Endothelial Cell Migration in Three Dimensions

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。 人物：罗伯特·费舍尔 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。在新生血管的萌发过程中，内皮细胞会根据血管内皮生长因子或低氧等生化信号，从现有血管定向分支，迁移并侵入周围的细胞外基质(ECM)，这一过程需要基质金属蛋白酶(MMPs)对ECM进行重塑。端部EC分支是由亚细胞伪足分支的定向突起介导的。在这里，我们试图了解EC伪足分支是如何被局部调控以定向指导血管生成的。我们开发了一个体外三维EC模型系统，其中迁移的EC显示出与活斑马鱼相似的分枝伪足形态动力学。使用这个系统，我们发现细胞外基质的僵硬和岩石介导的肌球蛋白II活性抑制了EC伪足分支的起始。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的局部耗竭先于分支的启动，而肌球蛋白II的局部抑制可以诱导分支的启动。因此，肌球蛋白II皮质收缩的局部下调允许伪足起始介导EC分支，从而引导定向迁移和血管生成。 2012年发表了一篇论文，描述了用来完成这项研究的细胞培养方法的发展。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。两家报纸都被邀请了。一篇描述这项研究结果的论文发表在《自然细胞生物学》杂志上 项目2：开发三维追踪细胞形态动力学的算法，并分析肌球蛋白II在三维细胞形状调节中的作用。。 人物：罗伯特·费舍尔 与高登兹·丹瑟和亨特·埃利奥特合作，在哈佛医学院演出，并在伍兹霍尔的MBL演出。人们已经开发出了追踪细胞在三维胶原凝胶中迁移的表面的算法。内皮细胞分支和极化迁移的调节决定了血管神经丛的形态发生，而血管丛形态发生对正常发育至关重要。在此之前，我们证明了内皮细胞中的皮质肌球蛋白II响应细胞外基质(ECM)中的机械信号，负向调节3D胶原凝胶中内皮细胞分支的起始和迁移。然而，如何在空间上控制分支起始的调控以产生3D中标准的树状内皮细胞形状仍不清楚。我们假设，3D环境中内皮细胞的形状可以由肌动蛋白聚合驱动的细胞突起和肌球蛋白II收缩驱动的皮质膜张力和收缩共同决定。为了了解这些成分如何共同作用来定义3D内皮细胞的细胞形状，我们对原代内皮细胞进行了4D成像，这些细胞在胶原凝胶中表达荧光标记的质膜标记，以及由Arp2/3或福尔马林或肌球蛋白II收缩能力驱动的药物操纵的肌动蛋白聚合。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。我们使用计算分析来量化与细胞相关的三个空间尺度上的细胞形态参数。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方向，作为细胞极化的衡量标准。在区域上，我们通过分支序数和相对直径来量化分支复杂性。在局部，我们量化了沿细胞表面的每个点的细胞表面曲率。我们发现，肌动蛋白聚合只调节分支数量，而肌球蛋白II在所有空间尺度上控制细胞形状。具体地说，福尔马林和Arp2/3促进细胞分枝数目和复杂性增加，而肌球蛋白II促进细胞极化，但限制分枝复杂性和细胞膜曲率。我们的结果揭示了肌动蛋白聚合和肌球蛋白II活性在生理学3D ECM中控制复杂细胞形态发生通路中的新作用。一篇描述这些研究的论文正在准备出版

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