Centrosomes and Cytoskeletal Mechanisms of Blood Vessel Dysfunction

血管功能障碍的中心体和细胞骨架机制

• 批准号: 8891096
• 负责人: Erich J Kushner
• 金额: $ 11.91万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-11 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8891096
• 关键词: Affect; Angiogenesis Inhibitors; Architecture; Behavior; Biological Preservation; Biology; Biomedical Research; Blood Vessels; Cardiovascular system; Cell Polarity; Cells; Centrosome; Characteristics; Chemicals; Collaborations; Colorado; Computer software; Cues; Data; Defect; Development; Diabetes Mellitus; Disease; Dynein ATPase; Educational Activities; Endothelial Cells; Engineering; Environment; Frequencies; Functional disorder; Funding; Goals; Golgi Apparatus; Growth Factor; Harvest; Human; Image; Image Analysis; In Vitro; Interphase; Investigation; Ischemia; Laboratories; Lasers; Lead; Learning; Life; Light; Link; Maintenance; Malignant Neoplasms; Mechanics; Medical; Membrane; Mentors; Methods; Microsurgery; Microtubules; Modeling; Modification; Molecular; Monitor; Morphogenesis; Morphology; Motor; Mus; Neoplasms in Vascular Tissue; North Carolina; Nutrient; Oncologist; Organelles; Outcome; Pathology; Patients; Phase; Population; Positioning Attribute; Process; Proteins; Recruitment Activity; Research; Research Training; Running; Signal Pathway; Signal Transduction; Structure; System; Techniques; Testing; Tissues; Training; Training Activity; Transgenes; Transgenic Mice; Universities; Ursidae Family; Vesicle; Work; abstracting; angiogenesis; base; blood vessel development; career; cell motility; cellular imaging; cohort; design; directional cell; genetic inhibitor; in vivo; knock-down; migration; model development; mortality; mouse model; neoplastic cell; novel; overexpression; programs; rac1 GTP-Binding Protein; response; screening; skills; three-dimensional modeling; tool; trafficking; tumor; tumor microenvironment; tumor progression

 DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT CANDIDATE- I completed my graduate work at the University of Colorado, Boulder, focusing on endothelial cell dysfunction in human cohorts. After graduation, I broadened my research training by seeking a postdoctoral position in Dr. Victoria Bautch's (mentor) lab at the University of North Carolina (UNC), Department of Biology. Here, I use transgenic mouse and cell-based models to study the developmental and molecular mechanisms of vessel formation and dysfunction. At UNC, I am uniquely situated to carry out the proposed training plan and research strategy with the aid of my co-mentors, Dr.'s James Bear and Alexey Khodjakov. Completion of the proposed aims, training and educational activities will provide me with the necessary skills and collaborations to reach my long-term career goal of running an independent, extramurally funded biomedical research lab at a research-one university. PROPOSED RESEARCH- Setup and maintenance of blood vessels requires the integration and coordination of signaling pathways and cytoskeletal programs. Much is known about how aberrant signaling contributes to formation of pathological vasculature; however, less is understood about how cytoskeletal programs become dysfunctional and impair blood vessel architecture. This notion is best exemplified by tumor blood vessels; although, it is not limited to cancer-related pathologies. Tumor vessels are abnormal, leaky and dilated, providing a venue for tumor cell escape. Even in the absence of the tumor microenvironment, isolated tumor endothelial cells (ECs) demonstrate a preservation of abnormal cellular behaviors. These findings suggest that permanent alterations occur in tumor ECs, independent of signaling influences, possibly due to cytoskeletal abnormalities. In this regard, our group has previously described a mechanism by which excessive pro- angiogenic growth factor signaling, akin to that found in cancers, promotes the formation of supernumerary centrosomes (more than two centrosomes) in ECs. This data provided a mechanism for how tumor ECs acquire excess centrosomes in the tumor compartment at very high frequencies (>1/3 of total EC population). Furthering this finding, I have recently provided a novel mechanism linking interphase supernumerary centrosomes to EC motility defects in 2D (Kushner et al.; JCB. 2014). Our results demonstrated that supernumerary centrosomes are mispolarized, causing a cascade of cytoskeletal changes, which culminates in loss of directional cell migration. However, this investigation has prompted many additional questions, which this proposal strives to better understand and significantly expand upon. Globally, this proposal aims to determine how supernumerary centrosomes influence blood vessel morphogenesis in 3D sprouting (mentored phase). Furthermore, because centrosome polarization is vital for proper EC migration, I will also explore unique mechanisms of centrosome polarization and tethering (independent phase). For the mentored phase, in multiple models of 3D angiogenesis (in vitro, ex vivo, and in vivo) blood vessels with and without supernumerary centrosomes via Plk4 overexpression will be analyzed for morphological defects. Previously, I demonstrated that supernumerary centrosomes affect microtubule (MT) dynamics in 2D. To examine if MT defects persist in 3D, live-cell imaging and MT analysis software will be employed to monitor MT dynamics in ECs in 3D sprouts. Additionally, I hypothesize that supernumerary centrosomes will also effect the Golgi complex and vesicle trafficking, as these organelles are MT-dependent. Accordingly, the Golgi complex and vesicular proteins will be marked in ECs with fluorescent proteins in order to visualize their dynamics with and without excess centrosomes. If perturbed, key EC polarity and junctional proteins will be examined for mislocalization downstream of disrupted post-Golgi vesicle trafficking due to the presence of excess centrosomes. Predicted results will shed light on how supernumerary centrosome promotes blood vessel dysmorphogenesis in 3D. For the independent phase, I will characterize a unique phenomenon in which centrosome pairs (two centrosomes connected by MTs) can differentially regulate their MT dynamics in response to pulling forces exerted at the cortex, such as in cell migration. In this aim, I will explore how/if centrosomes sense tension using photoactivable Rac1 protein to induced membrane tension, software-based MT tracking and MT laser severing techniques. Candidate proteins involved will be selectively knocked down, overexpressed and rescued to thoroughly interrogate signaling programs responsible for modulation of centrosomal-MTs in response to tension cues. Lastly, a new mouse will be generated for conditional, vascular- specific knock down of dynein (a MT-motor protein) to explore how disruption of centrosome tethering and polarization impacts vessel network formation. .

 描述（由适用提供）：项目摘要/摘要候选人 - 我在科罗拉多大学的博尔德分校完成了我的研究生工作，重点是人类同伙的内皮细胞功能障碍。毕业后，我通过在北卡罗来纳大学（UNC）的Victoria Bautch博士（UNC）的Victoria Bautch博士（Mentor）实验室中寻求博士后职位，扩大了研究培训。在这里，我使用转基因小鼠和基于细胞的模型来研究血管形成和功能障碍的发育和分子机制。在UNC上，我独一无二地借助我的院长James Bear和Alexey Khodjakov执行拟议的培训计划和研究策略。拟议的目标，培训和教育活动的完成将为我提供必要的技能和协作 在一所研究大学中实现我的长期职业目标，即在研究一所大学中运行独立的，外部资助的生物医学研究实验室。提出的研究 - 血管的设置和维持需要信号通路和细胞骨架程序的整合和协调。关于异常信号如何有助于病理性脉管系统的形成知之甚少。但是，关于细胞骨架程序如何变得功能失调并损害了血管结构的理解较少。虽然，它不仅限于与癌症相关的病理。肿瘤血管异常，漏水和扩张，为肿瘤细胞逃生提供了场所。即使在没有肿瘤微环境的情况下，分离的肿瘤内皮细胞（EC）也表现出异常的细胞行为。这些发现表明，由于细胞骨架异常而可能导致的肿瘤EC中发生永久性改变，与信号传导无关。在这方面，我们的小组先前已经描述了一种机制，通过这种机制，类似于癌症中发现的过量血管生成因子信号传导促进了ECS中超生物中心体（超过两个中心体）的形成。该数据为肿瘤ECS如何在非常高的频率（> EC总人群> 1/3）中获得过量的中心体提供了一种机制。进一步发现这一发现，我最近提供了 在2D中将相间超级中心体与EC运动缺陷联系起来的新型机制（Kushner等；JCB。2014）。我们的结果表明，超鼻中心体是杂极化的，导致一系列细胞骨架变化，这最终导致方向性细胞迁移的丧失。但是，这项投资引发了许多其他问题，该提案旨在更好地理解并大大扩展。在全球范围内，该提案旨在确定超努中心体如何影响3D发芽（指导阶段）中的血管形态发生。此外，由于中心体极化对于适当的EC迁移至关重要，因此我还将探索中心体极化和束缚的独特机制（独立阶段）。对于修补阶段，在多种模型中，通过PLK4过表达，将分析具有和不具有超代性中心体的3D血管生成（体内，体内和体内）的血管中的血管，以分析形态缺陷。以前，我证明了超鼻中心体影响2D中的微管（MT）动力学。为了检查MT缺陷是否在3D中持续存在，将采用活细胞成像和MT分析软件来监视3D芽孢中的EC中的MT动态。此外，我假设超级中心体也将影响高尔基体复合物和囊泡运输，因为这些细胞器依赖于MT。根据高尔基体复合物和囊泡蛋白，将在具有荧光蛋白的EC中标记，以便以有或没有过量的中心体的方式可视化其动力学。如果受到干扰，由于存在过量的中心体的存在，将检查关键的EC极性和连接蛋白是否在干扰后高尔基球囊泡运输的下游错误进行检查。预测的结果将阐明超代性中心体如何促进3D中的血管变形。对于独立阶段，我将表征一种独特的现象，其中中心体对（由MTS连接的两个中心体）可以不同地调节其MT动力学，以响应在皮层上施加的拉力，例如在细胞迁移中。在这个 目的，我将探索使用光活性Rac1蛋白诱导膜张力，基于软件的MT跟踪和MT激光器的几种技术的中心体张力。涉及的候选蛋白将被选择性地击倒，过表达并响应负责调节中心体MT的彻底询问的信号传导程序，以响应张力提示。最后，将生成一种新的小鼠，以降低有条件的，血管特异性的动力蛋白（MT运动蛋白），以探讨中心体束缚和极化的破坏如何影响血管网络的形成。