Lovelace Michael D, Powter Elizabeth E, Coleman Paul R, Zhao Yang, Parker Amelia, Chang Garry H, Lay Angelina J, Hunter Julie, McGrath Aaron P, Jormakka Mika, Bertolino Patrick, McCaughan Geoffrey, Kavallaris Maria, Vadas Mathew A, Gamble Jennifer R
Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia.
Tumour Biology and Targeting Program, Children's Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW 2052, Australia.
Mol Biol Cell. 2017 Apr 15;28(8):1066-1078. doi: 10.1091/mbc.E16-05-0285. Epub 2017 Mar 1.
RhoGTPases are important regulators of the cell cytoskeleton, controlling cell shape, migration and proliferation. Previously we showed that ARHGAP18 in endothelial cells is important in cell junctions. Here we show, using structured illumination microscopy (SIM), ground-state depletion (GSD), and total internal reflection fluorescence microscopy (TIRF) that a proportion of ARHGAP18 localizes to microtubules in endothelial cells, as well as in nonendothelial cells, an association confirmed biochemically. In endothelial cells, some ARHGAP18 puncta also colocalized to Weibel-Palade bodies on the microtubules. Depletion of ARHGAP18 by small interfering RNA or analysis of endothelial cells isolated from ARHGAP18-knockout mice showed microtubule destabilization, as evidenced by altered morphology and decreased acetylated α-tubulin and glu-tubulin. The destabilization was rescued by inhibition of ROCK and histone deacetylase 6 but not by a GAP-mutant form of ARHGAP18. Depletion of ARHGAP18 resulted in a failure to secrete endothelin-1 and a reduction in neutrophil transmigration, both known to be microtubule dependent. Thrombin, a critical regulator of the Rho-mediated barrier function of endothelial cells through microtubule destabilization, enhanced the plasma membrane-bound fraction of ARHGAP18. Thus, in endothelial cells, ARHGAP18 may act as a significant regulator of vascular homeostasis.
RhoGTPases是细胞骨架的重要调节因子，控制细胞形状、迁移和增殖。此前我们发现，内皮细胞中的ARHGAP18在细胞连接中起重要作用。在此我们利用结构光照显微镜（SIM）、基态耗尽（GSD）和全内反射荧光显微镜（TIRF）表明，一部分ARHGAP18定位于内皮细胞以及非内皮细胞的微管上，这种关联已通过生化方法得到证实。在内皮细胞中，一些ARHGAP18斑点也与微管上的Weibel-Palade小体共定位。通过小干扰RNA耗尽ARHGAP18或分析从ARHGAP18基因敲除小鼠分离的内皮细胞，显示微管不稳定，形态改变以及乙酰化α-微管蛋白和谷氨酰胺-微管蛋白减少证明了这一点。通过抑制ROCK和组蛋白脱乙酰酶6可挽救这种不稳定，但ARHGAP18的GAP突变形式则不能。ARHGAP18的耗尽导致内皮素-1分泌失败和中性粒细胞迁移减少，这两者都已知依赖于微管。凝血酶是通过微管不稳定调节Rho介导的内皮细胞屏障功能的关键调节因子，它增强了ARHGAP18的质膜结合部分。因此，在内皮细胞中，ARHGAP18可能是血管稳态的重要调节因子。
