P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2.

Upon proinflammatory challenges, endothelial cell surface presentation of the leukocyte receptor P-selectin, together with the stabilizing co-factor CD63, is needed for leukocyte capture and is mediated via demand-driven exocytosis from the Weibel-Palade bodies that fuse with the plasma membrane. We report that neutrophil recruitment to activated endothelium is significantly reduced in mice deficient for the endolysosomal cation channel TPC2 and in human primary endothelial cells with pharmacological TPC2 block. We observe less CD63 signal in whole-mount stainings of proinflammatory-activated cremaster muscles from TPC2 knockout mice. We find that TPC2 is activated and needed to ensure the transfer of CD63 from endolysosomes via Weibel-Palade bodies to the plasma membrane to retain P-selectin on the cell surface of human primary endothelial cells. Our findings establish TPC2 as a key element to leukocyte interaction with the endothelium and a potential pharmacological target in the control of inflammatory leukocyte recruitment.

Primary neurons lacking the SNAREs vti1a and vti1b show altered neuronal development.

BACKGROUND: Neurons are highly specialized cells with a complex morphology generated by various membrane trafficking steps. They contain Golgi outposts in dendrites, which are formed from somatic Golgi tubules. In trafficking membrane fusion is mediated by a specific combination of SNARE proteins. A functional SNARE complex contains four different helices, one from each SNARE subfamily (R-, Qa, Qb and Qc). Loss of the two Qb SNAREs vti1a and vti1b from the Golgi apparatus and endosomes leads to death at birth in mice with massive neurodegeneration in peripheral ganglia and defective axon tracts. METHODS: Hippocampal and cortical neurons were isolated from Vti1a-/- Vti1b-/- double deficient, Vti1a-/- Vti1b+/-, Vti1a+/- Vti1b-/- and Vti1a+/- Vti1b+/- double heterozygous embryos. Neurite outgrowth was determined in cortical neurons and after stimulation with several neurotrophic factors or the Rho-associated protein kinase ROCK inhibitor Y27632, which induces exocytosis of enlargeosomes, in hippocampal neurons. Moreover, postsynaptic densities were isolated from embryonic Vti1a-/- Vti1b-/- and Vti1a+/- Vti1b+/- control forebrains and analyzed by western blotting. RESULTS: Golgi outposts were present in Vti1a-/- Vti1b+/- and Vti1a+/- Vti1b-/- dendrites of hippocampal neurons but not detected in the absence of vti1a and vti1b. The length of neurites was significantly shorter in double deficient cortical neurons. These defects were not observed in Vti1a-/- Vti1b+/- and Vti1a+/- Vti1b-/- neurons. NGF, BDNF, NT-3, GDNF or Y27632 as stimulator of enlargeosome secretion did not increase the neurite length in double deficient hippocampal neurons. Vti1a-/- Vti1b-/- postsynaptic densities contained similar amounts of scaffold proteins, AMPA receptors and NMDA receptors compared to Vti1a+/- Vti1b+/-, but much more TrkB, which is the receptor for BDNF. CONCLUSION: The absence of Golgi outposts did not affect the amount of AMPA and NMDA receptors in postsynaptic densities. Even though TrkB was enriched, BDNF was not able to stimulate neurite elongation in Vti1a-/- Vti1b-/- neurons. Vti1a or vti1b function as the missing Qb-SNARE together with VAMP-4 (R-SNARE), syntaxin 16 (Qa-SNARE) and syntaxin 6 (Qc-SNARE) in induced neurite outgrowth. Our data show the importance of vti1a or vti1b for two pathways of neurite elongation.

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Late Endosomal/Lysosomal Cholesterol Accumulation Is a Host Cell-Protective Mechanism Inhibiting Endosomal Escape of Influenza A Virus.

To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral content into the cytosol. Although IFITM3 induction occurs concomitantly with late endosomal/lysosomal (LE/L) cholesterol accumulation, the functional significance of this process is not well understood. Here we report that LE/L cholesterol accumulation itself plays a pivotal role in the early antiviral defense. We demonstrate that inducing LE/L cholesterol accumulation is antiviral in non-IFN-primed cells, restricting incoming IAV particles and impairing mixing of IAV/endosomal membrane lipids. Our results establish a protective function of LE/L cholesterol accumulation and suggest endosomal cholesterol balance as a possible antiviral target.IMPORTANCE With annual epidemics occurring in all parts of the world and the risk of global outbreaks, influenza A virus (IAV) infections remain a major threat to public health. Infected host cells detect viral components and mount an interferon (IFN)-mediated response to restrict virus propagation and spread of infection. Identification of cellular factors and underlying mechanisms that establish such an antiviral state can provide novel strategies for the development of antiviral drugs. The contribution of LE/L cholesterol levels, especially in the context of the IFN-induced antiviral response, has remained controversial so far. Here, we report that accumulation of cholesterol in the LE/L compartment contributes to the IFN-induced host cell defense against incoming IAV. Our results establish cholesterol accumulation in LE/L per se as a novel antiviral barrier and suggest the endosomal cholesterol balance as a putative druggable host cell factor in IAV infection.

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.