Fredriksson-Lidman Karin, Van Itallie Christina M, Tietgens Amber J, Anderson James M
Laboratory of Tight Junction Structure and Function, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS One. 2017 Sep 29;12(9):e0185448. doi: 10.1371/journal.pone.0185448. eCollection 2017.
SORBS2 is a scaffolding protein associated with Abl/Arg non-receptor tyrosine kinase pathways and is known to interact with actin and several other cytoskeletal proteins in various cell types. Previous BioID proximity labeling of tight and adherens junction proteins suggested that SORBS2 is a component of the apical junction complex of epithelial cells. We asked whether SORBS2 plays a previously unappreciated role in controlling perijunctional actin and tight junction barrier function. Using super resolution imaging we confirmed that SORBS2 is localized at the apical junction complex but farther from the membrane than ZO-1 and located partially overlapping both the tight- and adherens junctions with a periodic concentration that alternates with myosin IIB in polarized epithelial cells. Overexpression of GFP-SORBS2 recruited alpha-actinin, vinculin and N-WASP, and possibly CIP4 to cellular junctions. However, CRISPR-Cas9 knock-out of SORBS2 did not alter the localization- or immunofluorescent staining intensity of these or several other junctional- and cytoskeletal proteins. SORBS2 knock-out also did not affect the barrier function as measured by TER and dextran flux; nor did it change actin-dependent junction re-assembly as measured by Ca2+-switch and Latrunculin-B wash-out assays. The kinetics of HGF-induced cell scattering and wound healing, and dextran flux increase induced by PDGF also were unaffected by SORBS2 knock-out. SORBS2 concentrates with apical junctional actin that accumulates in response to knock-down of ZO-1 and ZO-2. In spite of our finding that SORBS2 is clearly a component of the apical junction complex, it does not appear to be required for either normal tight- or adherens junction assembly, structure or function or for growth factor-mediated changes in tight junction dynamics.
SORBS2是一种与Abl/Arg非受体酪氨酸激酶途径相关的支架蛋白，已知在多种细胞类型中可与肌动蛋白和其他几种细胞骨架蛋白相互作用。先前对紧密连接和黏附连接蛋白的生物素邻近标记表明，SORBS2是上皮细胞顶端连接复合体的一个组成部分。我们研究了SORBS2在控制连接周围肌动蛋白和紧密连接屏障功能方面是否发挥了以前未被认识到的作用。通过超分辨率成像，我们证实SORBS2定位于顶端连接复合体，但比ZO-1离膜更远，并且在极化上皮细胞中部分重叠于紧密连接和黏附连接，其周期性聚集与肌球蛋白IIB交替出现。GFP-SORBS2的过表达将α-辅肌动蛋白、纽蛋白和N-WASP以及可能的CIP4募集到细胞连接部位。然而，SORBS2的CRISPR-Cas9敲除并未改变这些或其他几种连接和细胞骨架蛋白的定位或免疫荧光染色强度。SORBS2敲除也不影响通过跨上皮电阻（TER）和葡聚糖通量测量的屏障功能；通过钙离子切换和Latrunculin-B洗脱试验测量，它也没有改变肌动蛋白依赖性连接的重新组装。SORBS2敲除也不影响HGF诱导的细胞散射和伤口愈合的动力学，以及PDGF诱导的葡聚糖通量增加。SORBS2与顶端连接肌动蛋白聚集在一起，这种肌动蛋白在ZO-1和ZO-2敲低时会积累。尽管我们发现SORBS2显然是顶端连接复合体的一个组成部分，但它似乎对于正常的紧密连接或黏附连接的组装、结构或功能，或生长因子介导的紧密连接动力学变化都不是必需的。
