The novel lipid raft adaptor p18 controls endosome dynamics by anchoring the MEK–ERK pathway to late endosomes

The regulation of endosome dynamics is crucial for fundamental cellular functions, such as nutrient intake/digestion, membrane protein cycling, cell migration and intracellular signalling. Here, we show that a novel lipid raft adaptor protein, p18, is involved in controlling endosome dynamics by anchoring the MEK1–ERK pathway to late endosomes. p18 is anchored to lipid rafts of late endosomes through its N‐terminal unique region. p18^?/? mice are embryonic lethal and have severe defects in endosome/lysosome organization and membrane protein transport in the visceral endoderm. p18^?/? cells exhibit apparent defects in endosome dynamics through perinuclear compartment, such as aberrant distribution and/or processing of lysosomes and impaired cycling of Rab11‐positive recycling endosomes. p18 specifically binds to the p14–MP1 complex, a scaffold for MEK1. Loss of p18 function excludes the p14–MP1 complex from late endosomes, resulting in a downregulation of the MEK–ERK activity. These results indicate that the lipid raft adaptor p18 is essential for anchoring the MEK–ERK pathway to late endosomes, and shed new light on a role of endosomal MEK–ERK pathway in controlling endosome dynamics.     

α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α_1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α_1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α_1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α_1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α_1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).     

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

Rab2 is a conserved Rab GTPase with a well-established role in secretory pathway function and phagocytosis. Here we demonstrate that Drosophila Rab2 is recruited to late endosomal membranes, where it controls the fusion of LAMP-containing biosynthetic carriers and lysosomes to late endosomes. In contrast, the lysosomal GTPase Gie/Arl8 is only required for late endosome-lysosome fusion, but not for the delivery of LAMP to the endocytic pathway. We also find that Rab2 is required for the fusion of autophagosomes to the endolysosomal pathway, but not for the biogenesis of lysosome-related organelles. Surprisingly, Rab2 does not rely on HOPS-mediated vesicular fusion for recruitment to late endosomal membranes. Our work suggests that Drosophila Rab2 is a central regulator of the endolysosomal and macroautophagic/autophagic pathways by controlling the major heterotypic fusion processes at the late endosome.     

Sorting of membrane and fluid at the apical pole of polarized Madin-Darby canine kidney cells

When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (相似文献   

Rab9 GTPase regulates late endosome size and requires effector interaction for its stability

Rab9 GTPase resides in a late endosome microdomain together with mannose 6-phosphate receptors (MPRs) and the tail-interacting protein of 47 kDa (TIP47). To explore the importance of Rab9 for microdomain establishment, we depleted the protein from cultured cells. Rab9 depletion decreased late endosome size and reduced the numbers of multilamellar and dense-tubule-containing late endosomes/lysosomes, but not multivesicular endosomes. The remaining late endosomes and lysosomes were more tightly clustered near the nucleus, implicating Rab9 in endosome localization. Cells displayed increased surface MPRs and lysosome-associated membrane protein 1. In addition, cells showed increased MPR synthesis in conjunction with MPR missorting to the lysosome. Surprisingly, Rab9 stability on late endosomes required interaction with TIP47. Rabs are thought of as independent, prenylated entities that reside either on membranes or in cytosol, bound to GDP dissociation inhibitor. These data show that Rab9 stability is strongly influenced by a specific effector interaction. Moreover, Rab9 and the proteins with which it interacts seem critical for the maintenance of specific late endocytic compartments and endosome/lysosome localization.     

Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells

The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.     

Endosomal lipid accumulation in NPC1 leads to inhibition of PKC, hypophosphorylation of vimentin and Rab9 entrapment

Background information. Within the group of lysosomal storage diseases, NPC1 [NPC (Niemann‐Pick type C) 1] disease is a lipidosis characterized by excessive accumulation of free cholesterol as well as gangliosides, glycosphingolipids and fatty acids in the late E/L (endosomal/lysosomal) system (Chen et al., 2005 ) due to a defect in late endosome lipid egress. We have previously demonstrated that expression of the small GTPase Rab9 in NPC1 cells can rescue the lipid transport block phenotype (Walter et al., 2003 ), albeit by an undefined mechanism. Results. To investigate further the mechanism by which Rab9 facilitates lipid movement from late endosomes we sought to identify novel Rab9 binding/interacting proteins. In the present study, we report that Rab9 interacts with the intermediate filament phosphoprotein vimentin and this interaction is altered by lipid accumulation in late endosomes, which results in inhibition of PKC (protein kinase C) and hypophosphorylation of vimentin, leading to late endosome dysfunction. Intermediate filament hypophosphorylation, aggregation and entrapment of Rab9 ultimately leads to transport defects and inhibition of lipid egress from late endosomes. Conclusions. These results reveal a previously unappreciated interaction between Rab proteins and intermediate filaments in regulating intracellular lipid transport.     

Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.     

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55^Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4⁺ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P₂, therefore controlling Pr55^Gag membrane association. Rab27a also controls PI(4,5)P₂ levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55^Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P₂ production and, consequently, HIV-1 replication.     

Rab14 and its exchange factor FAM116 link endocytic recycling and adherens junction stability in migrating cells

Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions.     

Rab11 facilitates cross-talk between autophagy and endosomal pathway through regulation of Hook localization

During autophagy, double-membrane autophagosomes deliver sequestered cytoplasmic content to late endosomes and lysosomes for degradation. The molecular mechanism of autophagosome maturation is still poorly characterized. The small GTPase Rab11 regulates endosomal traffic and is thought to function at the level of recycling endosomes. We show that loss of Rab11 leads to accumulation of autophagosomes and late endosomes in Drosophila melanogaster. Rab11 translocates from recycling endosomes to autophagosomes in response to autophagy induction and physically interacts with Hook, a negative regulator of endosome maturation. Hook anchors endosomes to microtubules, and we show that Rab11 facilitates the fusion of endosomes and autophagosomes by removing Hook from mature late endosomes and inhibiting its homodimerization. Thus induction of autophagy appears to promote autophagic flux by increased convergence with the endosomal pathway.     

Phylogeny and evolution of Rab7 and Rab9 proteins

Background  

An important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network.     

Spatiotemporal Resolution of Rab9 and CI‐MPR Dynamics in the Endocytic Pathway

Rab9 is a small GTPase that localizes to the trans‐Golgi Network (TGN) and late endosomes. Its main function has long been connected to the recycling of mannose‐6‐phosphate receptors (MPRs). However, recent studies link Rab9 also to autophagy and lysosome biogenesis. In this paper, using confocal imaging, we characterize for the first time the live dynamics of the Rab9 constitutively active mutant, Rab9Q66L. We find that it localizes predominantly to late endosomes and that its expression in HeLa cells disperses TGN46 and cation‐independent (CI‐MPR) away from the Golgi yet, has no effect on the retrograde transport of CI‐MPR. We also show that CI‐MPR and Rab9 enter the endosomal pathway together at the transition stage between early, Rab5‐positive, and late, Rab7a‐positive, endosomes. CI‐MPR localizes transiently to separate domains on these endosomes, where vesicles carrying CI‐MPR attach and detach within seconds. Taken together, our results demonstrate that Rab9 mediates the delivery of CI‐MPR to the endosomal pathway, entering the maturing endosome at the early‐to‐late transition.      

Vaccinia Virus Infection Requires Maturation of Macropinosomes

The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live‐cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome‐associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow‐up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late‐penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration.      

Rabex-5 Is a Rab22 Effector and Mediates a Rab22-Rab5 Signaling Cascade in Endocytosis

Rabex-5 targets to early endosomes and functions as a guanine nucleotide exchange factor for Rab5. Membrane targeting is critical for Rabex-5 to activate Rab5 on early endosomes in the cell. Here, we report the identification of Rab22 as a binding site on early endosomes for direct recruitment of Rabex-5 and activation of Rab5, establishing a Rab22-Rab5 signaling relay to promote early endosome fusion. Rab22 in guanosine 5′-O-(3-thio)triphosphate-loaded form, but not guanosine diphosphate-loaded form, binds to the early endosomal targeting domain (residues 81-230) of Rabex-5 in pull-down assays. Rabex-5 targets to Rab22-containing early endosomes, and Rab22 knockdown by short hairpin RNA abrogates the membrane targeting of Rabex-5 in the cell. In addition, coexpression of Rab22 and Rab5 shows synergistic enlargement of early endosomes, and this synergy is dependent on Rabex-5, providing further support for the collaboration of the two Rab GTPases in regulation of endosome dynamics. This novel Rab22–Rabex-5–Rab5 cascade is functionally important for the endocytosis and degradation of epidermal growth factor.     

A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments

Rab5 is a small GTPase that plays roles in the homotypic fusion of early endosomes and regulation of intracellular vesicle transport. We show here that expression of GFP-tagged GTPase-deficient form of Rab5b (Rab5bQ79L) in NRK cells results in the sequential formation of three morphologically and functionally distinct types of endosomes. Expression of GFP-Rab5bQ79L initially caused a homotypic fusion of early endosomes accompanying a redistribution of the TGN-resident cargo molecules, and subsequent fusion with late endosomes/lysosomes, leading to the formation of giant hybrid organelles with features of early endosomes and late endosomes/lysosomes. Surprisingly, the giant endosomes gradually fragmented and shrunk, leading to the accumulation of early endosome clusters and concurrent reformation of late endosomes/lysosomes, a process accelerated by treatment with a phosphatidylinositol-3-kinase (PI(3)K) inhibitor, wortmannin. We postulate that such sequential processes reflect the biogenesis and maintenance of late endosomes/lysosomes, presumably via direct fusion with early endosomes and subsequent fission from hybrid organelles. Thus, our findings suggest a regulatory role for Rab5 in not only the early endocytic pathway, but also the late endocytic pathway, of membrane trafficking in coordination with PI(3)K activity.     

Cellular vacuolation induced by Clostridium perfringens epsilon-toxin

The epsilon-toxin of Clostridium perfringens forms a heptamer in the membranes of Madin-Darby canine kidney cells, leading to cell death. Here, we report that it caused the vacuolation of Madin-Darby canine kidney cells. The toxin induced vacuolation in a dose-dependent and time-dependent manner. The monomer of the toxin formed oligomers on lipid rafts in membranes of the cells. Methyl-β-cyclodextrin and poly(ethylene glycol) 4000 inhibited the vacuolation. Epsilon-toxin was internalized into the cells. Confocal microscopy revealed that the internalized toxin was transported from early endosomes (early endosome antigen 1 staining) to late endosomes and lysosomes (lysosomal-associated membrane protein 2 staining) and then distributed to the membranes of vacuoles. Furthermore, the vacuolation was inhibited by bafilomycin A1, a V-type ATPase inhibitor, and colchicine and nocodazole, microtubule-depolymerizing agents. The early endosomal marker green fluorescent protein-Rab5 and early endosome antigen 1 did not localize to vacuolar membranes. In contrast, the vacuolar membranes were specifically stained by the late endosomal and lysosomal marker green fluorescent protein-Rab7 and lysosomal-associated membrane protein 2. The vacuoles in the toxin-treated cells were stained with LysoTracker Red DND-99, a marker for late endosomes and lysosomes. A dominant negative mutant of Rab7 prevented the vacuolization, whereas a mutant form of Rab5 was less effective. These results demonstrate, for the first time, that: (a) oligomers of epsilon-toxin formed in lipid rafts are endocytosed; and (b) the vacuoles originating from late endosomes and lysosomes are formed by an oligomer of epsilon-toxin.     

Endosome to Golgi Transport of Ricin Is Independent of Clathrin and of the Rab9- and Rab11-GTPases

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.     

Tubular cell-derived exosomal miR-150-5p contributes to renal fibrosis following unilateral ischemia-reperfusion injury by activating fibroblast in vitro and in vivo

Unilateral ischemia reperfusion injury (UIRI) with longer ischemia time is associated with an increased risk of acute renal injury and chronic kidney disease. Exosomes can transport lipid, protein, mRNA, and miRNA to corresponding target cells and mediate intercellular information exchange. In this study, we aimed to investigate whether exosome-derived miRNA mediates epithelial-mesenchymal cell communication relevant to renal fibrosis after UIRI. The secretion of exosomes increased remarkably in the kidney after UIRI and in rat renal tubular epithelium cells (NRK-52E) after hypoxia treatment. The inhibition of exosome secretion by Rab27a knockout or GW4869 treatment ameliorates renal fibrosis following UIRI in vivo. Purified exosomes from NRK-52E cells after hypoxia treatment could activate rat kidney fibroblasts (NRK-49F). The inhibition of exosome secretion in hypoxic NRK-52E cells through Rab27a knockdown or GW4869 treatment abolished NRK-49F cell activation. Interestingly, exosomal miRNA array analysis revealed that miR-150-5p expression was increased after hypoxia compared with the control group. The inhibition of exosomal miR-150-5p abolished the ability of hypoxic NRK-52E cells to promote NRK-49F cell activation in vitro, injections of miR-150-5p enriched exosomes from hypoxic NRK-52E cells aggravated renal fibrosis following UIRI, and renal fibrosis after UIRI was alleviated by miR-150-5p-deficient exosome in vivo. Furthermore, tubular cell-derived exosomal miR-150-5p could negatively regulate the expression of suppressor of cytokine signaling 1 to activate fibroblast. Thus, our results suggest that the blockade of exosomal miR-150-5p mediated tubular epithelial cell-fibroblast communication may provide a novel therapeutic target to prevents UIRI progression to renal fibrosis.