Fadjukov, J. | Search bioRxiv

bioRxiv2023

CDK5 activity in retinal pigment epithelium contributes to gap junction dynamics during phagocytosis

Retinal pigment epithelium (RPE) at the back of the eye is a monolayer of cells with an extensive network of gap junctions that contributes to retinal health in a multitude of ways. One of those roles is the phagocytosis of photoreceptor outer segments. This renewal is under circadian regulation and peaks after light onset. Connexin 43 (Cx43) is the most predominantly expressed gap junction protein in RPE. In this study, we examine how gap junctions and specifically, Cx43 phosphorylation, contribute to phagocytosis in both human embryonic stem cell derived RPE and mouse RPE monolayers. We show that both Rac1 and CDK5 have differences in protein localization at different points in phagocytosis, and that by using their effectors, the capability of RPE for phagocytosis changes. CDK5 has not yet been reported in RPE tissue, and here we show that it likely regulates Cx43 localization and resulting electrical coupling. We find that gap junctions in RPE are temporally highly dynamic during phagocytosis and that regulation of gap junctions via phosphorylation is likely critical for maintaining eye health.

cell biology↗

bioRxiv2022

Light-induced nanoscale deformation in azobenzene thin film triggers rapid intracellular Ca2+ increase via mechanosensitive cation channels

Epithelial cells are in continuous dynamic biochemical and physical interaction with their extracellular environment. Ultimately, this interplay guides fundamental physiological processes. In these interactions, cells generate fast local and global transients of Ca2+ ions, which act as key intracellular messengers. However, the mechanical triggers initiating these responses have remained unclear. Light-responsive materials offer intriguing possibilities to dynamically modify the physical niche of the cells. Here, we use a light-sensitive azobenzene-based glassy material that can be micropatterned with visible light to undergo spatiotemporally controlled deformations. The material allows mechanical stimulation of single cells or multicellular assemblies, offering unique opportunities for experimental mechanobiology. Real-time monitoring of consequential rapid intracellular Ca2+ signals reveal that Piezo1 is the key mechanosensitive ion channel generating the Ca2+ transients after nanoscale mechanical deformation of the cell culture substrate. Furthermore, our studies indicate that Piezo1 preferably responds to lateral material movement at cell-material interphase rather than to absolute topographical change of the substrate. Finally, experimentally verified computational modeling of the signaling kinetics suggests that the lateral mechanical stimulus triggers multiplexed intercellular signaling that involves Na+, highlighting the complexity of mechanical signaling in multicellular systems. These results give mechanistic understanding on how cells respond to material dynamics and deformations.

bioengineering↗