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

Rho 和 Ras 家族 GTP 酶活性的分子调节控制血管腔的形成。

基本信息

批准号：
10545039
负责人：
Ondine B Cleaver
金额：
$ 39.19万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10545039
关键词：
3-Dimensional Acetylation Apical Binding Biological Assay Biological Models Blood Blood Vessels Cardiovascular Diseases Caveolins Cell Culture Techniques Cell Line Cell Polarity Collagen Complex Coupled Cytoskeleton Defect Development Diabetes Mellitus Disease Endothelial Cells Environment Epithelial Cells F-Actin Failure Family Funding GTPase-Activating Proteins Galium Goals Growth Guanine Guanine Nucleotide Exchange Factors Guanosine Triphosphate Phosphohydrolases Human Image In Vitro Individual Intracellular Transport Joints KRAS2 gene Label Laboratories Malignant Neoplasms Maps Membrane Microtubules Modeling Molecular Mus Play Process Regulation Role Shapes Signal Pathway Signal Transduction Signaling Molecule Standardization Surface Testing Time Tissue Viability Tissues Transmembrane Transport Tube Tubulin Vacuole Vesicle Visualization Work apical membrane blood vessel development candidate identification candidate selection human disease in vivo in vivo Model insight membrane assembly mouse model novel novel therapeutic intervention polarized cell rab GTP-Binding Proteins recruit rho rho GTP-Binding Proteins stem cells trafficking vesicle transport vesicle/vacuole

项目摘要

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.

概括在这项经过修订的协作续签建议中，我们调查了有关Rho的能力的新发现， RAS和RAB GTPase调节细胞骨架和膜顶基碱极化以及囊泡运输到根尖膜表面以控制人内皮细胞（EC）肾变生成。使用最新在体外和体内方法中，我们在此期间证明了CDC42的基本作用与人类EC的过程以及小鼠血管发育和血管生长的过程。失活 Cdc42功能导致体内和体外EC两极分化的破坏，从而导致未正确形成或组织EC管腔和管网络。此外，Rhoa在体内与体外的失活与相反 CDC42，其中腔形成增加。重要的是，我们已经表明RASIP1及其结合合作伙伴ARHGAP29一起起作用以抑制RhoA信号传导以允许发生EC管的形成。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作用。我们可以直接评估单个分子还是信号分别控制细胞内液泡的形成，细胞骨架极化，液态运输沿微管蛋白的细胞骨架朝着顶端表面或副圆形区域的液泡融合以形成顶端膜。由于我们有能力调节密钥的表达或活性，我们可以进行这些研究分子以及可视化细胞内液泡，极化细胞骨架和顶端的能力使用EC顶级标签（例如GFP-Caveolin1）表面（在静态或实时视频图像中）。在一起，这些结果提供了一个分子路线图，以阐明EC如何改变形状，极化，重新定位连接和将膜移至顶部表面；所有这些都是最终的目标，即形成带有血液的功能管血管形成，组织生存力和组织发育所必需的能力。在这里，我们检验了以下假设：EC管腔形成取决于GTPase信号级联促进膜在顶部表面和极化亚细胞的细胞内转运招募关键效应子。我们提出了三个特定的目标，以进一步研究这些新颖的见解进入体内和体外的EC微管发生的基本过程，它们是：目标＃1。阐明负责抑制RhoA的基本分子和机制 EC中的顶膜；目标＃2。研究RAB GTPase对液泡/囊泡形成，运输和融合的控制，导致极化EC顶膜组件和管腔形成；目标＃3。研究关键上游鸟嘌呤交换因子（GEF）与GTPase的特定作用在EC流明形成过程中调节CDC42，RAC，K-RAS和RAP1B的激活蛋白质（GAP）。