The role of tubedown in ocular endothelial permeability

Tubedown (N aa15) is a protein that associates with the acetyltransferase Arrest defective protein 1 (Naal 0) and is involved in the regulation of vascular homeostasis of the retina. In endothelial cells Tubedown interacts with the actin-binding protein Cortactin. Cortactin is a known regulator of the permeability of molecules through cells (transcytosis) and between cells (paracytosis). Transcytosis is enhanced by the phosphorylation of Cortactin at tyrosine residues. Recent studies have shown that stable suppression of Tubedown expression leads to increased endothelial cell permeability. Increased permeability leading to pathological angiogenesis is observed within patients with wet age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), and retinopathy of prematurity (ROP). Previous studies have revealed that patients with wet AMD, PDR, and ROP, have decreased levels of Tubedown in correlation with areas of pathological angiogenesis. The present work aims to investigate Tubedown's mechanistic involvement in retinal permeability pathways, to further elucidate its role in ocular neovascular diseases. Immunoprecipitation and western blotting techniques were used to explore possible interactions between Tubedown and known regulators of permeability pathways: Dynamin, Afadin-6 (AF-6), Y421-Phospho-Cortactin, c-Src, and Zona Occludins-1 (ZO- 1 ). These results provided evidence that the mechanism by which permeability is regulated by Tubedown does not likely involve an interaction with Dynamin, AF -6, c- Src, or Z0-1. The specific interaction of Tubedown with non-phosphorylated Cortactin but not Y421-Phospho-Cortactin suggests that Tubedown may attenuate phosphorylation of Cortactin and subsequently decrease transcytosis. Furthermore, in vitro transient and stable knockdown techniques for Tubedown expression were combined with immunofluorescence microscopy to determine if reduced Tubedown levels affect the distribution of the paracytosis regulator Z0-1 in cultured rhesus macaque retinalchoroidal endothelial cells. While stable Tubedown knockdowns showed decreased Z0-1 cell perimeter expression, transient Tubedown knockdowns had no effect on Z0-1 cell perimeter distribution. Tubedowns effect on Z0-1 perimeter distribution remains inconclusive