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[superscript 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.National Institutes of Health (U.S.) (Grant GM17151

Annexin A8 promotes VEGF-A driven endothelial cell sprouting

<p>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 β1 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 β1 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/β1 integrin complex formation, leading to hyperactivation of the VEGF-A signal transduction pathway, and severely disturbed VEGF-A-driven angiogenic sprouting.</p

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