Huen Arthur C, Park Jung K, Godsel Lisa M, Chen Xuejun, Bannon Leslie J, Amargo Evangeline V, Hudson Tracie Y, Mongiu Anne K, Leigh Irene M, Kelsell David P, Gumbiner Barry M, Green Kathleen J
Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
J Cell Biol. 2002 Dec 23;159(6):1005-17. doi: 10.1083/jcb.200206098.
By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF-membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF-membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.
通过将中间丝（IFs）拴系到细胞间粘附位点，桥粒促进了超细胞支架的形成，该支架赋予组织机械强度。然而，IF-膜附着在增强粘附中所起的作用尚未得到直接研究。为了解决这个问题，我们构建了可诱导表达缺乏杆状和IF结合结构域的桥粒斑蛋白（DP）突变体（DPNTP）的Tet-On A431细胞。DPNTP定位于质膜，导致IFs从连接斑解离，而不改变黏附连接或桥粒蛋白的总量或细胞表面分布。然而，桥粒芯糖蛋白的去污剂不溶性组分的特异性降低表明与IF细胞骨架的结合减少。表达DPNTP的细胞聚集体在悬浮液中或从底物释放的细胞片在受到机械应力时容易解离，而对照基本保持完整。解离发生时没有乳酸脱氢酶释放，表明组织完整性的丧失是由于粘附力降低而不是细胞溶解增加。来自一名患有DP羧基末端截短的患者的JD-1细胞与正常角质形成细胞相比，粘附力也更弱。当与DPNTP联合使用时，破坏肌动蛋白丝并破坏黏附连接的拉春库林A导致的解离比单独任何一种处理大一个数量级。这些数据提供了直接的体外证据，表明IF-膜附着调节粘附强度，并且进一步表明基于肌动蛋白和IF的连接协同作用以增强粘附。
