Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic.

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.

Recruitment of PI4KIIIß to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins.

ACBD3 is a protein localised to the Golgi apparatus and recruits other proteins, such as PI4KIIIß, to the Golgi. However, the mechanism through which ACBD3 itself is recruited to the Golgi is poorly understood. This study demonstrates there are two mechanisms for ACBD3 recruitment to the Golgi. First, we identified that an MWT374-376 motif in the unique region upstream of the GOLD domain in ACBD3 is essential for Golgi localization. Second, we use unbiased proteomics to demonstrate that ACBD3 interacts with SCFD1, a Sec1/Munc-18 (SM) protein, and a SNARE protein, SEC22B. CRISPR-KO of SCFD1 causes ACBD3 to become cytosolic. We also found that ACBD3 is redundantly recruited to the Golgi apparatus by two golgins: golgin-45 and giantin, which bind to ACBD3 through interaction with the MWT374-376 motif. Taken together, our results suggest that ACBD3 is recruited to the Golgi in a two-step sequential process, with the SCFD1-mediated interaction occurring upstream of the interaction with the golgins.

The exocyst complex is an essential component of the mammalian constitutive secretory pathway.

Secreted proteins fulfill a vast array of functions, including immunity, signaling, and extracellular matrix remodeling. In the trans-Golgi network, proteins destined for constitutive secretion are sorted into post-Golgi carriers which fuse with the plasma membrane. The molecular machinery involved is poorly understood. Here, we have used kinetic trafficking assays and transient CRISPR-KO to study biosynthetic sorting from the Golgi to the plasma membrane. Depletion of all canonical exocyst subunits causes cargo accumulation in post-Golgi carriers. Exocyst subunits are recruited to and co-localize with carriers. Exocyst abrogation followed by kinetic trafficking assays of soluble cargoes results in intracellular cargo accumulation. Unbiased secretomics reveals impairment of soluble protein secretion after exocyst subunit knockout. Importantly, in specialized cell types, the loss of exocyst prevents constitutive secretion of antibodies in lymphocytes and of leptin in adipocytes. These data identify exocyst as the functional tether of secretory post-Golgi carriers at the plasma membrane and an essential component of the mammalian constitutive secretory pathway.

Liquid-liquid phase separation facilitates the biogenesis of secretory storage granules.

Insulin is synthesized by pancreatic ß-cells and stored into secretory granules (SGs). SGs fuse with the plasma membrane in response to a stimulus and deliver insulin to the bloodstream. The mechanism of how proinsulin and its processing enzymes are sorted and targeted from the trans-Golgi network (TGN) to SGs remains mysterious. No cargo receptor for proinsulin has been identified. Here, we show that chromogranin (CG) proteins undergo liquid-liquid phase separation (LLPS) at a mildly acidic pH in the lumen of the TGN, and recruit clients like proinsulin to the condensates. Client selectivity is sequence-independent but based on the concentration of the client molecules in the TGN. We propose that the TGN provides the milieu for converting CGs into a "cargo sponge" leading to partitioning of client molecules, thus facilitating receptor-independent client sorting. These findings provide a new receptor-independent sorting model in ß-cells and many other cell types and therefore represent an innovation in the field of membrane trafficking.

Clathrin adaptor AP-1-mediated Golgi export of amyloid precursor protein is crucial for the production of neurotoxic amyloid fragments.

One of the hallmarks of Alzheimer's disease is the accumulation of toxic amyloid-ß (Aß) peptides in extracellular plaques. The direct precursor of Aß is the carboxyl-terminal fragment ß (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer's disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of Aß. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and Aß release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

SNX19 restricts endolysosome motility through contacts with the endoplasmic reticulum.

The ability of endolysosomal organelles to move within the cytoplasm is essential for the performance of their functions. Long-range movement involves coupling of the endolysosomes to motor proteins that carry them along microtubule tracks. This movement is influenced by interactions with other organelles, but the mechanisms involved are incompletely understood. Herein we show that the sorting nexin SNX19 tethers endolysosomes to the endoplasmic reticulum (ER), decreasing their motility and contributing to their concentration in the perinuclear area of the cell. Tethering depends on two N-terminal transmembrane domains that anchor SNX19 to the ER, and a PX domain that binds to phosphatidylinositol 3-phosphate on the endolysosomal membrane. Two other domains named PXA and PXC negatively regulate the interaction of SNX19 with endolysosomes. These studies thus identify a mechanism for controlling the motility and positioning of endolysosomes that involves tethering to the ER by a sorting nexin.

The Parkinson's Disease Protein LRRK2 Interacts with the GARP Complex to Promote Retrograde Transport to the trans-Golgi Network.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). However, the precise function of LRRK2 remains unclear. We report an interaction between LRRK2 and VPS52, a subunit of the Golgi-associated retrograde protein (GARP) complex that identifies a function of LRRK2 in regulating membrane fusion at the trans-Golgi network (TGN). At the TGN, LRRK2 further interacts with the Golgi SNAREs VAMP4 and Syntaxin-6 and acts as a scaffolding platform that stabilizes the GARP-SNAREs complex formation. Therefore, LRRK2 influences both retrograde and post-Golgi trafficking pathways in a manner dependent on its GTP binding and kinase activity. This action is exaggerated by mutations associated with Parkinson's disease and can be blocked by kinase inhibitors. Disruption of GARP sensitizes dopamine neurons to mutant LRRK2 toxicity in C. elegans, showing that these pathways are interlinked in vivo and suggesting a link in PD.

Direct trafficking pathways from the Golgi apparatus to the plasma membrane.

In eukaryotic cells, protein sorting is a highly regulated mechanism important for many physiological events. After synthesis in the endoplasmic reticulum and trafficking to the Golgi apparatus, proteins sort to many different cellular destinations including the endolysosomal system and the extracellular space. Secreted proteins need to be delivered directly to the cell surface. Sorting of secreted proteins from the Golgi apparatus has been a topic of interest for over thirty years, yet there is still no clear understanding of the machinery that forms the post-Golgi carriers. Most evidence points to these post-Golgi carriers being tubular pleomorphic structures that bud from the trans-face of the Golgi. In this review, we present the background studies and highlight the key components of this pathway, we then discuss the machinery implicated in the formation of these carriers, their translocation across the cytosol, and their fusion at the plasma membrane.

The autophagy protein ATG9A promotes HIV-1 infectivity.

BACKGROUND: Nef is a multifunctional accessory protein encoded by HIV-1, HIV-2 and SIV that plays critical roles in viral pathogenesis, contributing to viral replication, assembly, budding, infectivity and immune evasion, through engagement of various host cell pathways. RESULTS: To gain a better understanding of the role of host proteins in the functions of Nef, we carried out tandem affinity purification-mass spectrometry analysis, and identified over 70 HIV-1 Nef-interacting proteins, including the autophagy-related 9A (ATG9A) protein. ATG9A is a transmembrane component of the machinery for autophagy, a catabolic process in which cytoplasmic components are degraded in lysosomal compartments. Pulldown experiments demonstrated that ATG9A interacts with Nef from not only HIV-1 and but also SIV (cpz, smm and mac). However, expression of HIV-1 Nef had no effect on the levels and localization of ATG9A, and on autophagy, in the host cells. To investigate a possible role for ATG9A in virus replication, we knocked out ATG9A in HeLa cervical carcinoma and Jurkat T cells, and analyzed virus release and infectivity. We observed that ATG9A knockout (KO) had no effect on the release of wild-type (WT) or Nef-defective HIV-1 in these cells. However, the infectivity of WT virus produced from ATG9A-KO HeLa and Jurkat cells was reduced by ~ fourfold and eightfold, respectively, relative to virus produced from WT cells. This reduction in infectivity was independent of the interaction of Nef with ATG9A, and was not due to reduced incorporation of the viral envelope (Env) glycoprotein into the virus. The loss of HIV-1 infectivity was rescued by pseudotyping HIV-1 virions with the vesicular stomatitis virus G glycoprotein. CONCLUSIONS: These studies indicate that ATG9A promotes HIV-1 infectivity in an Env-dependent manner. The interaction of Nef with ATG9A, however, is not required for Nef to enhance HIV-1 infectivity. We speculate that ATG9A could promote infectivity by participating in either the removal of a factor that inhibits infectivity or the incorporation of a factor that enhances infectivity of the viral particles. These studies thus identify a novel host cell factor implicated in HIV-1 infectivity, which may be amenable to pharmacologic manipulation for treatment of HIV-1 infection.

A neurodevelopmental disorder caused by mutations in the VPS51 subunit of the GARP and EARP complexes.

Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) are related heterotetrameric complexes that associate with the cytosolic face of the trans-Golgi network and recycling endosomes, respectively. At these locations, GARP and EARP function to promote the fusion of endosome-derived transport carriers with their corresponding compartments. GARP and EARP share three subunits, VPS51, VPS52 and VPS53, and each has an additional complex-specific subunit, VPS54 or VPS50, respectively. The role of these complexes in human physiology, however, remains poorly understood. By exome sequencing, we have identified compound heterozygous mutations in the gene encoding the shared GARP/EARP subunit VPS51 in a 6-year-old patient with severe global developmental delay, microcephaly, hypotonia, epilepsy, cortical vision impairment, pontocerebellar abnormalities, failure to thrive, liver dysfunction, lower extremity edema and dysmorphic features. The mutation in one allele causes a frameshift that produces a longer but highly unstable protein that is degraded by the proteasome. In contrast, the other mutant allele produces a protein with a single amino acid substitution that is stable but assembles less efficiently with the other GARP/EARP subunits. Consequently, skin fibroblasts from the patient have reduced levels of fully assembled GARP and EARP complexes. Likely because of this deficiency, the patient's fibroblasts display altered distribution of the cation-independent mannose 6-phosphate receptor, which normally sorts acid hydrolases to lysosomes. Furthermore, a fraction of the patient's fibroblasts exhibits swelling of lysosomes. These findings thus identify a novel genetic locus for a neurodevelopmental disorder and highlight the critical importance of GARP/EARP function in cellular and organismal physiology.

Molecular mechanism for the subversion of the retromer coat by the Legionella effector RidL.

Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29-VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.

Endosomal Trafficking: Retromer and Retriever Are Relatives in Recycling.

Transmembrane proteins are sorted from endosomes to avoid lysosomal degradation. A recent study has identified a new multimeric complex called retriever that is essential for recycling numerous cell-surface cargoes from endosomes and is structurally and functionally related to the well-characterised retromer complex.

Segregation in the Golgi complex precedes export of endolysosomal proteins in distinct transport carriers.

Biosynthetic sorting of newly synthesized transmembrane cargos to endosomes and lysosomes is thought to occur at the TGN through recognition of sorting signals in the cytosolic tails of the cargos by adaptor proteins, leading to cargo packaging into coated vesicles destined for the endolysosomal system. Here we present evidence for a different mechanism in which two sets of endolysosomal proteins undergo early segregation to distinct domains of the Golgi complex by virtue of the proteins' luminal and transmembrane domains. Proteins in one Golgi domain exit into predominantly vesicular carriers by interaction of sorting signals with adaptor proteins, but proteins in the other domain exit into predominantly tubular carriers shared with plasma membrane proteins, independently of signal-adaptor interactions. These findings demonstrate that sorting of endolysosomal proteins begins at an earlier stage and involves mechanisms that partly differ from those described by classical models.

Physical Removal of the Midbody Remnant from Polarised Epithelial Cells Using Take-Up by Suction Pressure (TUSP).

In polarised epithelial cells the midbody forms at the apical cell surface during cytokinesis. Once severed, the midbody is inherited by one of the daughter cells remaining tethered to the apical plasma membrane where it participates in non-cytokinetic processes, such as primary ciliogenesis. Here, we describe a novel method to physically remove the midbody remnant from cells and assess the possible effects caused by its loss (Bernabé- Rubio et al., 2016 ).

Structural Mechanism for Cargo Recognition by the Retromer Complex.

Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer's disease and Parkinson's disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes have remained elusive. Here, we present an X-ray crystallographic analysis of a four-component complex comprising the VPS26 and VPS35 subunits of retromer, the sorting nexin SNX3, and a recycling signal from the divalent cation transporter DMT1-II. This analysis identifies a binding site for canonical recycling signals at the interface between VPS26 and SNX3. In addition, the structure highlights a network of cooperative interactions among the VPS subunits, SNX3, and cargo that couple signal-recognition to membrane recruitment.

TSSC1 is novel component of the endosomal retrieval machinery.

Endosomes function as a hub for multiple protein-sorting events, including retrograde transport to the trans-Golgi network (TGN) and recycling to the plasma membrane. These processes are mediated by tubular-vesicular carriers that bud from early endosomes and fuse with a corresponding acceptor compartment. Two tethering complexes named GARP (composed of ANG2, VPS52, VPS53, and VPS54 subunits) and EARP (composed of ANG2, VPS52, VPS53, and Syndetin subunits) were previously shown to participate in SNARE-dependent fusion of endosome-derived carriers with the TGN and recycling endosomes, respectively. Little is known, however, about other proteins that function with GARP and EARP in these processes. Here we identify a protein named TSSC1 as a specific interactor of both GARP and EARP and as a novel component of the endosomal retrieval machinery. TSSC1 is a predicted WD40/ß-propeller protein that coisolates with both GARP and EARP in affinity purification, immunoprecipitation, and gel filtration analyses. Confocal fluorescence microscopy shows colocalization of TSSC1 with both GARP and EARP. Silencing of TSSC1 impairs transport of internalized Shiga toxin B subunit to the TGN, as well as recycling of internalized transferrin to the plasma membrane. Fluorescence recovery after photobleaching shows that TSSC1 is required for efficient recruitment of GARP to the TGN. These studies thus demonstrate that TSSC1 plays a critical role in endosomal retrieval pathways as a regulator of both GARP and EARP function.