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Project description:Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning-defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling.
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Project description:Apical microvilli are critical for the homeostasis of transporting epithelia, yet mechanisms that control the assembly and morphology of these protrusions remain poorly understood. Previous studies in intestinal epithelial cell lines suggested a role for the F-BAR domain protein PACSIN2 in normal microvillar assembly. Here we report the phenotype of PACSIN2 KO mice and provide evidence that through its role in promoting apical endocytosis, this molecule plays a role in controlling microvillar morphology. PACSIN2 KO enterocytes exhibit reduced numbers of microvilli and defects in the microvillar ultrastructure, with membranes lifting away from rootlets of core bundles. Dynamin2, a PACSIN2 binding partner, and other endocytic factors were also lost from their normal localization near microvillar rootlets. To determine whether loss of endocytic machinery could explain defects in microvillar morphology, we examined the impact of PACSIN2 KD and endocytosis inhibition on live intestinal epithelial cells. These assays revealed that when endocytic vesicle scission fails, tubules are pulled into the cytoplasm and this, in turn, leads to a membrane-lifting phenomenon reminiscent of that observed at PACSIN2 KO brush borders. These findings lead to a new model where inward forces generated by endocytic machinery on the plasma membrane control the membrane wrapping of cell surface protrusions.
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Project description:Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P(2) is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (?, ? or ?). PIP5KI? localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KI? whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P(2)-containing liposomes of the PtdIns(4,5)P(2) binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P(2) on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KI? have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P(2) mediated by PIP5KI? is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P(2) may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis.
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Project description:BACKGROUND:Retinitis pigmentosa (RP) is an inherited retinal disease characterized by progressive loss of photoreceptor cells. This study aim at exploring the effect of retinal pigment epithelium (RPE) derived from human-induced pluripotent stem cell (hiPSC-RPE) on the retina of retinal degeneration 10 (rd10) mice, which are characterized with progressive photoreceptor death. METHODS:We generated RPE from hiPSCs by sequential supplementation with retinal-inducing factors and RPE specification signaling factors. The three-dimensional (3D) spheroid culture method was used to obtain optimal injectable hiPSC-RPE cells. Subretinal space transplantation was conducted to deliver hiPSC-RPE cells into the retina of rd10 mice. Neurotrophic factor secretion from transplanted hiPSC-RPE cells was detected by enzyme-linked immunosorbent assay (ELISA). Immunostaining, Western blotting, electroretinography (ERG), and visual behavior testing were performed to determine the effects of hiPSC-RPE on the retinal visual function in rd10 mice. RESULTS:Our data demonstrated that hiPSC-RPE cells exhibited classic RPE properties and phenotype after the sequential RPE induction from hiPSCs. hiPSC-RPE cells co-cultured with mouse retinal explants or retinal ganglion cells 5 (RGC5) exhibited decreased apoptosis. The viability and functional properties of hiPSC-RPE cells were enhanced by 3D spheroid culture. Transplanted hiPSC-derived RPE cells were identified by immunostaining with human nuclear antigen staining in the retina of rd10 14?days after subretinal space injection. The pigment epithelium-derived factor level was increased significantly. The expression of CD68, microglial activation marker, reduced after transplantation. The light avoidance behavior and ERG visual function in rd10 mice improved by the transplantation of hiPSC-RPE cells. CONCLUSION:Our findings suggest that injectable hiPSC-RPE cells after 3D spheroid culture can rescue the structure and function of photoreceptors by sub-retinal transplantation, which lay the foundation for future clinical cell therapy to treat RP and other retinal degeneration diseases.
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