First record of lily thrips, Liothrips vaneeckei Priesner, in Australia (Thysanoptera: Phlaeothripidae)

The first Australian record of the lily thrips, Liothrips vaneeckei Priesner, is reported from a bulb farm in Warragul South, Victoria. It is an occasional pest of Lilium bulbs, both in the field and in storage, particularly in the USA and several European countries, and is also infrequently found in considerable numbers on the corms of orchids.     

The C-terminal cytoplasmic tail of claudins 1 and 5 but not its PDZ-binding motif is required for apical localization at epithelial and endothelial tight junctions

Claudins are a family of tetraspan transmembrane proteins that represent the major constituents of epithelial and endothelial tight junctions (TJs). They form TJ strands representing the major barrier regulating paracellular transport of solutes and water. Intracellularly, claudins are connected via a C-terminal PDZ-binding motif with several TJ-associated proteins containing PDZ domains. Although these interactions can provide a link to the actin cytoskeleton, they appear to be dispensable for the TJ localization of claudins. To identify TJ-targeting elements in the C-terminal cytoplasmic domains of the claudins 1 and 5, we generated a series of C-terminal deletion mutants and analyzed their distribution in polarized epithelial (MDCK) and endothelial (HMEC-1) cells. TJ localization was revealed by establishing an in vivo cross-linking approach that stabilized claudin-TJ interactions. We show that residues located C-terminal to the last transmembrane domain are required for the proper targeting to apical TJ.s. While claudin derivatives lacking only the very C-terminal PDZ-binding motif continue to localize to TJs, mutants lacking the entire C-terminal juxtamembrane sequence do not associate with TJs and accumulate in intracellular structures. This indicates that crucial determinants for stable TJ incorporation of claudins reside in a cytoplasmic C-terminal sequence which up to now has not been implicated in specific protein-protein interactions.     

Rab3D and annexin A2 play a role in regulated secretion of vWF, but not tPA, from endothelial cells

von-Willebrand factor (vWF) and tissue-type plasminogen activator (tPA) are products of endothelial cells acutely released into the vasculature following cell activation. Both factors are secreted after intraendothelial Ca2+ mobilization, but exhibit opposing physiological effects with vWF inducing coagulation and tPA triggering fibrinolysis. To identify components that could regulate differentially the release of pro- and antithrombogenic factors, we analyzed the contribution of Rab3D and the annexin A2/S100A10 complex, proteins implicated in exocytotic events in other systems. We show that mutant Rab3D proteins interfere with the formation of bona fide Weibel-Palade bodies (WPbs), the principal storage granules of multimeric vWF, and consequently the acute, histamine-induced release of vWF. In contrast, neither appearance nor exocytosis of tPA storage granules is affected. siRNA-mediated downregulation of annexin A2/S100A10 and disruption of the complex by microinjection of peptide competitors result in a marked reduction in vWF but not tPA secretion, without affecting the appearance of WPbs. This indicates that distinct mechanisms underlie the acute secretion of vWF and tPA, enabling endothelial cells to fine-regulate the release of thrombogenic and fibrinolytic factors.     

Histidine phosphorylation of annexin I in airway epithelia

Although [Cl(-)](i) regulates many cellular functions including cell secretion, the mechanisms governing these actions are not known. We have previously shown that the apical membrane of airway epithelium contains a 37-kDa phosphoprotein (p37) whose phosphorylation is regulated by chloride concentration. Using metal affinity (chelating Fe(3+)-Sepharose) and anion exchange (POROS HQ 20) chromatography, we have purified p37 from ovine tracheal epithelia to electrophoretic homogeneity. Sequence analysis and immunoprecipitation using monoclonal and specific polyclonal antibodies identified p37 as annexin I, a member of a family of Ca(2+)-dependent phospholipid-binding proteins. Phosphate on [(32)P]annexin I, phosphorylated using both [gamma-(32)P]ATP and [gamma-(32)P]GTP, was labile under acidic but not alkaline conditions. Phosphoamino acid analysis showed the presence of phosphohistidine. The site of phosphorylation was localized to a carboxyl-terminal fragment of annexin I. Our data suggest that cAMP and AMP (but not cGMP) may regulate annexin I histidine phosphorylation. We propose a role for annexin I in an intracellular signaling system involving histidine phosphorylation.     

X-ray structure of full-length annexin 1 and implications for membrane aggregation

Annexins comprise a multigene family of Ca2+ and phospholipid- binding proteins. They consist of a conserved C-terminal or core domain that confers Ca2+-dependent phospholipid binding and an N-terminal domain that is variable in sequence and length and responsible for the specific properties of each annexin. Crystal structures of various annexin core domains have revealed a high degree of similarity. From these and other studies it is evident that the core domain harbors the calcium-binding sites that interact with the phospholipid headgroups. However, no structure has been reported of an annexin with a complete N-terminal domain. We have now solved the crystal structure of such a full-length annexin, annexin 1. Annexin 1 is active in membrane aggregation and its refined 1.8 A structure shows an alpha-helical N-terminal domain connected to the core domain by a flexible linker. It is surprising that the two alpha-helices present in the N-terminal domain of 41 residues interact intimately with the core domain, with the amphipathic helix 2-12 of the N-terminal domain replacing helix D of repeat III of the core. In turn, helix D is unwound into a flap now partially covering the N-terminal helix. Implications for membrane aggregation will be discussed and a model of aggregation based on the structure will be presented.     

Ca2+ -regulated secretion of tissue-type plasminogen activator and von Willebrand factor in human endothelial cells

von Willebrand factor (vWF) and tissue-type plasminogen activator (tPA) are products of endothelial cells which are secreted into the bloodstream upon a stimulus-induced rise in intracellular Ca(2+). Although the release of both factors appears to be regulated similarly, they exhibit opposing physiological effects in the vasculature with vWF inducing coagulation and platelet aggregation and tPA triggering fibrinolysis and thrombolysis. To analyze possible differences in the regulated secretion of vWF and tPA in more detail, we recorded the Ca(2+)-triggered exocytosis of both factors in cultured human endothelial cells. We demonstrate that vWF and tPA which are stored in different granules within endothelial cells are released with different kinetics following endothelial stimulation with histamine or the Ca(2+) ionophore A23187. While the stimulus-induced release of vWF increases with time over a course of 30 min, maximal acute secretion of tPA is observed 5 min following stimulation and subsequently drops to background levels. In the case of vWF, secretion can also be monitored indirectly through an antibody-reinternalization assay which indicates an incomplete release of vWF during single exocytotic fusion events. Our data thus point to differences in the Ca(2+)-triggered secretion of vWF and tPA which could allow a fine-tuning of their release thereby ensuring a balanced physiological action.     

Association of annexin 2 with recycling endosomes requires either calcium- or cholesterol-stabilized membrane domains

Annexin 2 is a Ca2+- and phospholipid-binding protein previously identified on endosomal membranes and the plasma membrane. Inferred from this location and its stimulatory effect on membrane transport annexin 2 has been proposed to play a role in the structural organization and dynamics of endosomal membranes. Validation of this view requires a detailed analysis of the distribution of annexin 2 over the endosomal compartment and a characterization of the parameters governing this distribution. Towards this end we have devised an immunoisolation protocol to purify annexin 2-positive membrane vesicles from subcellular fractions of BHK cells containing early endosomes. We show that this approach leads to the isolation of intact endosomal vesicles containing internalized fluid-phase marker and that the immunoisolated membranes are positive for the transferrin receptor and Rab4 but not for the early endosomal antigen EEA1. A distinct and non-uniform distribution of annexin 2 over the early endosomal compartment is also observed in immunoelectron microscopy analyses of whole-mount specimens of BHK cells. Annexin 2 antibodies labeled transferrin receptor-containing tubular early endosomal structures, but not EEAl-positive endosomal vacuoles. We also observed that the Ca2+-independent association of annexin 2 with endosomal membranes was disrupted by the cholesterol-binding glycerid saponin, while Ca2+ could trigger annexin 2 binding to saponin-treated endosomal membranes. Thus, either Ca2+- or cholesterol-stabilized membrane domains are required for the binding of annexin 2 to endosomes suggesting that both factors may regulate this interaction.