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How do cells make efficient collective decisions during tissue morphogenesis? Humans and other organisms use feedback between movement and sensing known as 'sensorimotor coordination' or 'active perception' to inform behaviour, but active perception has not before been investigated at a cellular level within organs. Here we provide the first proof of concept in silico/in vivo study demonstrating that filopodia (actin-rich, dynamic, finger-like cell membrane protrusions) play an unexpected role in speeding up collective endothelial decisions during the time-constrained process of 'tip cell' selection during blood vessel formation (angiogenesis). We first validate simulation predictions in vivo with live imaging of zebrafish intersegmental vessel growth. Further simulation studies then indicate the effect is due to the coupled positive feedback between movement and sensing on filopodia conferring a bistable switch-like property to Notch lateral inhibition, ensuring tip selection is a rapid and robust process. We then employ measures from computational neuroscience to assess whether filopodia function as a primitive (basal) form of active perception and find evidence in support. By viewing cell behaviour through the 'basal cognitive lens' we acquire a fresh perspective on the tip cell selection process, revealing a hidden, yet vital time-keeping role for filopodia. Finally, we discuss a myriad of new and exciting research directions stemming from our conceptual approach to interpreting cell behaviour. This article is part of the theme issue 'Basal cognition: multicellularity, neurons and the cognitive lens'. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration. [ABSTRACT FROM AUTHOR]

Release of endothelial cells from contact-inhibition and cell cycle re-entry is required for the induction of new blood vessel formation by angiogenesis. Using a combination of chemical inhibition, loss of function, and gain of function approaches, we demonstrate that endothelial cell cycle re-entry, S phase progression, and subsequent angiogenic tubule formation are dependent upon the activity of cytosolic phospholipase A2-α (cPLA2α). Inhibition of cPLA2α activity and small interfering RNA (siRNA)-mediated knockdown of endogenous cPLA2α reduced endothelial cell proliferation. In the absence of cPLA2α activity, endothelial cells exhibited retarded progression from G1 through S phase, displayed reduced cyclin A/cdk2 expression, and generated less arachidonic acid. In quiescent endothehal cells, cPLA2α is inactivated upon its sequestration at the Golgi apparatus. Upon the stimulation of endothelial cell proliferation, activation of cPLA2α by release from the Golgi apparatus was critical to the induction of cyclin A expression and efficient cell cycle progression. Consequently, inhibition of cPLA2α was sufficient to block angiogenic tubule formation in vitro. Furthermore, the siRNA-mediated retardation of endothelial cell cycle re-entry and proliferation was reversed upon over- expression of an siRNA-resistant form of cPLA2α. Thus, activation of cPLA2α acts as a novel mechanism for the regulation of endothehial cell cycle re-entry, cell cycle progression, and angiogenesis. [ABSTRACT FROM AUTHOR]

Abstract: The association of cytosolic phospholipase A2-α (cPLA2α) with intracellular membranes is central to the generation of free arachidonic acid and thromboxane A2 in activated platelets. Despite this, the site and nature of this membrane association has not been fully characterised upon platelet activation. High resolution imaging showed that cPLA2α was distributed in a partly structured manner throughout the resting platelet. Upon glass activation or thrombin stimulation, cPLA2α relocated to a peripheral region corresponding to the platelet plasma membrane. Upon thrombin stimulation of platelets a major pool of cPLA2α was associated with the plasma membrane in an EGTA-resistant manner. EGTA-resistant membrane binding was abolished upon de-polymerisation of actin filaments by DNase I and furthermore, cPLA2α co-immunoprecipitated with actin upon thrombin stimulation of platelets. Immunofluorescence microscopy studies revealed that, upon platelet activation, cPLA2α and actin co-localised at the plasma membrane. Thus we have identified a novel mechanism for the interaction of cPLA2α with its membrane substrate via interaction with actin. [Copyright &y& Elsevier]

The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A2-α (cPLA2α) cleaves phospholipids in a Ca2+-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca2+-independent association of cPLA2α with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA2α and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA2α is associated with the phospholipid-binding protein annexin A1, Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA2α and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA2α activity and endothelial cell prostaglandin generation. [ABSTRACT FROM AUTHOR]

Arachidonic acid and its metabolites have been previously implicated in the regulation of endothelial cell proliferation. Arachidonic acid may be liberated from cellular phospholipids by the action of group VIA calcium-independent phospholipase A2 (iPLA2-VIA). Consequently, we tested the hypothesis that iPLA2-VIA activity is linked to the regulation of endothelial cell proliferation. Inhibition of iPLA2 activity by bromoenol lactone (BEL) was sufficient to entirely block endothelial cell growth. BEL dose-dependently inhibited endothelial cell DNA synthesis in a manner that was reversed upon the exogenous addition of arachidonic acid. DNA synthesis was inhibited by the S-isomer and not by the R-isomer of BEL, demonstrating that endothelial cell proliferation is mediated specifically by iPLA2-VIA. iPLA2-VIA activity was critical to the progression of endothelial cells through S phase and is required for the expression of the cyclin A/cdk2 complex. Thus, inhibition of iPLA2-VIA blocks S phase progression and results in exit from the cell cycle. Inhibition of iPLA2-VIA-mediated endothelial cell proliferation is sufficient to block angiogenic tubule formation in co-culture assays. Consequently, iPLA2-VIA is a novel regulator of endothelial cell S phase progression, cell cycle residence, and angiogenesis. [ABSTRACT FROM AUTHOR]

Cytosolic phospholipase A2-α (cPLA2-α) is a calcium-activated enzyme that plays an important role in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid can be converted subsequently into prostacyclin, a potent vasodilator and inhibitor of platelet activation, through the action of cyclooxygenase (COX) enzymes. Here we study the relocation of cPLA2-α in human EA.hy.926 endothelial cells following stimulation with the calcium-mobilizing agonist, A23187. Relocation of cPLA2-α was seen to be highly cell specific, and in EA.hy.926 cells occurred primarily to intracellular structures resembling the endoplasmic reticulum (ER) and Golgi. In addition, relocation to both the inner and outer surfaces of the nuclear membrane was observed. Colocalization studies with markers for these subcellular organelles, however, showed colocalization of cPLA2-α with nuclear membrane markers but not with ER or Golgi markers, suggesting that the relocation of cPLA2-α occurs to sites that are separate from these organelles. Colocalization with annexin V was also observed at the nuclear envelope, however, little overlap with staining patterns for the potential cPLA2-α interacting proteins, annexin I, vimentin, p11 or actin, was seen in this cell type. In contrast, cPLA2-α was seen to partially colocalize specifically with the COX-2 isoform at the ER-resembling structures, but not with COX-1. These studies suggest that cPLA2-α and COX-2 may function together at a distinct and novel compartment for eicosanoid signalling. [ABSTRACT FROM AUTHOR]