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Concept: Endocytosis

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The LDL receptor (LDLR) supports efficient uptake of both LDL and VLDL remnants by binding lipoprotein at the cell surface, internalizing lipoprotein through coated pits and releasing lipoprotein in endocytic compartments before returning to the surface for further rounds of uptake. While many aspects of lipoprotein binding and receptor entry are well understood, it is less clear where, when and how the LDLR releases lipoprotein. To address these questions, the current study employed quantitative fluorescence imaging to visualize the uptake and endosomal processing of LDL and the VLDL remnant, β-VLDL. We find that lipoprotein release is rapid with most release occurring prior to entry of lipoprotein into early endosomes. Published biochemical studies have identified two mechanisms of lipoprotein release: one that involves the β-propeller module of the LDLR and a second that is independent of this module. Quantitative imaging comparing uptake supported by the normal LDLR or by an LDLR variant incapable of β-propeller-dependent release show that the β-propeller-independent process is sufficient for release for both lipoproteins, but that the β-propeller process accelerates both LDL and β-VLDL release. Together these findings define where, when and how lipoprotein release occurs and provide a generalizable methodology for visualizing endocytic handling in situ.

Concepts: Cholesterol, Low density lipoprotein receptor gene family, Lipoproteins, Endocytosis, Endosome, Release, LDL receptor

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In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

Concepts: Cholesterol, Atherosclerosis, Heart, Blood vessel, Endothelium, Endocytosis, Caveolae, LDL receptor

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Tight regulation of signalling activity is crucial for proper tissue patterning and growth. Here we investigate the function of Pentagone (Pent), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. We show that Pent internalises the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and propose that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, we establish a requirement of Pent for Wg signalling. Our data propose a novel mechanism by which morphogen signalling is regulated.

Concepts: Cell, Signal transduction, Cytosol, Cell membrane, Regulation, Endocytosis, Administrative law, Development approvals

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ZIKA virus (ZIKV) is an emerging pathogen responsible for neurological disorders and congenital microcephaly. However, the molecular basis for ZIKV neurotropism remains poorly understood. Here, we show that Axl is expressed in human microglia and astrocytes in the developing brain and that it mediates ZIKV infection of glial cells. Axl-mediated ZIKV entry requires the Axl ligand Gas6, which bridges ZIKV particles to glial cells. Following binding, ZIKV is internalized through clathrin-mediated endocytosis and traffics to Rab5+ endosomes to establish productive infection. During entry, the ZIKV/Gas6 complex activates Axl kinase activity, which downmodulates interferon signaling and facilitates infection. ZIKV infection of human glial cells is inhibited by MYD1, an engineered Axl decoy receptor, and by the Axl kinase inhibitor R428. Our results highlight the dual role of Axl during ZIKV infection of glial cells: promoting viral entry and modulating innate immune responses. Therefore, inhibiting Axl function may represent a potential target for future antiviral therapies.

Concepts: Immune system, Bacteria, Virus, Vaccination, Innate immune system, Glial cell, Endocytosis, Microglia

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Endocytosis, like many dynamic cellular processes, requires precise temporal and spatial orchestration of complex protein machinery to mediate membrane budding. To understand how this machinery works, we directly correlated fluorescence microscopy of key protein pairs with electron tomography. We systematically located 211 endocytic intermediates, assigned each to a specific time window in endocytosis, and reconstructed their ultrastructure in 3D. The resulting virtual ultrastructural movie defines the protein-mediated membrane shape changes during endocytosis in budding yeast. It reveals that clathrin is recruited to flat membranes and does not initiate curvature. Instead, membrane invagination begins upon actin network assembly followed by amphiphysin binding to parallel membrane segments, which promotes elongation of the invagination into a tubule. Scission occurs on average 9 s after initial bending when invaginations are ∼100 nm deep, releasing nonspherical vesicles with 6,400 nm2 mean surface area. Direct correlation of protein dynamics with ultrastructure provides a quantitative 4D resource.

Concepts: Protein, Cell, Cell membrane, Yeast, Correlation and dependence, Endocytosis, Cellular processes

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Adaptor protein-2 (AP2), a central component of clathrin-coated vesicles (CCVs), is pivotal in clathrin-mediated endocytosis, which internalizes plasma membrane constituents such as G protein-coupled receptors (GPCRs). AP2, a heterotetramer of α, β, μ and σ subunits, links clathrin to vesicle membranes and binds to tyrosine- and dileucine-based motifs of membrane-associated cargo proteins. Here we show that missense mutations of AP2 σ subunit (AP2S1) affecting Arg15, which forms key contacts with dileucine-based motifs of CCV cargo proteins, result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular calcium homeostasis disorder affecting the parathyroids, kidneys and bone. We found AP2S1 mutations in >20% of cases of FHH without mutations in calcium-sensing GPCR (CASR), which cause FHH1. AP2S1 mutations decreased the sensitivity of CaSR-expressing cells to extracellular calcium and reduced CaSR endocytosis, probably through loss of interaction with a C-terminal CaSR dileucine-based motif, whose disruption also decreased intracellular signaling. Thus, our results identify a new role for AP2 in extracellular calcium homeostasis.

Concepts: Calcitonin, Signal transduction, Cell membrane, Cell biology, Membrane biology, G protein-coupled receptor, Endocytosis, Calcium-sensing receptor

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The ErbB2 receptor is a clinically validated cancer target whose internalization and trafficking mechanisms remain poorly understood. HSP90 inhibitors, such as Geldanamycin (GA), have been developed to target the receptor to degradation, or to modulate downstream signaling. Despite intense investigations, the entry route and postendocytic sorting of ErbB2 upon GA stimulation have remained controversial. We report that ErbB2 levels inversely impact on the cell clathrin-mediated endocytosis capacity. Indeed, the high levels of the receptor are responsible for its own low internalization rate. GA treatment does not directly modulate ErbB2 clathrin-mediated endocytosis rate but it affects ErbB2 recycling fate, routing the receptor to modified multivesicular (MVBs) and lysosomal compartments, by perturbing early/recycling endosome structure and sorting capacity. This activity occurs irrespective of the cargo interaction with HSP90, as both ErbB2 and the constitutively recycled, HSP90-independent, Transferrin receptor are found within modified endosomes, and within aberrant elongated recycling tubules leading to modified MVBs/lysosomes. We propose that GA, as part of its anti-cancer activity, perturbs early/recycling endosome sorting, routing recycling cargoes toward mixed endosomal compartments.

Concepts: Cell, Golgi apparatus, Inhibitor, Endocytosis, Recycling, Endosome, Receptor-mediated endocytosis, Exosome

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The early steps of human parvovirus B19 (B19V) infection were investigated in UT7/Epo cells. B19V and its receptor globoside (Gb4Cer) associate with lipid rafts, predominantly of the noncaveolar type. Pharmacological disruption of the lipid rafts inhibited infection when the drug was added prior to virus attachment but not after virus uptake. B19V is internalized by clathrin-dependent endocytosis and spreads rapidly throughout the endocytic pathway, reaching the lysosomal compartment within minutes, where a substantial proportion is degraded. B19V did not permeabilize the endocytic vesicles, indicating a mechanism of endosomal escape without apparent membrane damage. Bafilomycin A(1) (BafA1) and NH(4)Cl, which raise endosomal pH, blocked the infection by preventing endosomal escape, resulting in a massive accumulation of capsids in the lysosomes. In contrast, in the presence of chloroquine (CQ), the transfer of incoming viruses from late endosomes to lysosomes was prevented; the viral DNA was not degraded; and the infection was boosted. In contrast to the findings for untreated or BafA1-treated cells, the viral DNA was progressively associated with the nucleus in CQ-treated cells, reaching a plateau by 3 h postinternalization, a time coinciding with the initiation of viral transcription. At this time, more than half of the total intracellular viral DNA was associated with the nucleus; however, the capsids remained extranuclear. Our studies provide the first insight into the early steps of B19V infection and reveal mechanisms involved in virus uptake, endocytic trafficking, and nuclear penetration.

Concepts: AIDS, Protein, Virus, Cell membrane, Endocytosis, Parvovirus B19, Parvovirus, Parvoviridae

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To ensure spread from one cell to another, exocytosed vaccinia virions recruit cellular actin polymerization machinery to blast off from the cell surface on actin tails. Humphries et al. (2012) now show that the virus exploits clathrin to organize viral factors into a launch pad that facilitates efficient actin tail formation.

Concepts: DNA, Protein, Gene, Organism, Virus, Vaccinia, Endocytosis, Radio resource management

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Dynamic actin filaments are a crucial component of clathrin-mediated endocytosis when endocytic proteins cannot supply enough energy for vesicle budding. Actin cytoskeleton is thought to provide force for membrane invagination or vesicle scission, but how this force is transmitted to the plasma membrane is not understood. Here we describe the molecular mechanism of plasma membrane-actin cytoskeleton coupling mediated by cooperative action of epsin Ent1 and the HIP1R homolog Sla2 in yeast Saccharomyces cerevisiae. Sla2 anchors Ent1 to a stable endocytic coat by an unforeseen interaction between Sla2’s ANTH and Ent1’s ENTH lipid-binding domains. The ANTH and ENTH domains bind each other in a ligand-dependent manner to provide critical anchoring of both proteins to the membrane. The C-terminal parts of Ent1 and Sla2 bind redundantly to actin filaments via a previously unknown phospho-regulated actin-binding domain in Ent1 and the THATCH domain in Sla2. By the synergistic binding to the membrane and redundant interaction with actin, Ent1 and Sla2 form an essential molecular linker that transmits the force generated by the actin cytoskeleton to the plasma membrane, leading to membrane invagination and vesicle budding.

Concepts: Protein, Cell, Cytosol, Cell membrane, Yeast, Saccharomyces cerevisiae, Cytoskeleton, Endocytosis