Effects of Brefeldin A on Synthesis and Intracellular Transport of Ganglioside GT3 by Chick Embryo Retina Cells

Abstract: Ganglioside GT3 is the precursor of c-series gangliosides. It is synthesized by sialylation of GD3 and is expressed in nervous tissue of birds and mammals at early stages of development. In this study we examined the sub-Golgi location of GT3 synthesis and the mechanism of its transport from the site of synthesis to the plasma membrane in chicken embryo retina cells in culture. Neural retina cells from 10-day-old chick embryo were cultured with [³H]galactose in the absence (control cells) or in the presence of 1 µg/ml brefeldin A (BFA). At the end of the labeling period, the fraction of labeled gangliosides transported to the plasma membrane was determined. For this, cells were treated with C . perfringens neuraminidase in conditions to desialylate only those gangliosides that were transported to the plasma membrane and consequently accessible to the enzyme. After neuraminidase treatment of cells, gangliosides were isolated, purified, and the pattern of radioactivity analyzed by HPTLC-fluorography. It was found that BFA blocked the synthesis of complex gangliosides without affecting the synthesis of GM3, GD3, and GT3. Furthermore, in BFA-treated cells, GM3, GD3, and GT3 were protected from the action of added neuraminidase, indicating an intracellular localization and, hence, an inhibition of their transport to the plasma membrane. The results indicate that synthesis of the first intermediates of a-, b-, and c- series gangliosides occurs in a proximal Golgi compartment and that the proximal Golgi-synthesized gangliosides (GM3, GD3, and GT3) use a transport mechanism that is dependent on ADP ribosylation factor and coatomer proteins.     

Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein

We have investigated the intracellular traffic of PrP(c), a glycosylphosphatidylinositol (GPI)-anchored protein implicated in spongiform encephalopathies. A fluorescent functional green fluorescent protein (GFP)-tagged version of PrP(c) is found at the cell surface and in intracellular compartments in SN56 cells. Confocal microscopy and organelle-specific markers suggest that the protein is found in both the Golgi and the recycling endosomal compartment. Perturbation of endocytosis with a dynamin I-K44A dominant-negative mutant altered the steady-state distribution of the GFP-PrP(c), leading to the accumulation of fluorescence in unfissioned endocytic intermediates. These pre-endocytic intermediates did not seem to accumulate GFP-GPI, a minimum GPI-anchored protein, suggesting that PrP(c) trafficking does not depend solely on the GPI anchor. We found that internalized GFP-PrP(c) accumulates in Rab5-positive endosomes and that a Rab5 mutant alters the steady-state distribution of GFP-PrP(c) but not that of GFP-GPI between the plasma membrane and early endosomes. Therefore, we conclude that PrP(c) internalizes via a dynamin-dependent endocytic pathway and that the protein is targeted to the recycling endosomal compartment via Rab5-positive early endosomes. These observations indicate that traffic of GFP-PrP(c) is not determined predominantly by the GPI anchor and that, different from other GPI-anchored proteins, PrP(c) is delivered to classic endosomes after internalization.     

The transport of low density lipoprotein-derived cholesterol to the plasma membrane is defective in NPC1 cells

Niemann-Pick disease type C (NPC) is characterized by lysosomal storage of cholesterol and gangliosides, which results from defects in intracellular lipid trafficking. Most studies of NPC1 have focused on its role in intracellular cholesterol movement. Our hypothesis is that NPC1 facilitates the egress of cholesterol from late endosomes, which are where active NPC1 is located. When NPC1 is defective, cholesterol does not exit late endosomes; instead, it is carried along to lysosomal storage bodies, where it accumulates. In this study, we addressed whether cholesterol is transported from endosomes to the plasma membrane before reaching NPC1-containing late endosomes. Our study was conducted in Chinese hamster ovary cell lines that display the classical NPC biochemical phenotype and belong to the NPC1 complementation group. We used three approaches to test whether low density lipoprotein (LDL)-derived cholesterol en route to NPC1-containing organelles passes through the plasma membrane. First, we used cyclodextrins to measure the arrival of LDL cholesterol at the plasma membrane and found that the arrival of LDL cholesterol in a cyclodextrin-accessible pool was significantly delayed in NPC1 cells. Second, the movement of LDL cholesterol to NPC1-containing late endosomes was assessed and found to be normal in Chinese hamster ovary mutant 3-6, which exhibits defective movement of plasma membrane cholesterol to intracellular membranes. Third, we examined the movement of plasma membrane cholesterol to the endoplasmic reticulum and found that this pathway is intact in NPC1 cells, i.e. it does not pass through NPC1-containing late endosomes. Our data suggest that in NPC1 cells LDL cholesterol traffics directly through endosomes to lysosomes, bypassing the plasma membrane, and is trapped there because of dysfunctional NPC1.     

A novel assay to demonstrate an intersection of the exocytic and endocytic pathways at early endosomes

The mechanism of transport of membrane proteins from the trans-Golgi to the cell surface is still poorly understood. Previous studies suggested that basolateral membrane proteins, such as the transferrin receptor and the asialoglycoprotein receptor H1, take an indirect route to the plasma membrane via an intracellular, most likely endosomal intermediate. To define this compartment we developed a biochemical assay based on the very definition of endosomes. The assay is based on internalizing anti-H1 antibodies via the endocytic cycle of the receptor itself. Internalized antibody formed immune complexes with newly synthesized H1, which had been pulse-labeled with [(35)S]sulfate and chased out of the trans-Golgi for a period of time that was insufficient for H1 to reach the surface. Hence, antibody capture occurred intracellularly. Double-immunofluorescence labeling demonstrated that antibody-containing compartments also contained transferrin and thus corresponded to early and recycling endosomes. The results therefore demonstrate an intracellular intersection of the exocytic and endocytic pathways with implications for basolateral sorting.     

Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases

Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli.     

Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and immortalized hypothalamic neurons

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds alpha-melanocyte-stimulating hormone (alpha-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of alpha-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toalpha-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that alpha-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.     

Sialidase NEU3 is a peripheral membrane protein localized on the cell surface and in endosomal structures

Sialidase NEU3 is also known as the plasma-membrane-associated form of mammalian sialidases, exhibiting a high substrate specificity towards gangliosides. In this respect, sialidase NEU3 modulates cell-surface biological events and plays a pivotal role in different cellular processes, including cell adhesion, recognition and differentiation. At the moment, no detailed studies concerning the subcellular localization of NEU3 are available, and the mechanism of its association with cellular membranes is still unknown. In the present study, we have demonstrated that sialidase NEU3, besides its localization at the plasma membrane, is present in intracellular structures at least partially represented by a subset of the endosomal compartment. Moreover, we have shown that NEU3 present at the plasma membrane is internalized and locates then to the recycling endosomal compartment. The enzyme is associated with the outer leaflet of the plasma membrane, as shown by selective cell-surface protein biotinylation. This evidence is in agreement with the ability of NEU3 to degrade gangliosides inserted into the plasma membrane of adjacent cells. Moreover, the mechanism of the protein association with the lipid bilayer was elucidated by carbonate extraction. Under these experimental conditions, we have succeeded in solubilizing NEU3, thus demonstrating that the enzyme is a peripheral membrane protein. In addition, Triton X-114 phase separation demonstrates further the hydrophilic nature of the protein. Overall, these results provide important information about the biology of NEU3, the most studied member of the mammalian sialidase family.     

Multiple pathways of exocytosis, endocytosis, and membrane recycling: validation of a Golgi route

A number of pathways for intracellular membrane traffic have been detected in various cell types. The major established routes are: 1) the lysosomal pathway, which is the major route utilized in phagocytic and cultured cells; 2) the transcellular route, which represents the major type of traffic in nonfenestrated, capillary endothelial cells and which also appears to be the preferred route for the transport of immunoglobulins (intact) across cells; 3) the exocytosis pathway, utilized in secretory cells for discharge of secretory products, and which is also believed to be used for delivery of intrinsic membrane glycoproteins; 4) the plasmalemma to Golgi route, also highly developed in secretory cells, which is believed to be utilized for the recycling of secretory granule membranes; and 5) the biosynthetic pathways for transport of secretory products, lysosomal enzymes, and membrane proteins from the endoplasmic reticulum to the Golgi complex and for transport of lysosomal enzymes from the Golgi complex to lysosomes. It has become clear that cells repeatedly reutilize or recycle the membranes used in these various transport operations. Clathrin-coated vesicles have been found to be involved in transport along all these routes, which suggests that there are multiple populations of coated vesicles with different transport functions in every cell. It has become clear that the Golgi complex is the site where the membrane and product traffic converges and is sorted and directed to its correct destinations. The validation of a transport route from the cell surface to the Golgi complex raises the possibility that bound ligands and membrane constituents could be modified or repaired in transit during recycling through the Golgi complex, which is a biosynthetic compartment.     

Birbeck granules are subdomains of endosomal recycling compartment in human epidermal Langerhans cells, which form where Langerin accumulates.

Birbeck granules are unusual rod-shaped structures specific to epidermal Langerhans cells, whose origin and function remain undetermined. We investigated the intracellular location and fate of Langerin, a protein implicated in Birbeck granule biogenesis, in human epidermal Langerhans cells. In the steady state, Langerin is predominantly found in the endosomal recycling compartment and in Birbeck granules. Langerin internalizes by classical receptor-mediated endocytosis and the first Birbeck granules accessible to endocytosed Langerin are those connected to recycling endosomes in the pericentriolar area, where Langerin accumulates. Drug-induced inhibition of endocytosis results in the appearance of abundant open-ended Birbeck granule-like structures appended to the plasma membrane, whereas inhibition of recycling induces Birbeck granules to merge with a tubular endosomal network. In mature Langerhans cells, Langerin traffic is abolished and the loss of internal Langerin is associated with a concomitant depletion of Birbeck granules. Our results demonstrate an exchange of Langerin between early endosomal compartments and the plasma membrane, with dynamic retention in the endosomal recycling compartment. They show that Birbeck granules are not endocytotic structures, rather they are subdomains of the endosomal recycling compartment that form where Langerin accumulates. Finally, our results implicate ADP-ribosylation factor proteins in Langerin trafficking and the exchange between Birbeck granules and other endosomal membranes.     

Expression of GD2 and GD3 gangliosides in human embryonic neural stem cells

NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.     

Constitutive endocytosis and recycling of the neuronal glutamate transporter, excitatory amino acid carrier 1

The neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), has a diverse array of physiologic and metabolic functions. There is evidence that there is a relatively large intracellular pool of EAAC1 both in vivo and in vitro, that EAAC1 cycles on and off the plasma membrane, and that EAAC1 cell surface expression can be rapidly regulated by intracellular signals. Despite the possible relevance of EAAC1 trafficking to both physiologic and pathologic processes, the cellular machinery involved has not been defined. In the present study, we found that agents that disrupt clathrin-dependent endocytosis or plasma membrane cholesterol increased steady-state levels of biotinylated EAAC1 in C6 glioma cells and primary neuronal cultures. Acute depletion of cholesterol increased the V(max) for EAAC1-mediated activity and had no effect on Na(+)-dependent glycine transport in the same system. These agents also impaired endocytosis as measured using a reversible biotinylating reagent. Co-expression with dominant-negative variants of dynamin or the clathrin adaptor, epidermal growth factor receptor pathway substrate clone 15, increased the steady-state levels of biotinylated myc-EAAC1. EAAC1 immunoreactivity was found in a subcellular fraction enriched in early endosome antigen 1 (EEA1) isolated by differential centrifugation and partially co-localized with EEA1. Co-expression of a dominant-negative variant of Rab11 (Rab11 S25N) reduced steady-state levels of biotinylated myc-EAAC1 and slowed constitutive delivery of myc-EAAC1 to the plasma membrane. Together, these observations suggest that EAAC1 is constitutively internalized via a clathrin- and dynamin-dependent pathway into early endosomes and that EAAC1 is trafficked back to the cell surface via the endocytic recycling compartment in a Rab11-dependent mechanism. As one defines the machinery required for constitutive trafficking of EAAC1, it may be possible to determine how intracellular signals regulate EAAC1 cell surface expression.     

Antagonistic roles of ESCRT and Vps class C/HOPS complexes in the recycling of yeast membrane proteins

In Saccharomyces cerevisiae, deficiencies in the ESCRT machinery trigger the mistargeting of endocytic and biosynthetic ubiquitinated cargoes to the limiting membrane of the vacuole. Surprisingly, impairment of this machinery also leads to the accumulation of various receptors and transporters at the plasma membrane in both yeast and higher eukaryotes. Using the well-characterized yeast endocytic cargo uracil permease (Fur4p), we show here that the apparent stabilization of the permease at the plasma membrane in ESCRT mutants results from an efficient recycling of the protein. Whereas several proteins as well as internalized dyes are known to be recycled in yeast, little is known about the machinery and molecular mechanisms involved. The SNARE protein Snc1p is the only cargo for which the recycling pathway is well characterized. Unlike Snc1p, endocytosed Fur4p did not pass through the Golgi apparatus en route to the plasma membrane. Although ubiquitination of Fur4p is required for its internalization, deubiquitination is not required for its recycling. In an attempt to identify actors in this new recycling pathway, we found an unexpected phenotype associated with loss of function of the Vps class C complex: cells defective for this complex are impaired for recycling of Fur4p, Snc1p, and the lipophilic dye FM4-64. Genetic analyses indicated that these phenotypes were due to the functioning of the Vps class C complex in trafficking both to and from the late endosomal compartment.     

Subcellular distribution and biosynthesis of rat liver gangliosides

Gangliosides have generally been assumed to be localized primarily in the plasma membrane. Analysis of gangliosides from isolated subcellular membrane fractions of rat liver indicated that 76% of the total ganglioside sialic acid was present in the plasma membrane. Mitochondria and endoplasmic reticulum fractions, while containing only low levels of gangliosides on a protein basis, each contained approx. 10% of total ganglioside sialic acid. Gangliosides also were present in the Golgi apparatus and nuclear membrane fractions, and soluble gangliosides were in the supernatant. Individual gangliosides were non-homogeneously distributed and each membrane fraction was characterized by a unique ganglioside composition. Plasma membrane contained only 14 and 28% of the total GD1a and GD3, respectively, but 80-90% of the GM1, GD1b, GT1b and GQ1b. Endoplasmic reticulum, when corrected for plasma membrane contamination, contained only trace amounts of GM1, GD1b, GT1b and GQ1b, but 11 and 5% of the total GD1a and GD3, respectively. The ganglioside composition of highly purified endoplasmic reticulum was similar. Ganglioside biosynthetic enzymes were concentrated in the Golgi apparatus. However, low levels of these enzymes were present in the highly purified endoplasmic reticulum fractions. Pulse-chase experiments with [3H]galactose revealed that total gangliosides were labeled first in the Golgi apparatus, mitochondria and supernatant within 10 min. Labeled gangliosides were next observed at 30 min in the endoplasmic reticulum, plasma membrane and nuclear membrane fractions. Analysis of the individual gangliosides also revealed that GM3, GM1, GD1a and GD1b were labeled first in the Golgi apparatus at 10 min. These studies indicate that gangliosides synthesized in the Golgi apparatus may be transported not only to the plasma membrane, but to the endoplasmic reticulum and to other internal endomembranes as well.     

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.     

Dynamin- and Rab5-Dependent Endocytosis of a Ca(2+)-Activated K(+) Channel, KCa2.3

Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca(2+)-activated K(+) channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane.     

Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recycling compartment en route to the trans-Golgi network and a subpopulation of late endosomes

Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. After endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment. A large fraction of the receptors return to the plasma membrane, but some are delivered to the trans-Golgi network and/or late endosomes. Over the course of an hour, the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the endocytic recycling compartment. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes.     

The Plasma Membrane Sialidase NEU3 Regulates the Malignancy of Renal Carcinoma Cells by Controlling β1 Integrin Internalization and Recycling

The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy.     

Investigating Endocytic Pathways to the Endoplasmic Reticulum and to the Cytosol Using SNAP‐Trap

Cholera toxin enters cells via an unusual pathway that involves trafficking through endosomes to the endoplasmic reticulum (ER). Whether the toxin induces its own pathway or travels along a physiological retrograde route is not known. To study its trafficking, we labeled cholera toxin B (CTB) or endogenous plasma membrane proteins with a small chemical compound, benzylguanine, which covalently reacts with the protein SNAP‐tag. Using ER‐targeted SNAP‐tag as reporter, we found that transport of CTB to the ER depends on dynamin‐2 and syntaxin 5. Plasma membrane proteins and a fluid‐phase marker added to the medium were also transported to the ER. This flux was not affected by exposing cells to CTB but was inhibited by depleting syntaxin 5 and increased by depleting dynamin‐2. As a control for confined intracellular localization of ER‐targeted SNAP‐tag we used adenovirus‐5, which traffics to endosomes and then escapes into the cytosol. The virus did not react with ER‐targeted SNAP but with cytosolic SNAP. Together, our results establish a new method (SNAP‐trap) to study trafficking of different cargo to the ER and the cytosol and provide evidence for the existence of a constitutive pathway from the cell surface to the ER .