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Actin 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. [ABSTRACT FROM AUTHOR]

Background. The gene encoding cortactin, CTTN (locus 11q13), an actin-binding substrate of Src kinases, is frequently amplified in breast and head and neck squamous cell carcinomas (HNSCC) and cortactin overexpression is thought to contribute in a significant way to the invasive phenotype of these tumors. Elevated Epidermal Growth Factor receptor (EGFR) expression is also commonly observed in HNSCC and has been associated with poor prognosis and resistance to cytotoxic agents, including ionizing radiation. It has been suggested that cortactin overexpression may increase EGFR levels in these tumors by affecting receptor downregulation, however we recently found by multivariate analysis, that cortactin expression status remained an independent prognostic factor for local recurrence, disease-free survival, and overall survival. Material and Methods. To examine the potential link between cortactin overexpression and EGFR status, we compared cortactin and EGFR levels in a series of tumor lines derived from HNSCC. RNAi-mediated silencing was performed in cortactin overexpressing cells and in vivo tumoral potential with respect to cortactin and EGFR status was analyzed. Results and Discussion. Cortactin and EGFR levels were not strictly coupled in these lines and cortactin depletion did not decrease steady state receptor levels, although it did affect the epithelial to mesenchymal phenotypic conversion of cells. These results, together with clinical findings point to the existence of an EGFR-independent role of cortactin in HNSCC that may have important implications regarding the design of targeted therapies to combat tumor spread. [ABSTRACT FROM AUTHOR]

Integrin-linked kinase (ILK) and cytoplasmic adaptors of the PINCH and parvin families form a ternary complex, termed IPP, that localizes to integrin adhesions. We show here that deletion of the genes encoding ILK or PINCH1 similarly blocks maturation of focal adhesions to tensin-rich and phosphotyrosine-poor fibrillar adhesions (FBs) by downregulating expression or recruitment of tensin and destabilizing α5β1-integrin-cytoskeleton linkages. As IPP components are interdependent for integrin targeting and protein stability, functional dissection of the complex was achieved by fusing ILK, PINCH, parvin or their individual motifs to the cytoplasmic tail of β3 integrin, normally excluded from FBs. Using this novel gain-of-function approach, we demonstrated that expression of the C-terminal kinase domain of ILK can restore tensin recruitment and prompt focal-adhesion maturation in IPP-null cells. Debilitating mutations in the paxillin- or ATP-binding sites of ILK, together with α-parvin silencing, revealed a determinant role for ILK-parvin association, but not for direct paxillin binding, in this function. We propose a model in which the C-terminal domain of ILK promotes integrin sorting by reinforcing α5β1-integrin-actin linkage and controls force transmission by targeting tensin to maturing adhesions. [ABSTRACT FROM AUTHOR]

Summary During blood vessel formation, endothelial cells (ECs) establish cell-cell junctions and rearrange to form multicellular tubes. Here, we show that during lumen formation, the actin nucleator and elongation factor, formin-like 3 (fmnl3), localizes to EC junctions, where filamentous actin (F-actin) cables assemble. Fluorescent actin reporters and fluorescence recovery after photobleaching experiments in zebrafish embryos identified a pool of dynamic F-actin with high turnover at EC junctions in vessels. Knockdown of fmnl3 expression, chemical inhibition of formin function, and expression of dominant-negative fmnl3 revealed that formin activity maintains a stable F-actin content at EC junctions by continual polymerization of F-actin cables. Reduced actin polymerization leads to destabilized endothelial junctions and consequently to failure in blood vessel lumenization and lumen instability. Our findings highlight the importance of formin activity in blood vessel morphogenesis. [ABSTRACT FROM AUTHOR]