Endothelial Cell Migration in Three Dimensions

内皮细胞的三维迁移

• 批准号: 9157350
• 负责人: Clare Michal Waterman
• 金额: $ 24.69万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9157350
• 关键词: 4D Imaging; Actins; Algorithms; Biochemical; Biological Models; Blood Vessels; Caliber; Cell Culture Techniques; Cell Shape; Cell membrane; Cell model; Cell surface; Cells; Cellular biology; Collaborations; Collagen; Complex; Computer Analysis; Cues; Development; Dimensions; Down-Regulation; Endothelial Cells; Environment; Extracellular Matrix; Gel; Goals; Hypoxia; Image; Image Analysis; Immigration; In Vitro; Invaded; Life; Measures; Mechanics; Mediating; Membrane; Metalloproteases; Methods; Morphogenesis; Movement; Myosin Type II; Nature; Paper; Pathway interactions; Physiological; Process; Proteins; Pseudopodia; Publications; Publishing; Regulation; Relative (related person); Research; Resolution; Role; Structure; System; Techniques; Tissues; Translating; Vascular Endothelial Growth Factors; Wood material; Work; Zebrafish; angiogenesis; cell cortex; cell motility; light microscopy; medical schools; migration; novel; polymerization; response; tissue/cell culture

Project 1;Myosin II mediates local cortical tension to guide endothelial cell branching morphogenesis and migration in 3D. Personell: Robert Fischer A key feature of angiogenesis is directional control of endothelial cell (EC) morphogenesis and movement. During angiogenic sprouting, endothelial tip cells directionally branch from existing vessels in response to biochemical cues such as VEGF or hypoxia, and migrate and invade the surrounding extracellular matrix (ECM) in a process that requires ECM remodeling by matrix metalloproteases (MMPs). Tip EC branching is mediated by directional protrusion of subcellular pseudopodial branches. Here we sought to understand how EC pseudopodial branching is locally regulated to directionally guide angiogenesis. We develop an in vitro 3D EC model system where migrating ECs display branched pseudopodia morphodynamics similar to those in living zebrafish. Using this system, we find that ECM stiffness and ROCK-mediated myosin II activity inhibit EC pseudopodial branch initiation. Myosin II is dynamically localized to the EC cortex, and is partially released under conditions that promote branching. Local depletion of cortical myosin II precedes branch initiation, and initiation can be induced by local inhibition of myosin II activity. Thus, local downregulation of myosin II cortical contraction allows pseudopodium initiation to mediate EC branching and hence guide directional migration and angiogenesis. A paper describing the deveoplment of cell culture methods that were utilized to complete this study was published in 2012. In addition, a review paper describing the development of 3d light microscopy methods for studies like this one was also published in 2011. Both papers were invited. A paper describing the results of this study was published in Nature Cell Biology Project 2: Development of algorithms for tracking cell morphodynamics in three dimensions and analysis of the role of myosin II in regulating cell shape in 3d. . Personell: Robert Fischer In collaboration with Gaudenz Danuser and Hunter Elliot at Harvard Medical School and performed at MBL at Woods Hole. Algorithms have been developed to track the surface of cells migrating in 3-d collagen gels. Regulation of endothelial cell branching and polarized migration defines vascular plexus morphogenesis which is critical to normal development. Previously we demonstrated that cortical myosin II in endothelial cells responds to mechanical cues in the extracellular matrix (ECM) and negatively regulates endothelial cell branch initiation and migration in 3D collagen gels. However, how regulation of branch initiation is spatially controlled to generate the canonical arboreal endothelial cell shape in 3D is still unclear. We hypothesize that endothelial cell shape in 3D environments can be determined by a combination of cell protrusion driven by actin polymerization, and cortical membrane tension and retraction driven by myosin II contraction. To understand how these components work together to define cell shape in 3D EMCs, we performed 4D imaging of primary endothelial cells expressing a fluorescently tagged plasma membrane marker in collagen gels and pharmacologically manipulated actin polymerization driven by Arp2/3 or formins or myosin II contractility. We developed novel algorithms to track the cell surface and to define cell shape and branching structure. We used computational analysis to quantify cell morphometric parameters at three spatial scales relative to the cell. Globally, we quantified the overall cell branch orientation relative to the direction of cell movement as a measure of cell polarization. Regionally, we quantified branch complexity as measured by branch order number and relative diameter. Locally, we quantified cell surface curvature at each point along the cell surface. We find that actin polymerization regulates only branch number, while myosinII controls cell shape at all spatial scales. Specifically, formins and Arp2/3 promote increased cell branch number and complexity, while myosin II promotes cell polarization but limits branch complexity and cell membrane curvature. Our results reveal new roles for actin polymerization and myosin II activity in control of complex cell morphogenic pathways in physiologic, 3D ECMs. A paper describing these studies is being prepared for publication

项目1：肌球蛋白II介导局部皮质张力以引导内皮细胞分支 三维形态发生和迁移。 个人：Robert Fischer 血管生成的一个关键特征是对内皮细胞（EC）形态发生和运动的定向控制。在血管生成出芽过程中，内皮尖端细胞响应于生物化学信号（如VEGF或缺氧）从现有血管定向分支，并在需要基质金属蛋白酶（MMP）重塑ECM的过程中迁移和侵入周围的细胞外基质（ECM）。 顶端EC分支由亚细胞伪足分支的定向突起介导。 在这里，我们试图了解EC伪足分支是如何局部调节，定向引导血管生成。我们开发了一个体外三维EC模型系统，迁移EC显示分支伪足形态动力学类似于那些在活斑马鱼。使用这个系统，我们发现ECM刚度和ROCK介导的肌球蛋白II活性抑制EC伪足分支的启动。肌球蛋白II动态定位于EC皮质，并在促进分支的条件下部分释放。 皮质肌球蛋白II的局部消耗先于分支起始，并且起始可以通过肌球蛋白II活性的局部抑制来诱导。因此，肌球蛋白II皮质收缩的局部下调允许伪足启动介导EC分支，从而引导定向迁移和血管生成。 2012年发表了一篇描述用于完成本研究的细胞培养方法开发的论文。 此外，2011年还发表了一篇综述论文，描述了用于此类研究的3d光学显微镜方法的发展。 两份报纸都受到了邀请。 描述这项研究结果的论文发表在《自然细胞生物学》上 项目二：开发三维细胞形态动力学跟踪算法，并分析肌球蛋白II在三维细胞形状调节中的作用。 . 个人：Robert Fischer 与哈佛医学院的高登兹·达努瑟和亨特·埃利奥特合作，并在伍兹霍尔的MBL演出。 已经开发了算法来跟踪细胞在3-d胶原凝胶中迁移的表面。 内皮细胞分支和极化迁移的调节定义了血管丛形态发生，这对正常发育至关重要。以前我们证明了内皮细胞中的皮质肌球蛋白II对细胞外基质（ECM）中的机械信号作出反应，并在3D胶原凝胶中负调节内皮细胞分支的起始和迁移。然而，分支起始的调节如何在空间上被控制以产生3D中的典型树状内皮细胞形状仍然不清楚。我们假设，在3D环境中的内皮细胞的形状可以确定由肌动蛋白聚合驱动的细胞突起，和皮质膜张力和收缩肌球蛋白II收缩驱动的组合。为了理解这些成分如何共同作用以定义3D EMCs中的细胞形状，我们对胶原凝胶中表达荧光标记的质膜标记物的原代内皮细胞进行了4D成像，并对Arp 2/3或formins或肌球蛋白II收缩性驱动的肌动蛋白聚合进行了微操纵。 我们开发了新的算法来跟踪细胞表面并定义细胞形状和分支结构。我们使用计算分析，以量化细胞形态学参数在三个空间尺度相对于细胞。在全球范围内，我们量化了相对于细胞运动方向的整体细胞分支取向，作为细胞极化的量度。在区域上，我们量化了分支的复杂性，通过分支顺序数和相对直径来衡量。在局部，我们量化了细胞表面沿着每个点的细胞表面曲率。我们发现，肌动蛋白聚合调节只有分支的数量，而肌球蛋白II控制细胞的形状在所有的空间尺度。 具体而言，formins和Arp 2/3促进细胞分支数量和复杂性增加，而肌球蛋白II促进细胞极化，但限制分支复杂性和细胞膜曲率。 我们的研究结果揭示了新的作用，肌动蛋白聚合和肌球蛋白II的活动在控制复杂的细胞形态发生途径的生理，三维ECM。 目前正在编写一份介绍这些研究的文件，