A "cell-free" system to study regulation of focal adhesions and of the connected actin cytoskeleton.

The exact composition of focal adhesions and the role of single components in their assembly and regulation during dynamic adhesive processes are poorly understood. I have characterised and used a "cell-free" system consisting of ventral plasma membranes (VPMs) prepared from adherent fibroblasts, and found that this subcellular fraction preserves intact focal adhesion components and the connected actin cytoskeleton. Moreover, VPMs show an accumulation of several tyrosine phosphorylated proteins, including tyrosine phosphorylated focal adhesion kinase and paxillin. In particular, a pool of higly phosphorylated paxillin is found in focal adhesions, suggesting an important role for the phosphorylated polypeptide in the mechanism of integrin-mediated adhesion. With the aim of obtaining new important tools for my studies, I have produced and characterised monoclonal antibodies (mAbs) raised against VPM preparations. The use of VPM preparations as a "cell-free" system has shown that changes in [Ca2+] can affect integrin behaviour within VPMs. I observed a correlation between integrin localisation and the functional state of the receptors, which can be reversibly modulated either by changes in free [Ca2+], or by function modulating anti-integrin pi mAbs. [Ca2+]-induced integrin redistribution is dependent on the presence of the pi cytoplasmic domain, whereas it is independent from the presence of filamentous actin (F- actin) and focal adhesion components in this experimental system, thus implicating the uncoupling of events relevants for focal adhesion assembly under "cell-free" conditions. Moreover, reconstitution experiments show that a-actinin colocalises and redistributes with pi receptors on VPMs depleted of actin, implicating binding of a-actinin to the receptors. Finally, I found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerisation is inhibited. These data attest the value of the system for further analysis of the molecular mechanisms regulating integrin function and focal adhesions

β-Catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse

During heart development endocardial cells within the atrio-ventricular (AV) region undergo TGFβ-dependent epithelial-mesenchymal transformation (EMT) and invade the underlying cardiac jelly. This process gives rise to the endocardial cushions from which AV valves and part of the septum originate. In this paper we show that in mouse embryos and in AV explants TGFβ induction of endocardial EMT is strongly inhibited in mice deficient for endothelial β-catenin, leading to a lack of heart cushion formation. Using a Wnt-signaling reporter mouse strain, we demonstrated in vivo and ex vivo that EMT in heart cushion is accompanied by activation of β-catenin/TCF/Lef transcriptional activity. In cultured endothelial cells, TGFβ2 induces α-smooth muscle actin (αSMA) expression. This process was strongly reduced in β-catenin null cells, although TGFβ2 induced smad phosphorylation was unchanged. These data demonstrate an involvement of β-catenin/TCF/Lef transcriptional activity in heart cushion formation, and suggest an interaction between TGFβ and Wnt-signaling pathways in the induction of endothelial-mesenchymal transformation

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, β-catenin, and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in β-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. β-Catenin–null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density–enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin–β-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell–cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin–null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor

The conditional inactivation of the β-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility

Using the Cre/loxP system we conditionally inactivated β-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured β-catenin −/− endothelial cells showed a different organization of intercellular junctions with a decrease in α-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell–cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of β-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages

Complementary resource use by tree species in a rain forest tree plantation

Mixed-species tree plantations, composed of high-value native rain forest timbers, are potential forestry systems for the subtropics and tropics that can provide ecological and production benefits. Choices of rain forest tree species for mixtures are generally based on the concept that assemblages of fast-growing and light-demanding species are less productive than assemblages of species with different shade tolerances. We examined the hypothesis that mixtures of two fast-growing species compete for resources, while mixtures of shade-tolerant and shade-intolerant species are complementary. Ecophysiological characteristics of young trees were determined and analyzed with a physiology-based canopy model (MAESTRA) to test species interactions. Contrary to predictions, there was evidence for complementary interactions between two fast-growing species with respect to nutrient uptake, nutrient use efficiency, and nutrient cycling. Fast-growing Elaeocarpus angustifolius had maximum demand for soil nutrients in summer, the most efficient internal recycling of N, and low P use efficiency at the leaf and whole-plant level and produced a large amount of nutrient-rich litter. In contrast, fast-growing Grevillea robusta had maximum demand for soil nutrients in spring and highest leaf nutrient use efficiency for N and P and produced low-nutrient litter. Thus, mixtures of fast-growing G. robusta and E. angustifolius or G. robusta and slow-growing, shade-tolerant Castanospermum australe may have similar or even greater productivity than monocultures, as light requirement is just one of several factors affecting performance of mixed-species plantations. We conclude that the knowledge gained here will be useful for designing large-scale experimental mixtures and commercial forestry systems in subtropical Australia and elsewhere

Preferential localization of tyrosine-phosphorylated paxillin in focal adhesions

Focal adhesions are sites for integrin-mediated attachment of cultured cells to the extracellular matrix. Localization studies have shown that focal adhesions can be stained by antiphosphotyrosine antibodies, but the role of tyrosine-phosphorylated proteins in focal adhesions is not known. By using ventral plasma membranes prepared from chicken embryo fibroblasts spread on the substrate, we present evidence for the preferential localization of a minor pool of tyrosine-phosphorylated paxillin in focal adhesions. Ventral plasma membranes showed an enrichment in β1-integrins, and in several tyrosine-phosphorylated polypeptides, while focal adhesion proteins like vinculin and paxillin, although localized to focal adhesions in ventral plasma membranes, were not particularly enriched in these preparations compared to whole cell lysates. Biochemical and morphological analysis of ventral plasma membranes showed a dramatic increase in the level of tyrosine-phosphorylation of the pool of paxillin localized to the adhesive sites, when compared to the paxillin present in whole cell lysates. The observed preferential localization of tyrosine-phosphorylated paxillin to focal adhesions may represent a general mechanism to compartmentalize focal adhesion components from large non-phosphorylated, cytosolic pools