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. This work was performed in collaboration with Bob Adelstein and Xuefei Ma (NHLBI) and Margaret Gardel (U Chicago) and was published in 2009 Project 2: Development of algorithms for tracking cell morphodynamics in three dimensions. Personell: Robert Fischer In collaboration with Gaudenz Danuser and Sam Ching at Scripps and performed at MBL at Woods Hole. This is ongoing.

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National Heart, Lung, and Blood Institute
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Elliott, Hunter; Fischer, Robert S; Myers, Kenneth A et al. (2015) Myosin II controls cellular branching morphogenesis and migration in three dimensions by minimizing cell-surface curvature. Nat Cell Biol 17:137-47
Winter, Peter W; Chandris, Panagiotis; Fischer, Robert S et al. (2015) Incoherent structured illumination improves optical sectioning and contrast in multiphoton super-resolution microscopy. Opt Express 23:5327-34
Lam, Pui-Ying; Fischer, Robert S; Shin, William D et al. (2014) Spinning disk confocal imaging of neutrophil migration in zebrafish. Methods Mol Biol 1124:219-33
Braun, Alexander; Dang, Kyvan; Buslig, Felinah et al. (2014) Rac1 and Aurora A regulate MCAK to polarize microtubule growth in migrating endothelial cells. J Cell Biol 206:97-112
Wu, Yicong; Wawrzusin, Peter; Senseney, Justin et al. (2013) Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat Biotechnol 31:1032-8
Kanchanawong, Pakorn; Waterman, Clare M (2013) Localization-based super-resolution imaging of cellular structures. Methods Mol Biol 1046:59-84
Fischer, Robert S; Myers, Kenneth A; Gardel, Margaret L et al. (2012) Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior. Nat Protoc 7:2056-66
Myers, Kenneth A; Applegate, Kathryn T; Danuser, Gaudenz et al. (2011) Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis. J Cell Biol 192:321-34
Fischer, Robert S; Gardel, Margaret; Ma, Xuefei et al. (2009) Local cortical tension by myosin II guides 3D endothelial cell branching. Curr Biol 19:260-5