Temporal and spatial modulation of Rho GTPases during in vitro formation of capillary vascular network - Adherens junctions and myosin light chain as targets of Rac1 and RhoA
Temporal and spatial modulation of Rho GTPases during in vitro formation of capillary vascular network - Adherens junctions and myosin light chain as targets of Rac1 and RhoA
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DOI：
10.1074/jbc.m307234200
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发表时间：
2003-12-12
影响因子：
4.8
通讯作者：
Bussolino, F
中科院分区：
文献类型：
--
作者：
Cascone, I;Giraudo, E;Bussolino, F
内皮细胞(ECs)在细胞外基质上自组织形成毛细血管网络。在这个过程中，Rho GTP酶介导的细胞骨架动力学控制着细胞到基质和细胞到细胞接触的运动和组织。RhoA和rac1激活的时间进程分析符合新生模式的特定形态特征。RhoA-GTP在EC黏附过程中早期增加，并在膜皱缩部位蓄积。Rac1后来被激活，并定位于片状脂膜和有组织的细胞链的细胞对细胞接触处。当内皮细胞拉伸和重塑形成毛细血管结构时，RhoA-GTP再次增加，并与沿主细胞轴运动的应力纤维联系在一起。N17rac1和N19RhoA突变体破坏了模式的形成。细胞间接触和肌球蛋白轻链(MLC)分别是rac1和RhoA的靶标。N17rac1减少β-连环蛋白和血管内皮细胞钙粘连蛋白向Triton X-100不溶部分的移动，并破坏粘连连接处的β-连环蛋白分布，提示Rac1控制钙粘连蛋白-连环蛋白与F-肌动蛋白复合体的动力学。在网络形成的重塑阶段，内皮细胞在质膜上显示磷酸化的MLC的强烈染色；相反，MLC的磷酸化程度较低，并在N19RhoA内皮细胞中广泛弥散分布。N17rac1和N19RhoA已被用于研究野生型分子在体外血管生成的主要步骤中的作用：(I)细胞与底物的粘连，(Ii)细胞的运动，(Iii)基质的机械重塑。N17rac1对内皮细胞的亲和力有显著的抑制作用，而N19RhoA对EC的黏附和运动有轻微的抑制作用，但对Matrigel收缩的抑制作用更明显。我们得出结论，不同的Rho GTP酶控制着血管形态发生的不同方面。
Endothelial cells (ECs) self-organize into capillary networks when plated on extracellular matrix. In this process, Rho GTPases-mediated cytoskeletal dynamics control cell movement and organization of cell-to-matrix and cell-to-cell contacts. Time course analysis of RhoA and Rac1 activation matches specific morphological aspects of nascent pattern. RhoA-GTP increases early during EC adhesion and accumulates at sites of membrane ruffling. Rac1 is activated later and localizes in lamellipodia and at cell-to-cell contacts of organized cell chains. When ECs stretch and remodel to form capillary structures, RhoA-GTP increases again and associates with stress fibers running along the major cell axis. N17Rac1 and N19RhoA mutants impair pattern formation. Cell-to-cell contacts and myosin light chains (MLC) are targets of Rac1 and RhoA, respectively. N17Rac1 reduces the shift of beta-catenin and vascular endothelial cadherin to Triton X-100-insoluble fraction and impairs beta-catenin distribution at adherens junctions, suggesting that Rac1 controls the dynamics of cadherin-catenin complex with F-actin. During the remodeling phase of network formation, ECs show an intense staining for phosphorylated MLC along the plasma membrane; in contrast, MLC is less phosphorylated and widely diffused in N19RhoA ECs. Both N17Rac1 and N19RhoA have been used to investigate the role of wild type molecules in the main steps characterizing in vitro angiogenesis: (i) cell adhesion to the substrate, (ii) cell movement, and (iii) mechanical remodeling of matrix. N17Rac1 has a striking inhibitory effect on haptotaxis, whereas N19RhoA slightly inhibits EC adhesion and motility but more markedly Matrigel contraction. We conclude that different Rho GTPases control distinct morphogenetic aspects of vascular morphogenesis.