In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

Retrograde transport allows proteins and lipids to leave the endocytic pathway to reach other intracellular compartments, such as TGN/Golgi membranes, the endoplasmic reticulum, and in some instances, the cytosol. Here, we have used RNA interference against the SNARE proteins syntaxin 5 and syntaxin 16 combined with recently developed quantitative trafficking assays, morphological approaches, and cell intoxication analysis to show that these SNARE proteins are not only required for efficient retrograde transport of Shiga toxin, but also for that of an endogenous cargo protein, the mannose 6-phosphate receptor, and for the productive trafficking into cells of cholera toxin and ricin. We have found that the function of syntaxin 16 was specifically required for, and restricted to the retrograde pathway. Strikingly, syntaxin 5 RNA interference protected cells particularly strongly against Shiga toxin. Since our trafficking analysis showed that apart from inhibiting retrograde endosomes-to-TGN transport, the silencing of syntaxin 5 had no additional effect on Shiga toxin endocytosis or trafficking from TGN/Golgi membranes to the endoplasmic reticulum, we hypothesize that syntaxin 5 also has trafficking-independent functions. In summary, our data demonstrate that several cellular and exogenous cargo proteins use elements of the same SNARE machinery for efficient retrograde transport between early/recycling endosomes and TGN/Golgi membranes.

Progressive familial intrahepatic cholestasis type 3 is caused by biallelic variations of ABCB4, most often ( 70%) missense. In this study, we examined the effects of 12 missense variations identified in progressive familial intrahepatic cholestasis type 3 patients. We classified these variations on the basis of the defects thus identified and explored potential rescue of trafficking-defective mutants by pharmacological means. Variations were reproduced in the ABCB4 complementary DNA and the mutants, thus obtained, expressed in HepG2 and HEK293 cells. Three mutants were either fully (I541F and L556R) or largely (Q855L) retained in the endoplasmic reticulum, in an immature form. Rescue of the defect, i.e., increase in the mature form at the bile canaliculi, was obtained by cell treatments with cyclosporin A or C and, to a lesser extent, B, D, or H. Five mutations with little or no effect on ABCB4 expression at the bile canaliculi caused a decrease (F357L, T775M, and G954S) or almost absence (S346I and P726L) of phosphatidylcholine secretion. Two mutants (T424A and N510S) were normally processed and expressed at the bile canaliculi, but their stability was reduced. We found no defect of the T175A mutant or of R652G, previously described as a polymorphism. In patients, the most severe phenotypes appreciated by the duration of transplant-free survival were caused by ABCB4 variants that were markedly retained in the endoplasmic reticulum and expressed in a homozygous status. Conclusion: ABCB4 variations can be classified as follows: nonsense variations (I) and, on the basis of current findings, missense variations that primarily affect the maturation (II), activity (III), or stability (IV) of the protein or have no detectable effect (V); this classification provides a strong basis for the development of genotype-based therapies.

a These authors contributed equally to this work.

Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively. INTRODUCTIONIn eukaryotic cells, molecules of the plasma membrane, ligands and solutes are internalized into early endosomes, from where they can be recycled back to the plasma membrane, transported to the trans-Golgi network, or targeted to late endosomes and lysosomes (Gruenberg, 2001;Maxfield and McGraw, 2004;Mayor and Pagano, 2007). The latter pathway is followed in particular by ubiquitinated signaling receptors that need to be down-regulated. These receptors are incorporated into invaginations of the early endosomal membrane that form within their lumen (Hurley and Emr, 2006), thus giving rise to nascent multivesicular bodies (MVBs) or endosomal carrier vesicles (ECVs; Gruenberg and Stenmark, 2004;Piper and Katzmann, 2007), which function as intermediates between early and late endosomes. Eventually, intralumenal vesicles are delivered to lysosomes, where they are degraded together with their receptor cargo.Major progress has been made in our understanding of the molecular mechanisms that control the sorting of ubiquitinated receptors, in particular the epidermal growth factor (EGF) receptor (Hurley and Emr, 2006;Slagsvold et al., 2006;Piper and Katzmann, 2007;Williams and Urbe, 2007). These receptors interact via the ubiquitin moiety with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), which in turn binds clathrin and phosphatidyl-inositol-3-phosphate (PtdIns3P), thereby concentrating the receptor in early endosomal membrane domains (Raiborg et al., 2001;Urbe et al., 2003;Raiborg et al., ...

The targeting of solid tumors requires delivery tools that resist intracellular and extracellular inactivation, and that are taken up specifically by tumor cells. We have shown previously that the recombinant nontoxic B-subunit of Shiga toxin (STxB) can serve as a delivery tool to target digestive tumors in animal models. The aim of this study was to expand these experiments to human colorectal cancer. Tissue samples of normal colon, benign adenomas, colorectal carcinomas, and liver metastases from 111 patients were obtained for the quantification of the expression of the cellular STxB receptor, the glycosphingolipid globotriaosyl ceramide (Gb 3 or CD77). We found that compared with normal tissue, the expression of Gb 3 was strongly increased in colorectal adenocarcinomas and their metastases, but not in benign adenomas. Short-term primary cultures were prepared from samples of 43 patients, and STxB uptake was studied by immunofluorescence microscopy. Of a given tumor sample, on average, 80% of the cells could visibly bind STxB, and upon incubation at 37°C, STxB was transported to the Golgi apparatus, following the retrograde route. This STxB-specific intracellular targeting allows the molecule to avoid recycling and degradation, and STxB could consequently be detected on tumor cells even 5 days after initial uptake. In conclusion, the targeting properties of STxB could be diverted for the delivery of contrast agents to human colorectal tumors and their metastases, whose early detection and specific targeting remains one of the principal challenges in oncology. [Mol Cancer Ther 2008;7(8):2498 -508]

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Efficient methods for tumor targeting are eagerly awaited and must satisfy several challenges: molecular specificity, transport through physiologic barriers, and capacity to withstand extracellular or intracellular degradation and inactivation by the immune system. Through interaction with its hosts, the intestinal pathogen-produced Shiga toxin has evolved molecular properties that are of interest in this context. Its nontoxic B-subunit binds to the cellular toxin receptor, glycosphingolipid Gb 3 , which is highly expressed on human cancers and has recently been reported to be involved in the formation of metastasis in colorectal cancers. Its function as a target for cancer therapy has already been addressed in xenograft experiments. We here show that after oral or i.v. injections in mice, the B-subunit targets spontaneous digestive Gb 3 -expressing adenocarcinomas. The nontumoral mucosa is devoid of labeling, with the exception of rare enteroendocrine and CD11b-positive cells. As opposed to other delivery tools that are often degraded or recycled on cancer cells, the B-subunit stably associates with these cells due to its trafficking via the retrograde transport route. This can be exploited for the in vivo delivery of contrast agents to tumors, as exemplified using fibered confocal fluorescence endoscopy and positron emission tomography (PET) imaging. In conclusion, the data presented in this manuscript lay the groundwork for a novel delivery technology that, in addition to its use for molecular imaging applications such as noninvasive PET, could also be exploited for targeted tumor therapies.