Tip-end fusion of a rod-shaped secretory organelle.

Weibel-Palade bodies (WPB) are elongated, rod-like secretory organelles unique to endothelial cells that store the pro-coagulant von-Willebrand factor (VWF) and undergo regulated exocytosis upon stimulation with Ca2+- or cAMP-raising agonists. We show here that WPB preferentially initiate fusion with the plasma membrane at their tips and identify synaptotagmin-like protein 2-a (Slp2-a) as a positive regulator of VWF secretion most likely mediating this topological selectivity. Following secretagogue stimulation, Slp2-a accumulates at one WPB tip before fusion occurs at this site. Depletion of Slp2-a reduces Ca2+-dependent secretion of highly multimeric VWF and interferes with the formation of actin rings at WPB-plasma membrane fusion sites that support the expulsion of the VWF multimers and most likely require a tip-end fusion topology. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] binding via the C2A domain of Slp2-a is required for accumulation of Slp2-a at the tip ends of fusing WPB, suggesting that Slp2-a mediates polar exocytosis by initiating contacts between WPB tips and plasma membrane PI(4,5)P2.

Induction of Ca2+-Dependent Exocytotic Processes by Laser Ablation of Endothelial Cells.

Ca2+ regulates a variety of cellular processes that are essential to maintain cell integrity and function. Different methods have been used to study these processes by increasing intracellular Ca2+ levels. Here, we describe a protocol to initiate Ca2+-dependent membrane-related events, using laser ablation by near-infrared irradiation. This creates a rupture in the plasma membrane that allows the extracellular Ca2+ to enter the cell and thereby induce a receptor-independent Ca2+ increase. We report laser ablation protocols to study two different Ca2+-induced processes in human endothelial cells-membrane resealing and exocytosis of secretory granules called Weibel-Palade bodies (WPBs). Thus, laser ablation represents a technique that permits the analysis of different Ca2+-regulated processes at high spatiotemporal resolution in a controlled manner.

Plasma membrane phosphatidylinositol (4,5)-bisphosphate promotes Weibel-Palade body exocytosis.

Weibel-Palade bodies (WPB) are specialized secretory organelles of endothelial cells that control vascular hemostasis by regulated, Ca2+-dependent exocytosis of the coagulation-promoting von-Willebrand factor. Some proteins of the WPB docking and fusion machinery have been identified but a role of membrane lipids in regulated WPB exocytosis has so far remained elusive. We show here that the plasma membrane phospholipid composition affects Ca2+-dependent WPB exocytosis and von-Willebrand factor release. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] becomes enriched at WPB-plasma membrane contact sites at the time of fusion, most likely downstream of phospholipase D1-mediated production of phosphatidic acid (PA) that activates phosphatidylinositol 4-phosphate (PI4P) 5-kinase γ. Depletion of plasma membrane PI(4,5)P2 or down-regulation of PI4P 5-kinase γ interferes with histamine-evoked and Ca2+-dependent WPB exocytosis and a mutant PI4P 5-kinase γ incapable of binding PA affects WPB exocytosis in a dominant-negative manner. This indicates that a unique PI(4,5)P2-rich environment in the plasma membrane governs WPB fusion possibly by providing interaction sites for WPB-associated docking factors.

Annexins and plasma membrane repair.

Plasma membrane wound repair is a cell-autonomous process that is triggered by Ca2+ entering through the site of injury and involves membrane resealing, i.e., re-establishment of a continuous plasma membrane, as well as remodeling of the cortical actin cytoskeleton. Among other things, the injury-induced Ca2+ elevation initiates the wound site recruitment of Ca2+-regulated proteins that function in the course of repair. Annexins are a class of such Ca2+-regulated proteins. They associate with acidic phospholipids of cellular membranes in their Ca2+ bound conformation with Ca2+ sensitivities ranging from the low to high micromolar range depending on the respective annexin protein. Annexins accumulate at sites of plasma membrane injury in a temporally controlled manner and are thought to function by controlling membrane rearrangements at the wound site, most likely in conjunction with other repair proteins such as dysferlin. Their role in membrane repair, which has been evidenced in several model systems, will be discussed in this chapter.

A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel-Palade body exocytosis in endothelial cells.

Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated WPB exocytosis critically depends on their proper recruitment to the plasma membrane, but factors involved in WPB-plasma membrane tethering are not known. Here we identify Munc13-4, a protein mutated in familial hemophagocytic lymphohistiocytosis 3, as a WPB-tethering factor. Munc13-4 promotes histamine-evoked WPB exocytosis and is present on WPBs, and secretagogue stimulation triggers an increased recruitment of Munc13-4 to WPBs and a clustering of Munc13-4 at sites of WPB-plasma membrane contact. We also identify the S100A10 subunit of the annexin A2 (AnxA2)-S100A10 protein complex as a novel Munc13-4 interactor and show that AnxA2-S100A10 participates in recruiting Munc13-4 to WPB fusion sites. These findings indicate that Munc13-4 supports acute WPB exocytosis by tethering WPBs to the plasma membrane via AnxA2-S100A10.

Annexin A8 promotes VEGF-A driven endothelial cell sprouting.

The physiological and pathological process of angiogenesis relies on orchestrated endothelial cell (EC) adhesion, migration and formation of new vessels. Here we report that human umbilical vein endothelial cells (HUVECs) deficient in Annexin A8 (AnxA8), a member of the annexin family of Ca2+- and membrane binding proteins, are strongly deficient in their ability to sprout in response to vascular endothelial growth factor (VEGF)-A, and are strongly impaired in their ability to migrate and adhere to ß1 integrin-binding extracellular matrix (ECM) proteins. We find that these cells are defective in the formation of complexes containing the tetraspanin CD63, the main VEGF-A receptor VEGFR2, and the ß1 integrin subunit, on the cell surface. We observe that upon VEGF-A activation of AnxA8-depleted HUVECs, VEGFR2 internalization is reduced, phosphorylation of VEGFR2 is increased, and the spatial distribution of Tyr577-phosphorylated focal adhesion kinase (pFAK577) is altered. We conclude that AnxA8 affects CD63/VEGFR2/ß1 integrin complex formation, leading to hyperactivation of the VEGF-A signal transduction pathway, and severely disturbed VEGF-A-driven angiogenic sprouting.

Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases.

Dominant mutations in five tRNA synthetases cause Charcot-Marie-Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA(Gly) aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.