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
基本信息
- 批准号:8891096
- 负责人:
- 金额:$ 11.91万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AffectAngiogenesis InhibitorsArchitectureBehaviorBiological PreservationBiologyBiomedical ResearchBlood VesselsCardiovascular systemCell PolarityCellsCentrosomeCharacteristicsChemicalsCollaborationsColoradoComputer softwareCuesDataDefectDevelopmentDiabetes MellitusDiseaseDynein ATPaseEducational ActivitiesEndothelial CellsEngineeringEnvironmentFrequenciesFunctional disorderFundingGoalsGolgi ApparatusGrowth FactorHarvestHumanImageImage AnalysisIn VitroInterphaseInvestigationIschemiaLaboratoriesLasersLeadLearningLifeLightLinkMaintenanceMalignant NeoplasmsMechanicsMedicalMembraneMentorsMethodsMicrosurgeryMicrotubulesModelingModificationMolecularMonitorMorphogenesisMorphologyMotorMusNeoplasms in Vascular TissueNorth CarolinaNutrientOncologistOrganellesOutcomePathologyPatientsPhasePopulationPositioning AttributeProcessProteinsRecruitment ActivityResearchResearch TrainingRunningSignal PathwaySignal TransductionStructureSystemTechniquesTestingTissuesTrainingTraining ActivityTransgenesTransgenic MiceUniversitiesUrsidae FamilyVesicleWorkabstractingangiogenesisbaseblood vessel developmentcareercell motilitycellular imagingcohortdesigndirectional cellgenetic inhibitorin vivoknock-downmigrationmodel developmentmortalitymouse modelneoplastic cellnoveloverexpressionprogramsrac1 GTP-Binding Proteinresponsescreeningskillsthree-dimensional modelingtooltraffickingtumortumor microenvironmenttumor progression
项目摘要
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 - 我在科罗拉多大学博尔德分校完成了我的研究生工作,主要研究人类群体的内皮细胞功能障碍。毕业后,我通过寻求博士后职位来扩大我的研究培训。北卡罗来纳大学 (UNC) 生物系的维多利亚·鲍奇 (Victoria Bautch)(导师)实验室在这里使用转基因小鼠和细胞模型来研究发育和分子机制。在北卡罗来纳大学,我处于独特的地位,可以在我的合作导师 James Bear 博士和 Alexey Khodjakov 博士的帮助下完成拟议的目标、培训和研究策略。教育活动将为我提供必要的技能和协作
实现我在研究型大学运营一个独立的、校外资助的生物医学研究实验室的长期职业目标。 拟议的研究——血管的建立和维护需要信号通路和细胞骨架程序的整合和协调。异常的信号传导会导致病理性脉管系统的形成;然而,人们对细胞骨架程序如何变得功能失调并损害血管结构的了解较少。不限于癌症相关的病理,肿瘤血管异常、渗漏和扩张,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。研究结果表明,肿瘤内皮细胞发生永久性改变,与信号传导影响无关,可能是由于细胞骨架异常所致。在这方面,我们的小组之前描述了一种过度促血管生成生长因子信号传导的机制,类似于在中发现的机制。癌症,促进 EC 中多余中心体(超过两个中心体)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)获得多余中心体的机制。发现,我最近提供了一个
将间期多余中心体与二维 EC 运动缺陷联系起来的新机制(Kushner 等人;JCB.2014)我们的结果表明,多余中心体极化错误,导致细胞骨架发生级联变化,最终导致细胞定向迁移的丧失。这项调查还有许多其他问题,本提案旨在更好地理解这些问题并在全球范围内进行重大扩展。多余中心体如何影响 3D 萌芽(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,因此我还将在多个指导阶段探索中心体极化和束缚(独立阶段)的独特机制。将通过 Plk4 过表达分析具有或不具有多余中心体的 3D 血管生成(体外、离体和体内)血管模型之前,我证明了多余的中心体会影响 2D 中的微管 (MT) 动态。为了检查 3D 中的 MT 缺陷是否持续存在,我们将采用活细胞成像和 MT 分析软件来监测 3D 芽中的 MT 动态。 ,高尔基体复杂的和囊泡蛋白将在 EC 中用荧光蛋白进行标记,以便可视化它们在有或没有过量中心体的情况下的动态,如果受到干扰,将检查关键的 EC 极性和连接蛋白是否由于存在而被破坏的高尔基体后囊泡运输的下游错误定位。预测结果将揭示多余中心体如何促进 3D 血管畸形发生。其中中心体对(由 MT 连接的两个中心体)可以差异性地调节其 MT 动力学,以响应皮层施加的拉力,例如细胞迁移。
目标是,我将探索中心体如何/是否使用可光激活的 Rac1 蛋白感应张力来诱导膜张力,基于软件的 MT 跟踪和 MT 激光切断技术将选择性地敲低、过度表达和拯救所涉及的候选蛋白,以彻底询问负责的信号传导程序。最后,将产生一种新的小鼠,用于有条件地、血管特异性地敲低动力蛋白(一种 MT 马达)。蛋白)来探索中心体束缚和极化的破坏如何影响血管网络的形成。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Erich J Kushner其他文献
Erich J Kushner的其他文献
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血管发育过程中 Delta-like 4 内吞作用和 Notch 激活的机制
- 批准号:
10202195 - 财政年份:2021
- 资助金额:
$ 11.91万 - 项目类别:
Mechanisms of Basement Membrane Regulation During Angiogenesis
血管生成过程中基底膜的调节机制
- 批准号:
10002605 - 财政年份:2019
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$ 11.91万 - 项目类别:
Centrosome Over-duplication and Blood Vessel Function
中心体过度复制与血管功能
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8455123 - 财政年份:2013
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$ 11.91万 - 项目类别:
Centrosome Over-duplication and Blood Vessel Function
中心体过度复制与血管功能
- 批准号:
8627974 - 财政年份:2013
- 资助金额:
$ 11.91万 - 项目类别:
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