Tuning the endothelial response: differential release of exocytic cargos from Weibel-Palade bodies.

Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation. The molecules that contribute to these processes are co-stored in secretory granules. How can the cells control release of granule content to allow differentiated responses? Selected agonists recruit an exocytosis-linked actin ring to boost release of a subset of cargo. SUMMARY: Background Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease.

The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence.

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH(2)- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.

Comparison of the glycosyl-phosphatidylinositol cleavage/attachment site between mammalian cells and parasitic protozoa.

It was previously hypothesised that the requirements for glycosyl-phosphatidylinositol (GPI) anchoring in mammalian cells and parasitic protozoa are similar but not identical. We have investigated this by converting the GPI cleavage/attachment site in porcine membrane dipeptidase to that found in the trypanosomal variant surface glycoprotein 117 and expressing the resulting mutants in COS-1 cells. Changing the entire (omega), (omega)+1 and (omega)+2 triplet in membrane dipeptidase from Ser-Ala-Ala to Asp-Ser-Ser resulted in efficient GPI anchoring of the mutant proteins, as assessed by cell-surface activity assays and susceptibility to release by phosphatidylinositol-specific phospholipase C. Immunoelectrophoretic blot analysis with antibodies recognising epitopes either side of the native (omega) residue in porcine membrane dipeptidase, and expression of a mutant in which potential alternative cleavage/attachment sites were disrupted, indicated that alternative GPI cleavage/attachment sites had not been used. These results indicate that the requirements for GPI anchoring between mammalian and protozoal cells are not as different as previously suggested, and that rules for predicting the probability of a sequence acting as a GPI cleavage/attachment site need to be applied with caution.

Intrinsic pinning of vorticity by domain walls of l texture in superfluid 3He-A.

We present the first observation of substantial persistent flow in superfluid 3He-A in thick simply connected slabs in a zero magnetic field, but only in l textures with domain walls. The flow is induced in a rotating cryostat using a torsional oscillator as a probe. The hysteretic dependences of the trapped vorticity on the maximal angular velocity of rotation are fairly universal for different densities of domain walls and slab thicknesses. A model of a critical state set by either the critical velocity for vortex nucleation or pinning strength explains all observations.

A continuous fluorometric assay for leukotriene D4 hydrolase.

A fluorogenic substrate for assay of leukotriene D4 hydrolase (LTDase; EC 3.4.13.19) has been prepared and evaluated, using enzyme purified from porcine kidney. The compound is based on internal quenching of the synthetic, fluorescent amino acid d, l-2-amino-3-(7-methoxy-4-coumaryl)propanoic acid (d,l-Amp) by a 2, 4-dinitrophenyl (DNP) group. The compound is epsilon-DNP-l-Lys-d-Amp which incorporates the D-isomer of Amp to exploit the unique ability among mammalian peptidases for LTDase to hydrolyze peptides containing a d-amino acid in the C-terminal position. epsilon-DNP-l-Lys-d-Amp was found to be an excellent substrate for LTDase, with Km value of 370 microoffUnder the conditions of assay, the substrate was without noticeable quenching effect on the fluorescence of the product (d-Amp) liberated by the action of LTDase. Using porcine kidney microvillar membranes, which contain a battery of peptidases, the specific inhibitor of LTDase, cilastatin, completely inhibited the breakdown of epsilon-DNP-l-Lys-d-Amp, indicating that the substrate is selective for LTDase.