Ivanov Andrei I, McCall Ingrid C, Parkos Charles A, Nusrat Asma
Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, USA.
Mol Biol Cell. 2004 Jun;15(6):2639-51. doi: 10.1091/mbc.e04-02-0163. Epub 2004 Mar 26.
Disassembly of the epithelial apical junctional complex (AJC), composed of the tight junction (TJ) and adherens junction (AJ), is important for normal tissue remodeling and pathogen-induced disruption of epithelial barriers. Using a calcium depletion model in T84 epithelial cells, we previously found that disassembly of the AJC results in endocytosis of AJ/TJ proteins. In the present study, we investigated the role of the actin cytoskeleton in disassembly and internalization of the AJC. Calcium depletion induced reorganization of apical F-actin into contractile rings. Internalized AJ/TJ proteins colocalized with these rings. Both depolymerization and stabilization of F-actin inhibited ring formation and disassembly of the AJC, suggesting a role for actin filament turnover. Actin reorganization was accompanied by activation (dephosphorylation) of cofilin-1 and its translocation to the F-actin rings. In addition, Arp3 and cortactin colocalized with these rings. F-actin reorganization and disassembly of the AJC were blocked by blebbistatin, an inhibitor of nonmuscle myosin II. Myosin IIA was expressed in T84 cells and colocalized with F-actin rings. We conclude that disassembly of the AJC in calcium-depleted cells is driven by reorganization of apical F-actin. Mechanisms of such reorganization involve cofilin-1-dependent depolymerization and Arp2/3-assisted repolymerization of actin filaments as well as myosin IIA-mediated contraction.
由紧密连接（TJ）和黏着连接（AJ）组成的上皮顶端连接复合体（AJC）的解体，对于正常组织重塑和病原体诱导的上皮屏障破坏至关重要。我们之前利用T84上皮细胞中的钙耗竭模型发现，AJC的解体导致AJ/TJ蛋白的内吞作用。在本研究中，我们研究了肌动蛋白细胞骨架在AJC解体和内化中的作用。钙耗竭诱导顶端F-肌动蛋白重组成收缩环。内化的AJ/TJ蛋白与这些环共定位。F-肌动蛋白的解聚和稳定均抑制了环的形成和AJC的解体，提示肌动蛋白丝周转的作用。肌动蛋白重组伴随着丝切蛋白-1的激活（去磷酸化）及其向F-肌动蛋白环的转位。此外，肌动蛋白相关蛋白3（Arp3）和皮层肌动蛋白与这些环共定位。F-肌动蛋白重组和AJC的解体被非肌肉肌球蛋白II的抑制剂blebbistatin阻断。肌球蛋白IIA在T84细胞中表达并与F-肌动蛋白环共定位。我们得出结论，钙耗竭细胞中AJC的解体是由顶端F-肌动蛋白的重组驱动的。这种重组的机制涉及丝切蛋白-1依赖性的肌动蛋白丝解聚和Arp2/3辅助的肌动蛋白丝再聚合以及肌球蛋白IIA介导的收缩。
