SciCombinator

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Concept: Endosome

169

The actin-based molecular motor myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the myosin VI-binding partner Dab2 and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI.

Concepts: Fluorescence, Molecular biology, Function, Dynamics, Endosome, Receptor-mediated endocytosis, Dynamin, Fluorescence recovery after photobleaching

167

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

28

Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex which associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose-6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson’s disease-associated Vps35 variant, R524W, but not P316S, is a loss-of-function mutation as marked by a reduced association with this regulatory network and dysregulation of endosomal receptor sorting. Expression of Vps35 R524W containing retromer results in the accumulation of intracellular α-synuclein positive aggregates, a hallmark of Parkinson’s disease (PD). Overall the Vps35 R524W containing retromer has a decreased endosomal association which can be partially rescued by R55, a small molecule previously shown to stabilize the retromer complex, supporting the potential for future targeting of the retromer complex in the treatment of Parkinson’s disease.

Concepts: DNA, Protein, Cell, Cell membrane, Golgi apparatus, Cell biology, Endosome, Mannose 6-phosphate receptor

28

The AAA-ATPase Vps4 is critical for function of the multivesicular body sorting pathway, which impacts cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. Vps4 activity is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction is unclear. The fragment Vps60(128-186) was reported to display the full activity of Vps60. Vta1 interacts with Vps60 using its N-terminal domain (Vta1NTD). In this work, the structure of Vps60(128-186) in complex with Vta1NTD was determined using NMR techniques, demonstrating a novel recognition mode of the microtubule-interacting and transport (MIT) domain in which Vps60(128-186) interacts with Vta1NTD through helices α4' and α5', extending over Vta1NTD MIT2 domain helices 1-3. The Vps60 binding does not result in Vta1 conformational changes, further revealing the fact that Vps4 ATPase is enhanced by the interaction between Vta1 and Vps60 in an unanticipated manner.

Concepts: Protein, Protein structure, Function, Structure, C-terminus, N-terminus, Endosome, Downregulation and upregulation

28

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

27

The retromer is a trimeric cargo-recognition protein complex composed of Vps26, Vps29 and Vps35 associated with protein trafficking within endosomes. Recently, a pathogenic point mutation within the Vps35 subunit (D620N) was linked to the manifestation of Parkinson’s disease (PD). Here, we investigated details underlying the molecular mechanism by which the D620N mutation in Vps35 modulates retromer function, including examination of retromer’s subcellular localization and its capacity to sort cargo. We show that expression of the PD-linked Vps35 D620N mutant redistributes retromer-positive endosomes to a perinuclear subcellular localization and that these endosomes are enlarged in both model cell lines and fibroblasts isolated from a PD patient. Vps35 D620N is correctly folded and binds Vps29 and Vps26A with the same affinity as wild-type Vps35. While PD-linked point mutant Vps35 D620N interacts with the cation-independent mannose-6-phosphate receptor (CI-M6PR), a known retromer cargo, we find that its expression disrupts the trafficking of cathepsin D, a CI-M6PR ligand and protease responsible for degradation of α-synuclein, a causative agent of Parkinson’s disease. In summary, we find that the expression of Vps35 D620N leads to endosomal alterations and trafficking defects that may partly explain its action in PD.

Concepts: DNA, Gene, Cell, Function, Cell biology, Endosome, Mannose 6-phosphate receptor, Retromer

27

Endocytosis describes the processes by which proteins, peptides and solutes but also pathogens enter the cell. Endocytosed material continues its way to endosomes. Genetic studies in yeast, worms, flies, and mammals have identified a set of universally conserved proteins that are essential for early-to-late endosome transition, lysosome biogenesis, and for endolysosomal trafficking pathways, including autophagy. The two Vps-C complexes, CORVET and HOPS, are among those factors performing diverse biochemical functions: they tether membranes, interact with Rab GTPases, activate and proofread SNARE assembly to drive membrane fusion, and possibly attach endosomes to the cytoskeleton. In addition, several of the CORVET and HOPS subunits have diversified in metazoans and probably give rise to additional specialized complexes to accomodate the higher complexity of trafficking pathways in these cells. Recent studies offer new insights into the complex relationships between CORVET and HOPS complexes and various other factors of the endolysosomal pathway. Interactions with the V-ATPase, the ESCRT machinery, phosphoinositides, the cytoskeleton and the Rab switch suggest an intricate cooperative network for endosome maturation. Accumulating evidence supports the view that endosomal tethering complexes implement a regulatory logic that governs endomembrane identity and dynamics. © 2013 The Authors Journal compilation © 2013 FEBS.

Concepts: Protein, Cell, Cell membrane, Golgi apparatus, Endocytosis, Endosome, Receptor-mediated endocytosis, Exosome

25

In addition to supporting cell survival in response to starvation or stress, autophagy promotes basal protein and organelle turnover. Compared to our understanding of stress-induced autophagy, little is known about how basal autophagy is regulated and how its activity is coordinated with other cellular processes. We recently identified a novel interaction between the ATG12-ATG3 conjugate and the ESCRT-associated protein PDCD6IP/Alix that promotes basal autophagy and endolysosomal trafficking. Moreover, ATG12-ATG3 is required for diverse PDCD6IP-mediated functions including late endosome distribution, exosome secretion, and viral budding. Our results highlight the importance of late endosomes for basal autophagic flux and reveal distinct roles for the core autophagy proteins ATG12 and ATG3 in controlling late endosome function.

Concepts: Protein, Cell nucleus, Cell, Golgi apparatus, Endoplasmic reticulum, Cell biology, Endosome, Exosome

21

Sorting nexins anchor trafficking machines to membranes by binding phospholipids. The paradigm of the superfamily is sorting nexin 3 (SNX3), which localizes to early endosomes by recognizing phosphatidylinositol 3-phosphate (PI3P) to initiate retromer-mediated segregation of cargoes to the trans-Golgi network (TGN). Here we report the solution structure of full length human SNX3, and show that PI3P recognition is accompanied by bilayer insertion of a proximal loop in its extended Phox homology (PX) domain. Phosphoinositide (PIP) binding is completely blocked by cancer-linked phosphorylation of a conserved serine beside the stereospecific PI3P pocket. This “PIP-stop” releases endosomal SNX3 to the cytosol, and reveals how protein kinases control membrane assemblies. It constitutes a widespread regulatory element found across the PX superfamily and throughout evolution including of fungi and plants. This illuminates the mechanism of a biological switch whereby structured PIP sites are phosphorylated to liberate protein machines from organelle surfaces.

Concepts: DNA, Signal transduction, Adenosine triphosphate, Cell membrane, Golgi apparatus, Kinase, Endosome, Phosphatidylinositol

18

Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the, endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites.

Concepts: DNA, Bacteria, Microbiology, Cytosol, Golgi apparatus, Secretion, Messenger RNA, Endosome