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

摘要 在这份修订后的协作续签提案中，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的心尖-基底极化以及囊泡 转运到顶膜表面以控制人内皮细胞(EC)的小管生成。使用状态- 体外和体内的抗逆转录病毒治疗方法，我们已经证明了cdc42在这一过程中的基本作用。 在人内皮细胞和小鼠血管发育和血管生长的过程中。停用 CDC42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管腔和管路网络。此外，RhoA在体内灭活与在体外灭活的作用相反 Cdc42，管腔形成增多。重要的是，我们已经证明了Rasip1及其结合 合作伙伴Arhgap29共同发挥作用，抑制RhoA信号，允许EC管形成。 为了进一步研究这一过程，我们开发了一种高度明确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、空泡的沿 微管蛋白细胞骨架朝向根尖表面，或在根尖下区域的空泡融合，以创建根尖 薄膜。我们可以进行这些研究，因为我们有能力调节KEY的表达或活性 具有显示细胞内空泡、极化细胞骨架和根尖细胞的能力的分子 表面(在静态或实时视频图像中)，使用EC顶端标签，如GFP-CAVeolin1。加在一起，这些 结果为阐明内皮细胞如何改变形状、极化、重定向连接以及 将膜移动到根尖表面；所有这些都是为了形成输送血液的功能管， 血管形成、组织活力和组织发育所必需的能力。 在这里，我们测试了这样的假设，即EC管腔的形成依赖于GTPase信号级联 促进细胞内膜向根尖表面和极化亚细胞的转运 招募关键效应者。我们提出了三个具体目标来进一步研究这些新颖的见解 研究体内和体外EC小管发生的基本过程，它们是： 目的#1.阐明抑制RhoA的潜在分子和机制。 内皮细胞的顶膜； 目的#2.研究Rab GTP酶对空泡/囊泡的形成、运输和融合的控制，从而导致 极化EC根尖膜组装和管腔形成； 目的#3.研究关键的上游鸟嘌呤交换因子(GEF)与GTP酶的特定作用 在EC腔形成过程中调节CDc42、Rac、k-Ras和Rap1b的激活蛋白(GAP)。

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.