Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility.     

Through metal binding, curcumin protects against lead- and cadmium-induced lipid peroxidation in rat brain homogenates and against lead-induced tissue damage in rat brain

Curcumin, the major constituent of turmeric is a known, naturally occurring antioxidant. The present study examined the ability of this compound to protect against lead-induced damage to hippocampal cells of male Wistar rats, as well as lipid peroxidation induced by lead and cadmium in rat brain homogenate. The thiobarbituric assay (TBA) was used to measure the extent of lipid peroxidation induced by lead and cadmium in rat brain homogenate. The results show that curcumin significantly protects against lipid peroxidation induced by both these toxic metals. Coronal brain sections of rats injected intraperitoneally with lead acetate (20 mg/kg) in the presence and absence of curcumin (30 mg/kg) were compared microscopically to determine the extent of lead-induced damage to the cells in the hippocampal CA1 and CA3 regions, and to establish the capacity of curcumin to prevent such damage. Lead-induced damage to the neurons was significantly curtailed in the rats injected with curcumin. Possible chelation of lead and cadmium by curcumin as its mechanism of neuroprotection against such heavy metal insult to the brain was investigated using electrochemical, ultraviolet spectrophotometric and infrared spectroscopic analyses. The results of the study show that there is an interaction between curcumin and both cadmium and lead, with the possible formation of a complex between the metal and this ligand. These results imply that curcumin could be used therapeutically to chelate these toxic metals, thus potentially reducing their neurotoxicity and tissue damage.     

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

Background  

Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.     

ARNO through its coiled-coil domain regulates endocytosis at the apical surface of polarized epithelial cells

ARNO is a guanine-nucleotide exchange protein for the ARF family of GTPases. Here we show that in polarized epithelial cells, ARNO is localized exclusively to the apical plasma membrane, where it regulates endocytosis. Expression of ARNO stimulates apical endocytosis of the polymeric immunoglobulin receptor, and coexpression of ARF6 with ARNO leads to a synergistic stimulation of apical endocytosis. Expression of a dominant negative ARF6 mutant, ARF6-T27N, antagonizes this stimulatory effect. Deletion of the N-terminal coiled-coil (CC) domain of ARNO causes the mutant ARNO to localize to both the apical and basolateral plasma membranes. Expression of the CC domain alone abolishes ARNO-induced apical endocytosis as well as co-localization of IgA-receptor complexes with ARNO and clathrin. These results suggest that the CC domain contributes to the specificity of apical localization of ARNO through association with components of the apical plasma membrane. We conclude that ARNO acts together with ARF6 to regulate apical endocytosis.     

Chlamydomonas reinhardtii has a small family of purple acid phosphatase homologue genes that are differentially expressed in response to phytate

Purple acid phosphatases (PAPs) are metallophosphoesterase enzymes involved in the acquisition and recycling of phosphorus. PAP phytases from microorganisms and plants are responsible for the dephosphorylation of phytate. Phytate is the main storage form of phosphorus in plant seeds and constitutes the major form of organic phosphorus present in soil. Although some phosphatases have been studied in Chlamydomonas reinhardtii, no gene coding for PAPs have so far been characterized. In this study, six PAP homologue genes were identified and characterized in silico in C. reinhardtii (CrPAP1 to CrPAP6). A metallophosphoesterase domain including the seven conserved residues characteristic of PAP enzymes was found in all six CrPAPs. The phylogenetic tree comprising PAP homologue sequences from microalgae, plants, and animals showed nine major clades and CrPAPs resolved in four of them. A constitutive expression was found for CrPAP2, CrPAP3, CrPAP4, and CrPAP6 in all media tested, while CrPAP1 and CrPAP5 were induced by the addition of phytate in a medium without phosphate salts. Our results provide a starting point for further functional analysis of the CrPAP gene family, and the evaluation of their potential as phytases in C. reinhardtii.     

Structural and phylogenetic analysis of Pto-type disease resistance gene candidates in banana

The tomato Pto gene encodes a serine/threonine kinase (STK) whose molecular characterization has provided valuable insights into the disease resistance mechanism of tomato and it is considered as a promising candidate for engineering broad-spectrum pathogen resistance in this crop. In this study, a pair of degenerate primers based on conserved subdomains of plant STKs similar to the tomato Pto protein was used to amplify similar sequences in banana. A fragment of approximately 550 bp was amplified, cloned and sequenced. The sequence analysis of several clones revealed 13 distinct sequences highly similar to STKs. Based on their significant similarity with the tomato Pto protein (BLASTX E value <3e-53), seven of them were classified as Pto resistance gene candidates (Pto-RGCs). Multiple sequence alignment of the banana Pto-RGC products revealed that these sequences contain several conserved subdomains present in most STKs and also several conserved residues that are crucial for Pto function. Moreover, the phylogenetic analysis showed that the banana Pto-RGCs were clustered with Pto suggesting a common evolutionary origin with this R gene. The Pto-RGCs isolated in this study represent a valuable sequence resource that could assist in the development of disease resistance in banana.     

ACRP30 is secreted from 3T3-L1 adipocytes via a Rab11-dependent pathway

Adipocytes are now known to secrete a range of adipokines that exhibit distinct biological functions. Here, we sought to understand the secretory pathways utilised by ACRP30 to the surface of adipocytes. We find that ACRP30 overlaps with adipsin in intracellular compartments distinct from Glut4, but nonetheless exhibits insulin-stimulated secretion from cells. Both adipsin and ACRP30 overlap with transferrin receptor-positive membranes, implying that the pathway of secretion involves the transferrin receptor-positive endosomal system. Consistent with this, we show that ablation of endosomes significantly inhibited the secretion of ACRP30, as did treatment of cells with Brefeldin A. In order to further probe the role of recycling endosomes on the secretion of ACRP30, we over-expressed a mutant form of Rab11, Rab11-S25N, in 3T3-L1 adipocytes and found that expression of this mutant significantly reduced basal and insulin-stimulated secretion. We also demonstrate that Arf6 also plays a role in the secretion of ACRP30. Collectively, these data implicate both Arf6 and Rab11 as crucial mediators of constitutive and insulin-stimulated secretion of ACRP30 and further suggest that recycling endosomes may play a central role in this process.     

The DOCK180/Elmo complex couples ARNO-mediated Arf6 activation to the downstream activation of Rac1

Cell motility requires extensions of the plasma membrane driven by reorganization of the actin cytoskeleton. Small GTPases, particularly the Rho family, are key regulators of this process. A second class of GTPases, the ADP-ribosylation factors (ARFs), have also been implicated in the regulation of the actin cytoskeleton and motility. ARF6 is intimately involved in the regulation of Rac activity; however, the mechanisms by which ARF activation leads to activation of Rac remain poorly understood. We have previously shown that expression of the ARF-GEF ARNO in MDCK cells induces robust activation of Rac, the formation of large lamellipodia, and the onset of motility. We report here that ARNO-dependent activation of Rac is mediated by a bipartite Rac GEF, the Dock180/Elmo complex. Both DOCK180 and Elmo colocalize extensively with ARNO in migrating MDCK cells. Importantly, both a catalytically inactive Dock180 mutant and an Elmo mutant that fails to couple to Dock180 block ARNO-induced Rac activation and motility. In contrast, a similar mutant of the Rac GEF beta-PIX fails to inhibit ARNO-induced Rac activation or motility. Together, these data suggest that ARNO and ARF6 coordinate with the Dock180/Elmo complex to promote Rac activation at the leading edge of migrating cells.     

The NPR1 family of transcription cofactors in papaya: insights into its structure, phylogeny and expression

The NPR1 (non-expressor of pathogenesis related gene 1) gene was initially identified in Arabidopsis as a master regulator of the systemic acquired resistance (SAR). Five additional NPR1 homologues have been identified in Arabidopsis whose function range from regulators of SAR to plant development. In the present study, we characterized the structure, phylogeny and expression of the NPR1 family in papaya (Carica papaya L.), one of the most important tropical fruit crops. We identified four NPR1 homologues in the papaya genome sequence (CpNPR1 to CpNPR4). Overall, the four papaya predicted NPR1 proteins showed the characteristic BTB/POZ and ankyrin domains of the Arabidopsis NPR1 family. Twelve additional open reading frames showing homology to retrotransposon elements or genes involved in different physiological processes were found in close proximity to the papaya NPR1 homologues. The phylogenetic analysis revealed that the papaya NPR1 sequences resolved in three clades, each clade containing two Arabidopsis NPR1 homologues involved either in the positive regulation of SAR (clade I), negative regulation of SAR (clade II) or plant development (clade III), suggesting a similar function for the corresponding papaya NPR1 homologues. Furthermore, the expression of the four papaya NPR1 homologues was detected in both vegetative and reproductive tissues. The present study has provided the first comparative analysis of the NPR1 family in a tropical fruit crop and expanded our knowledge on this type of genes in dicotyledoneous plants. The identification of the full set of papaya NPR1 homologues will pave the way for their systematic functional analysis and new opportunities for engineering disease resistance in this crop.