Molecular regulation of Rho and Ras family GTPase activity controls vascular lumen formation.

总结 在这个修订后的合作更新提案中，我们调查了我们关于Rho能力的新发现， Ras和Rab GTP酶调节细胞骨架和膜顶-基底极化以及囊泡 运输至顶端膜表面以控制人内皮细胞（EC）小管生成。使用状态- 通过体外和体内方法的现有技术，我们已经证明了Cdc 42在这一过程中的基本作用。 在小鼠血管发育和血管生长过程中。失活 Cdc 42的功能导致体内和体外EC极化的破坏，导致不能正确形成 或组织EC管腔和管道网络。此外，体内RhoA失活与体外RhoA失活相反， cdc 42，其中管腔形成增加。重要的是，我们已经证明了Rasip 1及其结合 配偶体Arhgap 29共同起作用以抑制RhoA信号传导，从而允许EC管形成发生。 为了进一步研究这一过程，我们开发了一种高度定义的方法来阐明何时何地 特定的分子和信号通路起作用。我们可以直接评估单个分子或 信号分别控制细胞内空泡的形成、细胞骨架的极化、空泡沿 微管蛋白细胞骨架朝向顶端表面，或在亚顶端区域的液泡融合以产生顶端 膜的我们可以进行这些研究，因为我们有能力调节关键基因的表达或活性。 分子加上可视化细胞内空泡，极化细胞骨架和顶端的能力， 表面（静态或实时视频图像）使用EC顶端标签，如GFP-小窝蛋白1。所有这些 结果提供了一个分子路线图，以阐明EC如何改变形状，弯曲，重新定向连接， 将膜移到顶端表面;所有这些都是为了形成运送血液的功能性管道， 血管形成、组织活力和组织发育所必需的能力。 在这里，我们检验了EC管腔形成依赖于GT3信号级联的假设， 促进细胞膜向顶端表面和极化亚细胞的细胞内运输 招募关键效应器。我们提出了三个具体的目标，以进一步研究这些新的见解 在体内和体外EC小管形成的基本过程中，它们是： 目标1。为了阐明RhoA抑制的潜在分子和机制， 内皮细胞顶端膜; 目标2。为了研究Rab GT3对空泡/囊泡形成、运输和融合的控制， 极化EC顶端膜组装和管腔形成; 目标3。研究关键上游鸟嘌呤交换因子（GEF）与GT3的特定作用 激活蛋白（GAP）在EC管腔形成过程中调节Cdc 42、Rac、k-Ras和Rap 1b。

SUMMARY In this revised, collaborative renewal proposal, we investigate our novel findings regarding the ability of Rho, Ras and Rab GTPases to modulate cytoskeletal and membrane apical-basal polarization as well as vesicle trafficking to the apical membrane surface to control human endothelial cell (EC) tubulogenesis. Using state-of- the art in vitro and in vivo approaches, we have demonstrated a fundamental role for Cdc42 during this process with human ECs and during mouse vascular development and blood vessel growth. Inactivation of Cdc42 function leads to disruption of EC polarization in vivo and in vitro that results in a failure to properly form or organize EC lumen and tube networks. In addition, RhoA inactivation in vivo vs. in vitro does the opposite of Cdc42, where increased lumen formation occurs. Importantly, we have shown that Rasip1 and its binding partner, Arhgap29, function together to suppress RhoA signaling to allow EC tube formation to occur. To further investigate this process, we have developed a highly defined approach to elucidate when and where particular molecules and signaling pathways act. We can directly assess whether individual molecules or signals separately control intracellular vacuole formation, cytoskeletal polarization, vacuole trafficking along the tubulin cytoskeleton toward the apical surface, or vacuole fusion in the subapical region to create the apical membrane. We can perform these studies due to our ability to regulate the expression or activity of key molecules coupled with the ability to visualize intracellular vacuoles, the polarized cytoskeleton and the apical surface (in static or real-time video images) using EC apical labels such as GFP-caveolin1. Together, these results provide a molecular road map to elucidate how ECs change shape, polarize, reorient junctions, and move membranes to the apical surface; all with the ultimate goal of forming functional tubes that carry blood, a capacity essential for blood vessel formation, tissue viability, and tissue development. Here, we test the hypothesis that EC lumen formation depends on GTPase signaling cascades that promote the intracellular transport of membranes to the apical surface and polarized subcellular recruitment of critical effectors. We propose three specific aims to further investigate these novel insights into the fundamental process of EC tubulogenesis in vivo and in vitro and they are: Aim #1. To elucidate the underlying molecules and mechanisms responsible for suppression of RhoA at the apical membrane in ECs; Aim #2. To investigate Rab GTPase control of vacuole/vesicle formation, trafficking and fusion, leading to polarized EC apical membrane assembly and lumen formation; Aim #3. To investigate the specific roles of key upstream guanine exchange factors (GEFs) vs. GTPase activating proteins (GAPs) regulating Cdc42, Rac, k-Ras and Rap1b during EC lumen formation.