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.
|Commitee:||Adam, Alejandro, Barroso, Margarida, Mazurkiewicz, Joseph|
|School:||Albany Medical College|
|Department:||Molecular and Cellular Physiology (MCP)|
|School Location:||United States -- New York|
|Source:||MAI 58/03M(E), Masters Abstracts International|
|Subjects:||Morphology, Cellular biology, Physiology|
|Keywords:||Adherens junction, Fluorescence microscopy, VE-cadherin, mEos2|
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