High resolution imaging and interpretation of three-dimensional RPE sheet structure

bioRxiv [Preprint]. 2024 Dec 4:2024.12.04.626881. doi: 10.1101/2024.12.04.626881.

Abstract

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells which plays an essential role in visual function via its interaction with the adjacent neural retina. Typically hexagonal in shape and arranged in a mosaic-like pattern, RPE cells maintain a relatively uniform size and arrangement in healthy eyes. Under stress or disease conditions such as age-related macular degeneration (AMD) and other heritable vision disorders, individual RPE cell dysmorphia has been observed. This has led to investigation of potential cellular compensatory mechanisms which may be dysregulated, affecting proper barrier structure and function. A commonly observed dysmorphic trait is that of enlarged cells which appear to be multinucleated (containing more than two nuclei) when viewed in two-dimensional (2D), immunohistochemically labeled images from the apical surface perspective. One explanation for the multinucleation is that of ongoing cellular fusion which the RPE may be employing to maintain cell-to-cell contact while simultaneously conserving cellular resources in unhealthy tissue. While this may be the most likely interpretation, caution should be applied when interpreting traditional (2D) images which only use cell border outline markers in the absence of lateral markers. Here we present two examples of high-resolution confocal images which allow for three-dimensional (3D) viewing of a traditional apical border delineation marker (ZO-1) and nuclei as well as labeling of alpha catenin which can serve as a lateral cell membrane marker. We find multiple examples in two separate RPE damage models where enlarged, seemingly multinucleate, cells are in actuality not multinucleate and instead appear this way due to surrounding cell nuclei and lateral cell membrane displacement towards the central cell. When viewed from the apical surface, these nuclei appear contained by the ZO-1 border, however when viewed from multiple angles it becomes apparent that this is not the case. This approach calls for more careful analyses in future studies investigating RPE sheet dysmorphia as this could lead to potential misinterpretation of the multinucleation phenomenon and by extension, the potential underlying fusion mechanism.

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  • Preprint