<p> Regulation of barrier function is critical for patients who suffer from inflammatory diseases such as acute respiratory distress syndrome (ARDS) and sepsis. A major regulator of endothelial barrier function is vascular endothelial cadherin (VE-cad). Cellular levels of VE-cad are known to be regulated by p120 catenin. Loss of p120 leads to decreased barrier function as a result of the endocytosis of VE-cad. However, recent work from our lab shows that expression of an endocytic defective VE-cad mutant was not able to rescue barrier function, as measured using transendothelial electrical resistance (TEER). In contrast, expression of a non-phosphorylatable VE-cad mutant was able to restore barrier function independent of p120 binding. These results suggest that endocytosis is not the only mechanism regulating VE-cad localization to the cell-cell junctions, but rather the phosphorylation state of the protein may play a more critical role to stabilizing VE-cad at the junction. In order to investigate junctional stability of VE-cad, we created a recombinant form of VE-cad by cloning mEos2 into a plasmid containing the VE-cad gene. This fluorophore is photoconvertible, thus allowing for tracking protein movement at the cell-cell junction. The VE-cad proteins, labeled with mEos2 at the C-terminus, were introduced via adenoviral infection into human umbilical vein endothelial cells (HUVEC). Initially, mEos2 fluoresces green, in order to induce photoconversion, a 405nm laser is directed in a specific region of interest (ROI) at the junction. A conformational change in the mEos2 protein will cause irreversible red fluorescence. Tracking the change in fluorescence intensity in the ROI will provide insight into the localization of VE-cad at endothelial cell junctions. We now have a model that can be used to test junctional localization and stability of endocytic defective and non-phosphorylatable mutants of VE-cad.</p><p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:13422975 |
| Date | 19 December 2018 |
| Creators | Harvey, Taylor R. |
| Publisher | Albany Medical College |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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