Danièle Tenza

Shiga toxin induces tubular membrane invaginations for its uptake into cells

Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit–Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.

Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin

To investigate retrograde transport along the biosynthetic/secretory pathway, we have constructed a recombinant Shiga toxin B-fragment carrying an N-glycosylation site and a KDEL retrieval motif at its carboxyl terminus (B-Glyc-KDEL). After incubation with HeLa cells, B-Glyc-KDEL was progressively glycosylated in the endoplasmic reticulum (ER) and remained stably associated with this compartment. B-fragment with a nonfunctional KDEL sequence (B-Glyc-KDELGL) was glycosylated with about the same kinetics as B-Glyc-KDEL but localized at steady state to the Golgi apparatus. Morphological studies showed that B-Glyc-KDEL was delivered from the plasma membrane, via endosomes and the cisternae of the Golgi apparatus, to the ER. Moreover, the addition of a sulfation site allowed us to show that B-Glyc-KDEL on transit to the ER entered the Golgi apparatus through the trans-Golgi network. Transport of B-Glyc-KDEL to the ER was slowed down by nocodazole, indicating that microtubules are important for the retrograde pathway. Our results document the existence of a continuous pathway from the plasma membrane to the endoplasmic reticulum via the Golgi apparatus and show that a fully folded exogenous protein arriving in the endoplasmic reticulum via this pathway can undergo N-glycosylation.

Clathrin Adaptor epsinR Is Required for Retrograde Sorting on Early Endosomal Membranes

Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.

ARF6 controls post-endocytic recycling through its downstream exocyst complex effector

The small guanosine triphosphate (GTP)–binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP–bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.

Inhibition of Retrograde Transport Protects Mice from Lethal Ricin Challenge

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.

The retromer complex and clathrin define an early endosomal retrograde exit site

Previous studies have indicated a role for clathrin, the clathrin adaptors AP1 and epsinR, and the retromer complex in retrograde sorting from early/recycling endosomes to the trans Golgi network (TGN). However, it has remained unclear whether these protein machineries function on the same or parallel pathways. We show here that clathrin and the retromer subunit Vps26 colocalize at the ultrastructural level on early/recycling endosomes containing Shiga toxin B-subunit, a well-studied retrograde transport cargo. As previously described for clathrin, we find that interfering with Vps26 expression inhibits retrograde transport of the Shiga toxin B-subunit to the TGN. Under these conditions, endosomal tubules that take the Shiga toxin B-subunit out of transferrin-containing early/recycling endosomes appear to be stabilized. This situation differs from that previously described for low-temperature incubation and clathrin-depletion conditions under which Shiga toxin B-subunit labeling was found to overlap with that of the transferrin receptor. In addition, we find that the Shiga toxin B-subunit and the transferrin receptor accumulate close to multivesicular endosomes in clathrin-depleted cells, suggesting that clathrin initiates retrograde sorting on vacuolar early endosomes, and that retromer is then required to process retrograde tubules. Our findings thus establish a role for the retromer complex in retrograde transport of the B-subunit of Shiga toxin, and strongly suggest that clathrin and retromer function in consecutive retrograde sorting steps on early endosomes.

AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis

The clathrin adaptor protein AP-1 and the motor KIF13A work together to deliver cargo into maturing melanosomes.

Analysis of articulation between clathrin and retromer in retrograde sorting on early endosomes.

Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans‐Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor‐mediated endocytosis‐8 (RME‐8) protein, and that RME‐8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor‐binding B‐subunit of Shiga toxin (STxB). RME‐8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME‐8 or Hsc70 affects retrograde trafficking at the early endosomes–TGN interface of STxB and the cation‐independent mannose 6‐phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin‐binding protein hepatocyte growth factor‐regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069–7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME‐8, and possibly also by Hrs.

Myosin Ib modulates the morphology and the protein transport within multi-vesicular sorting endosomes

Members of at least four classes of myosin (I, II, V and VI) have been implicated in the dynamics of a large variety of organelles. Despite their common motor domain structure, some of these myosins, however, are non processive and cannot move organelles along the actin tracks. Here, we demonstrate in the human pigmented MNT-1 cell line that, (1) the overexpression of one of these myosins, myosin 1b, or the addition of cytochalasin D affects the morphology of the sorting multivesicular endosomes; (2) the overexpression of myosin 1b delays the processing of Pmel17 (the product of murine silver locus also named GP100), which occurs in these multivesicular endosomes; (3) myosin 1b associated with endosomes coimmunoprecipitates with Pmel17. All together, these observations suggest that myosin 1b controls the traffic of protein cargo in multivesicular endosomes most probably through its ability to modulate with actin the morphology of these sorting endosomes.

Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes

Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.