Sjö A, Magnusson K E, Peterson K H
Division of Medical Microbiology, Department of Molecular and Clinical Medicine, Faculty of Health Sciences, Linköping University, Linköping, 581 85, Sweden.
J Membr Biol. 2005 Jan;203(1):21-30. doi: 10.1007/s00232-004-0728-1.
Alpha-dystrobrevin (alpha-DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex in skeletal muscle cells. Isoforms of alpha-DB show different localization in cells and tissues; at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, alpha-DBs are assumed to be important in cell signalling and cell differentiation. We have primarily assessed the role of alpha-DB in two epithelial cell lines (MDCK I, HT 29), which represent different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-alpha-DB antibody, we have investigated its expression, localization and association with tight junction (TJ)- associated proteins (ZO-1, occludin) before and after protein kinase C (PKC) activation with phorbol myristate acetate. Distinct subsets of alpha-DB isoforms were detected in the two cell lines by immunoblotting. In both cell lines there was submembranous localization of alpha-DB both apically and basolaterally, shown with confocal imaging. PKC activation caused a reorganization of TJ, which was parallel to increased localization of alpha-DB to TJ areas, most pronounced in MDCK I cells. Moreover, actin and ZO-1 co-immunoprecipitated with a-DB, as displayed with immunoblotting. Our findings suggest that a-dystrobrevin specifically is associated with the tight junctions during their reorganization.
α-肌营养不良蛋白（α-DB）主要被描述为骨骼肌细胞中肌营养不良蛋白-糖蛋白复合物的一种细胞质成分。α-DB的同工型在细胞和组织中表现出不同的定位；在上皮细胞的基底外侧膜上，肌营养不良蛋白介导与细胞外基质、外周和跨膜蛋白以及丝状肌动蛋白细胞骨架的接触。除了其结构作用外，α-DB还被认为在细胞信号传导和细胞分化中起重要作用。我们主要评估了α-DB在两种上皮细胞系（MDCK I、HT 29）中的作用，这两种细胞系代表不同的发育阶段并表现出不同的通透性特征。使用多克隆抗α-DB抗体，我们研究了在用佛波酯肉豆蔻酸酯激活蛋白激酶C（PKC）之前和之后，其表达、定位以及与紧密连接（TJ）相关蛋白（ZO-1、闭合蛋白）的关联。通过免疫印迹在两种细胞系中检测到α-DB同工型的不同亚群。通过共聚焦成像显示，在两种细胞系中，α-DB在顶端和基底外侧均有膜下定位。PKC激活导致TJ重组，这与α-DB在TJ区域的定位增加平行，在MDCK I细胞中最为明显。此外，如免疫印迹所示，肌动蛋白和ZO-1与α-DB共免疫沉淀。我们的研究结果表明，α-肌营养不良蛋白在紧密连接重组过程中与紧密连接特异性相关。
