Speaker
Description
Epithelial polarity is essential for retinal pigment epithelium (RPE) function, yet how polarity signalling regulates membrane trafficking remains poorly understood. We previously identified the apical polarity determinant Dbl3, a Cdc42-specific GEF, and its effector kinase MRCKβ as key regulators of apical actomyosin contractility. Here, we investigate whether this signalling axis coordinates cytoskeletal dynamics with apical membrane trafficking. Using loss-of-function approaches in polarised MDCK and ARPE-19 cells, we combined quantitative fluid-phase uptake assays, antibody-based trafficking reporters, and confocal z-stack imaging to analyse apical endocytosis and recycling. Depletion or chemical inhibition of MRCKβ significantly reduced apical fluid-phase uptake. Using the FcLR 5-22 apical recycling reporter cell line, we showed that disruption of Dbl3-MRCKβ signalling impairs clathrin-mediated endocytosis and perturbs apical recycling, resulting in altered receptor distribution and increased endolysosomal accumulation. Colocalisation analysis further indicates receptor mis-sorting along the apical endosomal axis upon pathway downregulation. Our findings demonstrate that the Dbl3-MRCKβ pathway integrates polarity signalling with actomyosin contractility to regulate apical endocytic trafficking. This work expands the role of polarity-dependent cytoskeletal regulation beyond epithelial polarisation, identifying a mechanistic link between apical polarity and membrane trafficking dynamics. These results provide insights into how spatial signalling networks coordinate membrane organisation and intracellular transport in polarised epithelial cells.
Lay Abstract
Cells lining our organs have a distinct "top" and "bottom", a property called cell polarity that is essential for normal function. In the eye, the retinal pigment epithelium (RPE) relies on this organisation to perform critical tasks including clearing debris shed daily by photoreceptors, a process called phagocytosis. Our previous research showed that a molecular signalling pathway involving two proteins, Dbl3 and MRCKβ are essential for this, and that disrupting it impairs phagocytosis in the living eye, while restoring the pathway rescues it. Here we show this pathway does even more than we thought. Using retinal and kidney epithelial cell models, we disrupted Dbl3–MRCKβ signalling and tracked what happened to proteins being taken up and recycled at the apical cell surface. Without this pathway, cells internalised less material, recycling was disrupted, and proteins that should return to the cell surface instead became trapped inside and digested. This reveals that Dbl3–MRCKβ not only controls phagocytosis but actively regulates the broader movement of proteins at the apical surface. Given the RPE's central role in sustaining vision, these findings have implications for understanding how polarity breakdown contributes to retinal degenerative disease.
| Lay Title | When the eye's support cells lose their sense of direction, essential protein trafficking and vision itself may be at risk |
|---|---|
| Role | PhD Student |