GTPases as molecular gatekeepers of cytoskeletal and cellular polarization during endothelial tubulogenesis

 描述（由申请人提供）：在这项新的合作提案中，我们研究了Rho GTP酶调节细胞骨架和膜顶部-基底极化以控制内皮细胞（EC）小管生成的能力。我们之前的研究证明了Cdc 42和Rasip 1在这个过程中的关键作用。使用最先进的体外和体内方法，我们已经证明了Cdc 42在使用培养的人EC形成EC管腔期间以及在小鼠血管发育和血管生长期间的基本作用（先前的研究和初步数据显示在这里）。Cdc 42功能的失活导致体内和体外EC极化的破坏，这导致未能正确形成或组织EC管网。此外，我们一起表明，Rasip 1，一个小的GTdR功能的调节剂，是所需的EC tubulogenesis，在体内和体外。我们发现Rasip 1是激活GTP酶Cdc 42和Rac 1以及抑制EC中RhoA所必需的。总之，这项工作强调了在血管形态发生过程中GTdR活性的核心重要性。我们最近筛选了小管形成的内皮细胞，并确定了多种GT3活性调节剂作为这一过程的关键调节剂。我们已经确定了与Cdc 42一起控制血管小管形成的其他GTP酶，包括Rac亚型，k-Ras和Rap 1。GTP酶的关键功能作用是通过囊泡运输沿着极化微管轨道（其富含乙酰化微管蛋白并且存在于亚顶端膜结构域中以促进顶端表面处的膜融合事件）刺激从基底表面到顶端表面的膜运输。我们观察到强大的富集的RAC，激活Src和rasip 1在EC顶端膜和囊泡内被运输的顶端。我们还发现了新的微管发生调节因子，包括GT3效应子IQGAP 1、MRCKβ和β-Pix，以及关键的鸟嘌呤交换因子（GEF）Dock 6、Sos 1和FGD 5，它们在此过程中激活Cdc 42、Rac和k-Ras。此外，我们已经发现，Arhgap 31（失活Cdc 42和Rac）和Rasa 1（失活Ras）是关键的监管机构，揭示了为了正确控制和平衡EC tubulogenesis，负调控是必要的，而Rasip 1相关的Arhgap 29，失活Rho，是EC tubulogenesis所需的，在体内和体外。总之，这些结果提供了一个分子路线图，说明EC如何改变它们的形状，改变和重新定位连接点，所有这些都是为了形成携带血液的功能管，这是血管形成和组织活力所必需的能力。在这里，我们建议使用遗传和体外方法来剖析复杂的信号通路如何控制特定的细胞事件，以支持血管形态发生。本提案的重点是阐明Cdc 42和其他GTPases如何控制细胞骨架极化和囊泡运输，以驱动EC形态发生和微管发生的分子和细胞机制。我们提出了三个具体的目标，以检查在体内和体外EC tubulogenesis的过程：具体目标#1。阐明小的Rho GTdR Cdc 42如何通过细胞骨架极化、顶膜运输和EC连接形成来控制EC小管形成，以促进多细胞管组装和稳定。具体目标#2确定Rac-，k-Ras-和Rap 1如何与Cdc 42在EC微管发生过程中相互作用。具体目标#3鉴定和功能性表征积极和消极调节EC小管形成的关键GTGFs和GAP。

 DESCRIPTION (provided by applicant): In this new collaborative proposal, we investigate the ability of Rho GTPases to modulate both cytoskeletal and membrane apical-basal polarization to control endothelial cell (EC) tubulogenesis. Our previous studies demonstrated critical roles for Cdc42 and Rasip1 during this process. Using state-of-the art in vitro and in vivo approaches, we have demonstrated a fundamental role for Cdc42 during EC lumen formation using cultured human ECs and during mouse vascular development and blood vessel growth (previous studies and preliminary data shown here). 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 tube networks. In addition, together, we showed that Rasip1, a modulator of small GTPase function, is required for EC tubulogenesis, both in vivo and in vitro. We showed that Rasip1 is required to activate the GTPases Cdc42 and Rac1, as well as to suppress RhoA in ECs. Together, this work underlined the central importance of GTPase activity during blood vessel morphogenesis. We recently screened tubulogenic ECs and identified multiple modulators of GTPase activity as key regulators of this process. We have identified additional GTPases that work with Cdc42 to control vascular tubulogenesis, including Rac isoforms, k-Ras and Rap1. A key functional role of GTPases is to stimulate membrane trafficking from the basal to apical surface through vesicle transport along polarized microtubule tracks (which are enriched in acetylated tubulin and which are present in a subapical membrane domain to promote membrane fusion events at the apical surface). We observe strong enrichment of Rac, activated Src and Rasip1 at EC apical membranes and within vesicles that are being transported apically. We have also discovered novel regulators of tubulogenesis, including the GTPase effectors IQGAP1, MRCKβ, and beta-Pix, and critical guanine exchange factors (GEFs) Dock6, Sos1 and FGD5, that activate Cdc42, Rac, and k-Ras during this process. In addition, we have found that Arhgap31 (inactivates Cdc42 and Rac) and Rasa1 (which inactivates Ras) are critical regulators, revealing that in order to properly control and balance EC tubulogenesis, negative regulators are necessary, while the Rasip1-associated Arhgap29, which inactivates Rho, is required for EC tubulogenesis, both in vivo and in vitro. Together, these results provide a molecular road map for how ECs change their shape, polarize and reorient junctions, all with the ultimate goal of forming functional tubes that carry blood, a capacity essential for blood vessel formation and tissue viability. Here, we propose to use genetic and in vitro approaches to dissect how complicated signaling pathways control specific cellular events to support blood vessel morphogenesis. The focus of this proposal is to elucidate the molecular and cellular mechanisms underlying how Cdc42 and other GTPases control cytoskeletal polarization and vesicular trafficking to drive EC morphogenesis and tubulogenesis. We propose three specific aims to examine the process of EC tubulogenesis in vivo and in vitro: Specific Aim #1. To elucidate how the small Rho GTPase Cdc42 controls EC tubulogenesis via cytoskeletal polarization, apical membrane trafficking and EC junction formation to facilitate multicellular tube assembly and stability. Specific Aim #2. To determine how Rac-, k-Ras-, and Rap1 interact with Cdc42 during EC tubulogenesis. Specific Aim #3. To identify and functionally characterize critical GTPase GEFs and GAPs which positively and negatively regulate EC tubulogenesis.