Abstract
Purpose :
Previous studies suggest that occludin phosphorylation on Ser490 contributes to both VEGF-induced vascular permeability and angiogenesis in cell culture and in transgenic animals. Tight junction, transmembrane protein occludin localizes to centrosomes in a distinct vesicle compartment, required for VEGF-induced endothelial cell proliferation and neovascularization. Dynein motor is solely responsible for minus end trafficking of proteins and vesicles in cells. We hypothesized that dynein trafficks occludin to centrosomes and identified regions of occludin required to bind the dynein light intermedia chain 2 (LIC2).
Methods :
Co-immunoprecipitation (Co-IP) studies were performed in bovine retinal endothelial cells (BREC) and human osteosarcoma U2OS cells that have well-characterized centrosomes. A series of occludin mutants were generated to test centrosomal localization in U2OS cells by immunofluorescence (IF) confocal microscopy and VEGF-induced proliferation in BREC.
Results :
Accumulation of dynein and pS490 occludin on microtubules was observed in BREC treated with dynein inhibitor dynapyrazole A suggesting a requirement for dynein in trafficking. Co-IP and Western blotting from U2OS cells and BREC revealed that occludin and dynein interact. Mutational analysis of occludin revealed that the occludin coiled-coil domain and specifically the Ser490 phosphorylation site was required for centrosomal localization and VEGF-induced BREC proliferation. The occludin coiled-coil contained homology to the LIC2 binding adapter BICD, CC1 domain and additional mutational analysis and co-IP studies revealed an interaction site between the occludin coiled-coil and the dynein LIC2 with Ser471 contributing an essential role to binding while Ser490 was not required for this interaction but was required for centrosomal localization. Mutation of 7 amino acids in the occludin coiled-coil with homology to the CC1 domain completely blocked LIC2 binding.
Conclusions :
These results reveal a novel role for occludin interaction with dynein LIC2 and begin to provide an explanation for the role of occludin in both VEGF-induced vascular permeability and angiogenesis and suggest occludin may serve to link cargo to dynein.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.