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. 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 myosin II 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 Project 3: microtubule regulation of endothelial cell branching morphogenesis personell: bob fischer and ken myers Ctyoskeletal dynamics driving endothelial cell (EC) branching morphogenesis during angiogenesis are thought to be regulated in part by cellular signals elicited in response to compliance and topology of the extracellular matrix (ECM) via a process termed mechanosensing. We hypothesized that ECM mechanosensing of compliance or topology (2 dimensional vs 3 dimensional ECMs, i.e. ECM dimensionality) could elicit different responses of the microtubule (MT) cytoskeleton to mediate EC branching morphogenesis. To test this, we used novel MT end-tracking software to analyze spatial variations in MT dynamics in ECs in 2D and 3D compliance-controlled ECMs during branching morphogenesis. Pharmacological inhibition showed that MT dynamics negatively regulate EC branching, independent of ECM compliance or dimensionality. Analyzing MT dynamics incompliant 2D and 3D ECMs with or without myosinII inhibition indicated that myosinII down-regulation by compliance mechanosensing promotes fast MT assembly, but we found dimensionality-specific effects on MT growth persistence. Comparing MT dynamics in cell bodies versus cell branches in in 2D and 3D ECMs of varying stiffness revealed faster, more dynamically unstable MT growth in EC bodies and slower, more persistent MT growth in EC branches. Thus, distinct compliance and dimensionality ECM mechanosensing pathways regionally regulate MT dynamics in ECs to guide branching morphogenesis in physically complex ECMs. This work was published in the JCB in 2011 this is a collaboration with gaudenz danuser and kathryn applegate. some of the goals of this project are worked on by ken under the microtuybules and actin interaction work.
