Purchase this article with an account.
Tyler Pfister, Aman George, Kapil Bharti, Brian Patrick Brooks; Role of atypical cadherin FAT1 in establishing earliest cell-cell contact and junctional integrity of retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1915. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We had reported on the role of the atypical cadherin FAT1 in a syndromic form of coloboma and microphthalmia. While the binding domains of FAT1 suggest evolutionary divergence from the classical cadherin family, localization at cell junctions of non-retinal pigmented epithelial (RPE) cell types in previous reports suggests that FAT1 can function similar to classical cadherins in forming and maintaining junctional integrity. The present study was carried out to investigate the role of FAT1 in establishing earliest cell-cell contacts and in maintaining junctional integrity of RPE cells.
RPE cell were cultured on trans-well and 4 well chamber slides. Immunofluorescence staining and super-resolution confocal microscopy were used to study the protein localization pattern in RPE cells. Knock-down of FAT1 in RPE cells was performed using shRNA lentiviral transduction particles. Changes in protein expression and ultrastructure were analyzed using Western blotting and transmission electron microscopy (TEM), respectively.
We observed FAT1 protein at earliest cell-cell junctions in sub-confluent RPE cultures, co-localized with F-ACTIN fibers, ZO1, β-CATENIN and N-CADHERIN. These earliest cell-cell junctions were disrupted upon shRNA mediated knock-down of FAT1. Upon culturing on trans-wells, FAT1 expression was observed at apical junctions in monolayer RPE basal to the ZO1 and F-ACTIN staining. ShRNA-mediated knockdown of FAT1 in RPE monolayers resulted in significant loss of pan-CADHERIN immunostaining and reduced transepithelial resistance. When cell-cell junctions were disrupted and re-established by depleting and re-introducing Ca+2 and Mg+2. FAT1 displayed a temporal staining pattern consistent with other cadherins. ShRNA knock-down of FAT1 48hr prior to depletion and re-introduction of Ca+2 and Mg+2 resulted in failure to re-establish the cell-cell junctions as confirmed by N-CADHERIN, ZO1 and β-CATENIN staining and TEM.
Our study provides evidence that FAT1 has a similar expression pattern as other cadherins, but its role in organizing junctional proteins at earliest cell-cell contacts diverges from the classical cadherin protein family. Also, FAT1 is required to maintain RPE monolayer junctional integrity, and in its absence, classical cadherins fail to re-organize junctions between RPE cells.
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