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（由申请人提供）：项目摘要/摘要候选人-我在博尔德的科罗拉多大学完成了我的研究生工作，重点研究人类队列中的内皮细胞功能障碍。毕业后，我在北卡罗来纳州大学生物系维多利亚鲍奇博士（导师）的实验室寻求博士后职位，从而拓宽了我的研究训练。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。在斯坦福大学，我有着得天独厚的条件，可以在我的导师们的帮助下实施拟议的培训计划和研究战略。詹姆斯·贝尔和阿列克谢·霍贾科夫。完成拟议的目标，培训和教育活动将为我提供必要的技能和合作， 实现我的长期职业目标，在一所研究型大学里经营一个独立的、由校外资助的生物医学研究实验室。建议的研究-建立和维护血管需要整合和协调信号通路和细胞骨架程序。关于异常信号传导如何促进病理性脉管系统的形成，人们知道得很多;然而，关于细胞骨架程序如何变得功能障碍并损害血管结构，人们了解得很少。这一概念最好的例子是肿瘤血管;虽然，它不限于癌症相关的病理。肿瘤血管异常、渗漏和扩张，为肿瘤细胞逃逸提供了场所。即使在没有肿瘤微环境的情况下，分离的肿瘤内皮细胞（EC）也表现出异常细胞行为的保留。这些发现表明，永久性的改变发生在肿瘤EC，独立的信号影响，可能是由于细胞骨架异常。在这方面，我们的小组先前已经描述了一种机制，通过该机制，过度的促血管生成生长因子信号传导（类似于在癌症中发现的信号传导）促进EC中额外中心体（多于两个中心体）的形成。该数据提供了肿瘤EC如何以非常高的频率（>总EC群体的1/3）在肿瘤隔室中获得过量中心体的机制。为了进一步推动这一发现，我最近提供了一个 在2D中将间期额外中心体与EC运动缺陷联系起来的新机制（Kushner等; JCB 2014年）。我们的研究结果表明，多余的中心体被错误极化，导致级联的细胞骨架的变化，最终在定向细胞迁移的损失。然而，这一调查引发了许多其他问题，本提案力求更好地理解和大大扩展。 在全球范围内，该提案旨在确定额外中心体如何影响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正确的EC迁移至关重要，我还将探索中心体极化和束缚（独立相）的独特机制。对于指导阶段，在3D血管生成的多种模型（体外、离体和体内）中，将分析具有和不具有通过Plk 4过表达的额外中心体的血管的形态缺陷。以前，我证明了多余的中心体影响微管（MT）在2D的动力学。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分析软件来监测3D芽中EC中的MT动态。此外，我推测，额外的中心体也将影响高尔基复合体和囊泡贩运，因为这些细胞器是MT依赖。因此，高尔基复合体和囊泡蛋白将在EC中用荧光蛋白标记，以便在有和没有过量中心体的情况下可视化它们的动态。如果受到干扰，关键EC极性和连接蛋白将被检查由于过量中心体的存在而导致的破坏后高尔基体囊泡运输下游的错误定位。预测的结果将揭示多余中心体如何促进3D血管畸形发生。对于独立的阶段，我将描述一个独特的现象，其中中心体对（两个中心体连接的MT）可以差异调节其MT动态响应施加在皮层的拉力，如在细胞迁移。在这 本论文的目的是利用光激活的Rac 1蛋白诱导细胞膜张力、基于软件的MT跟踪和MT激光切割技术来探索中心体如何/是否感受张力。参与的候选蛋白质将被选择性地敲低、过表达和拯救，以彻底询问负责调节中心体-MT响应于张力线索的信号传导程序。最后，将产生新的小鼠用于动力蛋白（一种MT-运动蛋白）的条件性血管特异性敲低，以探索中心体束缚和极化的破坏如何影响血管网络形成。.