Some proteins and lipids traffic from the plasma membrane to the trans Golgi network (TGN)/Golgi apparatus and the endoplasmic reticulum, via the retrograde transport route. Endosomes are an ...obligatory through station. Whether early, recycling and late endosomes all hand off material to the TGN have remained a matter of debate. In this review, we give a short historical overview on how retrograde transport was discovered and explored. We then summarize and critically discuss data that have been put forward in favour of the existence of trafficking interfaces between each of the different endocytic localizations and the TGN. We finally point out some conceptual and technological challenges that will have to be met to establish definite conclusions for each of these scenarios.
The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in ...membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.
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► Acute osmotic swelling or cell stretching induces rapid caveola flattening and disassembly ► Caveola disassembly is mechanically driven in an actin- and ATP-independent manner ► Stress relaxation leads to caveola reassembly in an ATP-dependent manner ► Caveola flattening buffers membrane tension variations and prevents membrane rupture
Membrane traffic requires membrane deformation to generate vesicles and tubules. Strong evidence suggests that assembly of curvature-active proteins can drive such membrane shape changes. ...Well-documented pathways often involve protein scaffolds, in particular coats (clathrin or COP). However, membrane curvature should, in principle, be influenced by any protein binding asymmetrically on a membrane; large membrane morphological changes could result from their aggregation. In the case of Shiga toxin or viral matrix proteins, tubules and buds appear to result from the cargo-driven formation of protein–lipid nanodomains, showing that collective protein behaviour is crucial in the process. We argue here that a combination of in vitro experiments on giant unilamellar vesicles and theoretical modelling based on statistical physics is ideally suited to tackle these collective effects.
Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their ...cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.
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► Identification of two toxin inhibitors by cell-based high-throughput screening ► Cells are protected against the plant toxin ricin and bacterial Shiga-like toxins ► Inhibitors selectively block toxin trafficking at endosome-TGN interface ► One compound protects mice from lethal nasal challenge with ricin
Macromolecular drugs inefficiently cross membranes to reach their cytosolic targets. They require drug delivery vectors to facilitate their translocation across the plasma membrane or escape from ...endosomes. Optimization of these vectors has however been hindered by the difficulty to accurately measure cytosolic arrival. We have developed an exceptionally sensitive and robust assay for the relative or absolute quantification of this step. The assay is based on benzylguanine and biotin modifications on a drug delivery vector of interest, which allow, respectively, for selective covalent capture in the cytosol with a SNAP‐tag fusion protein and for quantification at picomolar sensitivity. The assay was validated by determining the absolute numbers of cytosolic molecules for two drug delivery vectors: the B‐subunit of Shiga toxin and the cell‐penetrating peptide TAT. We expect this assay to favor delivery vector optimization and the understanding of the enigmatic translocation process.
Macromolecular drug delivery vectors must translocate at the plasma membrane or escape from endosomes to reach the cytosol. This very inefficient process requires optimization, which is hindered by the difficulty to accurately measure cytosolic arrival. An exceptionally sensitive and robust assay to quantify this step was developed. The assay was validated by determining the absolute numbers of two macromolecular vectors reaching the cytosol.
The bacteria causing Legionnaires’ disease, Legionella pneumophila, replicate intracellularly within unique Legionella-containing vacuoles (LCVs). LCV formation involves a type IV secretion system ...(T4SS) that translocates effector proteins into host cells. We show that the T4SS effector RidL localizes to LCVs, supports intracellular bacterial growth, and alters retrograde trafficking, in which selected proteins are transported from endosomes to the Golgi. The retromer complex that mediates retrograde trafficking localizes to LCVs independently of RidL and restricts intracellular bacterial growth. RidL binds the Vps29 retromer subunit and the lipid PtdIns(3)P, which localizes retromer components to membranes. Additionally, specific retromer cargo receptors and sorting nexins that mediate protein capture and membrane remodeling preferentially localize to LCVs in the absence of ridL. Ectopic RidL production inhibits retrograde trafficking, and L. pneumophila blocks retrograde transport at endosome exit sites in a ridL-dependent manner. Collectively, these findings suggest that RidL inhibits retromer function to promote intracellular bacterial replication.
•The host retromer complex restricts Legionella replication and localizes to LCVs•The Legionella effector RidL targets the eukaryotic retromer Vps29 subunit•Ectopic production of RidL inhibits retrograde transport in eukaryotic cells•Deletion of ridL abrogates the inhibition of retrograde trafficking by Legionella
Nascent transport intermediates detach from donor membranes by scission. This process can take place in the absence of dynamin, notably in clathrin-independent endocytosis, by mechanisms that are yet ...poorly defined. We show here that in cells scission of Shiga toxin-induced tubular endocytic membrane invaginations is preceded by cholesterol-dependent membrane reorganization and correlates with the formation of membrane domains on model membranes, suggesting that domain boundary forces are driving tubule membrane constriction. Actin triggers scission by inducing such membrane reorganization process. Tubule occurrence is indeed increased upon cellular depletion of the actin nucleator component Arp2, and the formation of a cortical actin shell in liposomes is sufficient to trigger the scission of Shiga toxin-induced tubules in a cholesterol-dependent but dynamin-independent manner. Our study suggests that membranes in tubular Shiga toxin-induced invaginations are poised to undergo actin-triggered reorganization leading to scission by a physical mechanism that may function independently from or in synergy with pinchase activity.
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► Shiga toxin-induced membrane tubules can undergo spontaneous scission ► Membrane reorganization and domain formation correlate with scission ► Scission results from domain boundary forces and does not require dynamin activity ► Actin polymerization triggers scission by inducing membrane reorganization
Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo‐ to ...ganglio‐series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self‐contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo‐series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate‐limiting ganglioside‐producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo–AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.
Synopsis
Schematic representation of glycosphingolipid reprogramming circuit in neural differentiation.
Globo‐series glycosphingolipids inhibit the production of ganglio‐series glycosphingolipids.
AUTS2 expression is repressed by globo‐series glycosphingolipids.
AUTS2 activates the promoter of the first and rate limiting enzyme involved in ganglio‐series glycosphingolipids production i.e., GM3 synthase by inducing histone acetylation.
The globo‐AUTS2 axis regulates the expression of neuronal genes during neural differentiation.
The decrease of globo‐series glycosphingolipids is required for AUTS2 induction and for stem cell differentiation to neural cells.
The switch from globo‐ to ganglio‐series glycophospholipids during neurodevelopment involves a self‐contained regulatory circuit controlling expression of both neuronal and ganglioside‐producing genes.
The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of ...the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.
Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of ...the type-I interferon (IFN-α/β) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. Binding of the retromer VPS35 subunit to IFNAR2 results in IFNAR2 recycling to the plasma membrane, whereas IFNAR1 is sorted to the lysosome for degradation. Depletion of VPS35 leads to abnormally prolonged residency and association of the IFNAR subunits at the early endosome, resulting in increased activation of STAT1- and IFN-dependent gene transcription. These experimental data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and a new factor in type-I IFN-induced JAK/STAT signalling and gene transcription.
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