Endothelial cells show surprising cell rearrangement behaviour during angiogenic sprouting; however, the underlying mechanisms and functional importance remain unclear. By combining computational modelling with experimentation, we identify that Notch/VEGFR-regulated differential dynamics of VE-cadherin junctions drive functional endothelial cell rearrangements during sprouting. We propose that continual flux in Notch signalling levels in individual cells results in differential VE-cadherin turnover and junctional-cortex protrusions, which powers differential cell movement. In cultured endothelial cells, Notch signalling quantitatively reduced junctional VE-cadherin mobility. In simulations, only differential adhesion dynamics generated long-range position changes, required for tip cell competition and stalk cell intercalation. Simulation and quantitative image analysis on VE-cadherin junctional patterning in vivo identified that differential VE-cadherin mobility is lost under pathological high VEGF conditions, in retinopathy and tumour vessels. Our results provide a mechanistic concept for how cells rearrange during normal sprouting and how rearrangement switches to generate abnormal vessels in pathologies.

SUMMARYActin filaments are instrumental in driving processes such as migration, cytokinesis and endocytosis and provide cells with mechanical support. During angiogenesis, actin-rich filopodia protrusions have been proposed to drive endothelial tip cell functions by translating guidance cues into directional migration and mediating new contacts during anastomosis. To investigate the structural organisation, dynamics and functional importance of F-actin in endothelial cells (ECs) during angiogenesis in vivo, we generated a transgenic zebrafish line expressing Lifeact-EGFP in ECs. Live imaging identifies dynamic and transient F-actin-based structures, such as filopodia, contractile ring and cell cortex, and more persistent F-actin-based structures, such as cell junctions. For functional analysis, we used low concentrations of Latrunculin B that preferentially inhibited F-actin polymerisation in filopodia. In the absence of filopodia, ECs continued to migrate, albeit at reduced velocity. Detailed morphological analysis reveals that ECs generate lamellipodia that are sufficient to drive EC migration when filopodia formation is inhibited. Vessel guidance continues unperturbed during intersegmental vessel development in the absence of filopodia. Additionally, hypersprouting induced by loss of Dll4 and attraction of aberrant vessels towards ectopic sources of Vegfa165 can occur in the absence of endothelial filopodia protrusion. These results reveal that the induction of tip cells and the integration of endothelial guidance cues do not require filopodia. Anastomosis, however, shows regional variations in filopodia requirement, suggesting that ECs might rely on different protrusive structures depending on the nature of the environment or of angiogenic cues.

Interactions between tumor cells and the ECM strongly influence tumor development, affecting cell proliferation and survival, as well as the ability to migrate beyond the original location into other tissues to form metastases. Many of these interactions are mediated by integrins, a ubiquitously expressed family of adhesion receptors. Integrins are essential for cell attachment and control cell migration, cell cycle progression, and programmed cell death, responses that they regulate in synergy with other signal transduction pathways.This large group of transmembrane proteins is formed from 18 α and 8 β subunits, which dimerize to yield at least 24 different integrin heterodimers, each with distinct ligand binding and signaling properties. With their extracellular domain, integrins can bind to different ECM molecules, such as collagens and laminins, or to cellular receptors, such as VCAM-1. Their intracellular domains connect directly or indirectly to the actin cytoskeleton, thus linking the cytoskeleton to the ECM. Integrins also serve as bidirectional signaling receptors, inducing changes in protein activities or gene expression in response to ligand binding, while also modulating adhesive affinity on the cell surface in response to changes in cellular physiology. Here, we describe how integrins affect migration, proliferation, and survival of both transformed and normal cells, and we discuss how they modulate invasive growth in vivo. Throughout, we stress that many of these functions are restricted to particular cell types and may be altered upon transformation. Integrin avidity and affinity changes in cell migrationCell migration is essential not only for tissue infiltration and the formation of metastases, but also for nonpathological processes, such as angiogenesis and leukocyte extravasation. In order to migrate, a cell has to pass through a sequence of distinct processes. Migration is initiated by cell polarization and the formation of membrane protrusions at the leading edge. Integrins fix cellular protrusions to the ECM, interact with the actin cytoskeleton, and trigger the association of many different signaling molecules at the so-called focal contacts. Thereafter, integrin signals stimulate cell contraction, which allows the movement of the cell body on the adhesive contacts. Finally, the rear of the cell detaches from the substratum by inactivation of the integrins and disassembly of the adhesion complexes. Integrins further facilitate cell movement through the tissue by activating ECM-degrading enzymes. Although these basic mechanisms are considered to be similar among the various types of migratory cells, there are also clear distinctions, since, for example, fibroblasts are about 3-20 times more adhesive than nonactivated leukocytes, move 10-60 times more slowly, and have a different cytoskeletal organization. These differences in the migration mechanism might explain at least some of the known cell-specific effects of integrins on cell migration.Integrin-mediated cell attachment depends not jus...

Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning.DOI: http://dx.doi.org/10.7554/eLife.12167.001

Tumor cell resistance to ionizing radiation and chemotherapy is a major obstacle in cancer therapy. One factor contributing to this is integrin-mediated adhesion to ECM. The adapter protein particularly interesting new cysteine-histidine-rich 1 (PINCH1) is recruited to integrin adhesion sites and promotes cell survival, but the mechanisms underlying this effect are not well understood. Here we have shown that PINCH1 is expressed at elevated levels in human tumors of diverse origins relative to normal tissue. Furthermore, PINCH1 promoted cell survival upon treatment with ionizing radiation in vitro and in vivo by perpetuating Akt1 phosphorylation and activity. Mechanistically, PINCH1 was found to directly bind to protein phosphatase 1α (PP1α) -an Akt1-regulating protein -and inhibit PP1α activity, resulting in increased Akt1 phosphorylation and enhanced radioresistance. Thus, our data suggest that targeting signaling molecules such as PINCH1 that function downstream of focal adhesions (the complexes that mediate tumor cell adhesion to ECM) may overcome radio-and chemoresistance, providing new therapeutic approaches for cancer.