α-adducin accumulation along cell junctions paralleled AJ reorganization.

<p>Integrity and structural alterations in endothelial cell monolayers, subjected to a Ca²⁺-switch assay, were further evaluated either by TER measurements or by immunofuorescence analysis. (A-B) Time course of mean TER values showed that Ca²⁺-depletion (indicated by first arrow) induced a significant drop in TER which was largely recovered by Ca²⁺-repletion (second arrow) in HDMEC (A) and MyEnd (B) cells. *p ≤ 0.05 difference between control cells and cells depleted of Ca²⁺. Immunofluorescence analysis in HDMEC (C-E) and MyEnd cells (F-H) was performed. After subsequent fixation and permeabilization, cell monolayers were stained for VE-cadherin, α-adducin and α-adducin (pSer481). Additionally, Alexa Fluor 488 phalloidin was used for staining of F-actin. To identify the nuclei, cells were counterstained for DAPI (blue). (C and F) Immunofluorescence in HDMEC (C) and MyEnd cells (F) revealed that under control condition, in parallel to VE-cadherin, α-adducin and α-adducin (pSer481) are markedly localized along cell-cell borders (arrows). (D and J) In both cell lines Ca²⁺-depletion led to irregular (arrows) or absent VE-cadherin staining, associated with reduced localization or complete absence of α-adducin and α-adducin (pSer481) at cell-cell-junctions. This was accompanied with reduced staining of F-actin and induced intercellular gap formation (arrowhead). (E and H) In contrast, repletion of Ca²⁺ restored linear VE-cadherin staining as well as accumulation of α-adducin and α-adducin (pSer481) at cell-cell borders (stars). The staining of F-actin was increased as well. For all conditions, no morphological changes in the nuclei were observed. Images are representative of three or more independent experiments. Scale bar = 20 μm.