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 tRNAGly 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.

Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.

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Neutrophil extravasation requires opening of the endothelial barrier, but does not automatically cause plasma leakage. Leaks are prevented by contractile actin filaments surrounding the diapedesis-pore keeping this opening tightly closed around the transmigrating neutrophils. Here, we have identified the receptor system which is responsible for this. We show that silencing, or gene inactivation of endothelial Tie-2 each result in leak formation in postcapillary venules of the inflamed cremaster at sites of neutrophil extravasation as visualized by fluorescent microspheres. Leakage was dependent on neutrophil extravasation, since it was absent upon neutrophil depletion. As downstream target of Tie-2 we identified the Cdc42 GTPase exchange factor FGD5 as essential for leakage prevention during neutrophil extravasation. Seeking for the Tie-2 agonist and its source we found that platelet derived Angiopoietin-1 was required to prevent neutrophil-induced leaks. Intriguingly, blocking von Willebrand Factor (VWF) resulted in vascular leaks during transmigration, indicating that platelets interacting with endothelial VWF activate Tie‑2 by secreting Angiopoietin-1, thereby preventing diapedesis-induced leakiness.