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. 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 is being revised for resubmission to journal of cell biology this is a collaboration wit gaudenz danuser and kathryn applegate. some of the goals of this project are worked on by ken under the microtuybules and actin interaction work.
