Domeless (Dome) is an IL-6-related cytokine receptor that activates a conserved JAK/STAT signalling pathway during Drosophila development. Despite good knowledge of the signal transduction pathway in several models, the role of receptor endocytosis in JAK/STAT activation remains poorly understood. Using both in vivo genetic analysis and cell culture assays, we show that ligand binding of Unpaired 1 (Upd1) induces clathrin-dependent endocytosis of receptor-ligand complexes and their subsequent trafficking through the endosomal compartment towards the lysosome. Surprisingly, blocking trafficking in distinct endosomal compartments using mutants affecting either Clathrin heavy chain, rab5, Hrs or deep orange led to an inhibition of the JAK/STAT pathway, whereas this pathway was unchanged when rab11 was affected. This suggests that internalization and trafficking are both required for JAK/STAT activity. The requirement for clathrin-dependent endocytosis to activate JAK/STAT signalling suggests a model in which the signalling 'on' state relies not only on ligand binding to the receptor at the cell surface, but also on the recruitment of the complex into endocytic vesicles on their way to lysozomes. Selective activation of the pool of receptors marked for degradation thus provides a way to tightly control JAK/STAT activity.

The compartmentalized production of superoxide (*O(2)(-)) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-kappaB following interleukin 1beta (IL-1beta) stimulation. It remains unclear how *O(2)(-) produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated *O(2)(-) movement out of IL-1beta-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-kappaB activation. The relative outward permeability of NADPH-dependent *O(2)(-) from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for *O(2)(-). In these studies, approximately 60% of *O(2)(-) efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that *O(2)(-) and Cl(-) are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1beta-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF-kappaB following IL-1beta stimulation. Together, these results suggest that *O(2)(-) exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of *O(2)(-) at the endosomal surface may produce the localized H(2)O(2) required for redox-activation of NF-kappaB.

ALS2/alsin is a member of guanine nucleotide exchange factors for the small GTPase Rab5 (Rab5GEFs), which act as modulators in endocytic pathway. Loss-of-function mutations in human ALS2 account for a number of juvenile recessive motor neuron diseases (MNDs). However, the normal physiological role of ALS2 in vivo and the molecular mechanisms underlying motor dysfunction are still unknown. To address these issues, we have generated mice homozygous for disruption of the Als2 gene. The Als2-null mice observed through 21 months of age demonstrated no obvious developmental, reproductive or motor abnormalities. However, immunohistochemical and electrophysiological analyses identified an age-dependent, slowly progressive loss of cerebellar Purkinje cells and disturbance of spinal motor neurons associated with astrocytosis and microglial cell activation, indicating a subclinical dysfunction of motor system in Als2-null mice. Further, quantitative epidermal growth factor (EGF)-uptake analysis identified significantly smaller-sized EGF-positive endosomes in Als2-null fibroblasts, suggesting an alteration of endosome/vesicle trafficking in the cells. Collectively, while loss of ALS2 does not produce a severe disease phenotype in mice, these Als2-null animals should provide a useful model with which to understand the interplay between endosomal dynamics and the long-term viability of large neurons such as Purkinje cells and spinal motor neurons.

After exocytosis, synaptic vesicles rapidly endocytose and recycle but little is known about the molecular mechanisms involved. Rab5 is a ubiquitous low molecular weight GTP-binding protein required for endosomal fusion in fibroblasts. We have now raised polyclonal and monoclonal antibodies to rat Rab5 and show that in rat brain, Rab5 is a major synaptic vesicle protein. Immunoisolation of vesicular organelles from brain with antibodies to either Rab3A and Rab5 as small GTP-binding proteins or with synaptophysin as general synaptic vesicle marker demonstrates that there are overlapping populations of synaptic vesicles containing either Rab5 or Rab3A or both, suggesting a stage-specific association of these low-molecular weight GTP-binding proteins with synaptic vesicles. Our data provide the first biochemical evidence that synaptic vesicle recycling involves an endosomal intermediate similar to that of the receptor-mediated endocytosis pathway.

Clustering of macrophage Fc gamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fc gamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large >> or =3 microm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (< or =1 microm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.

The key pathological features of Alzheimer's disease include synaptic dysfunction, profound changes in the cholinergic system, and deposition of beta-amyloid peptides generated by proteolytic processing of the amyloid-beta precursor protein (APP). However, the pathways linking APP with synaptic activity and cholinergic neuronal function are poorly understood. We report here that APP is essential in regulating the presynaptic expression and activity of the high-affinity choline transporter (CHT), a molecule that mediates the rate-limiting step of cholinergic synaptic transmission in both the neuromuscular junction and central cholinergic neurons. Loss of APP leads to aberrant localization of CHT at the neuromuscular synapses and reduced CHT activity at cholinergic projections. At the cellular level, we show that APP and CHT can be found in Rab5-positive endosomal compartments and that APP affects CHT endocytosis. Furthermore, we demonstrate that APP interacts with CHT through the C-terminal domain, providing support for a specific and direct regulation of CHT by APP through protein-protein interactions. These results identify a physiological activity of APP in cholinergic neurons, and our data indicate that deregulation of APP function may contribute to cholinergic impairment and AD pathogenesis.

It is increasingly recognized that the compartmental organization of signaling processes has a profound influence on cellular behavior. However, our inability to influence these compartmental events in a spatially restricted and acute manner limits our understanding of causation. To determine whether local compartmental loss of a phosphoinositide disrupts the normal traffic of specific cargoes through endosomes, we developed the use of a regulated dimerization device, here designed to compartmentally modify the phosphoinositide content of Rab5-positive endosomes. This modification is effected through the specific regulated recruitment of the 3-phosphatase myotubularin to endosomal membranes in intact cells. The selective manipulation of endosomal phosphatidylinositols (PIs) demonstrates that it is the phosphatidylinositol 3-phosphate (PtdIns3P) or its metabolite PtdIns(3,5)P2 within this compartment that determines the normal maturation of the endosomal compartment and the flux of receptors through it. On local loss of PtdIns3P/PtdIns(3,5)P2, the endosomal compartment itself fails to continue its normal maturation process, leading to the microtubule-dependent tubularization of the endosomal network. Furthermore, it is shown that endosomal PtdIns3P/PtdIns(3,5)P2 is necessary for transferrin receptor traffic through this compartment while having an effect on EGF receptor (EGFR) entry into and sorting from this endosome compartment. The ability to acutely and selectively influence compartmental behavior as exemplified here for endomsomes clearly illustrates the power of the approach used to dissect the role of localized signals and events.

The structure and function of the primary cilium as a sensory organelle depends on a motor-protein-powered intraflagellar transport system (IFT); defective IFT results in retinal degeneration and pleiotropic disorders such as the Bardet Biedl syndrome (BBS) and defective hedgehog (HH) signaling. Protein transport to the cilium involves Rab GTPases. Rab8, together with a multi protein complex of BBS proteins, recruits cargo to the basal body for transport to the cilium. Loss of Rab23 in mice recapitulates the HH phenotype but its function in HH signaling is unknown. Here we established a novel protocol, based on fluorescence recovery after photo-bleaching (FRAP), allowing the quantitative analysis of protein transport into the cilium of MDCK cells. We compared the effect of Rab8, Rab5 and Rab23 on the ciliary transport of the HH-associated transmembrane receptor Smoothened, the microtubular tip protein EB1, and the receptor protein Kim1. Ciliary FRAP confirmed the role of Rab8 in protein entry to the cilium. Dominant negative Rab5 had no impact on the ciliary transport of Smoothened or EB1, but slowed the recovery of the apical protein Kim1 in the cilium. Depletion of Rab23 or expression of dominant-negative Rab23 decreased the ciliary steady state specifically of Smoothened but not EB1 or Kim1, suggesting a role of Rab23 in protein turnover in the cilium.

CXCR4, the stromal cell-derived factor-1 receptor, plays an important role in the migration of hematopoietic progenitor/stem cells. The surface and cytoplasmic expression of CXCR4 on human hematopoietic CD34(+) cells was investigated. We show that its surface expression is low, whereas a large part of CXCR4 protein is sequestered intracellularly. Using confocal microscopy, we demonstrated that CXCR4 is colocalized with EEA-1, Rab5, Rab4, and Rab11, which are localized in early and recycling endosomes. No significant colocalization of CXCR4 with lysosomal markers CD63 and Lamp-1 was detected. Using antibody feeding experiments, we report a role for CXCR4 constitutive endocytosis in subcellular localization in stably transduced UT7-CXCR4-GFP and CD34(+) cells. Agonist-independent endocytosis of CXCR4 occurs through clathrin-coated vesicles. These data implicate a constitutive endocytosis in the regulation of CXCR4 membrane expression and suggest that constitutive endocytosis may be involved in the regulation of trafficking the human hematopoietic progenitor/stem cells to and in the bone marrow microenvironment.

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.

We demonstrate the presence of a glycosylphosphatidylinositol (GPI) anchor-specific endosomal pathway in the protozoan pathogen Trypanosoma brucei. In higher eukaryotes evidence indicates that GPI-anchored proteins are transported in both the endocytic and exocytic systems by mechanisms involving sequestration into specific membrane microdomains and consequently sorting into distinct compartments. This is potentially extremely important in trypanosomatids as the GPI anchor is the predominant mechanism for membrane attachment of surface macromolecules, including the variant surface glycoprotein (VSG). A highly complex developmentally regulated endocytic network, vital for nutrient uptake and evasion of the immune response, exists in T. brucei. In common with mammalian cells an early endosomal compartment is defined by Rab5 small GTPases, which control transport processes through the endosomal system. We investigate the function of two trypanosome Rab5 homologues. TbRAB5A and TbRAB5B, which colocalize in the procyclic stage, are distinct in the bloodstream form of the parasite. TbRAB5A endosomes contain VSG and transferrin, endocytosed by the T. brucei GPI-anchored transferrin receptor, whereas TbRAB5B endosomes contain the transmembrane protein ISG(100) but neither VSG nor transferrin. These findings indicate the presence of trypanosome endosomal pathways trafficking proteins through specific routes depending on the mode of membrane attachment. Ectopic expression of mutant TbRAB5A or -5B indicates that TbRAB5A plays a role in LDL endocytosis, whereas TbRAB5B does not, but both have a role in fluid phase endocytosis. Hence TbRAB5A and TbRAB5B have distinct functions in the endosomal system of T. brucei. A developmentally regulated GPI-specific endosomal pathway in the bloodstream form suggests that specialized transport of GPI-anchored proteins is required for survival in the mammalian host.

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-adaptin regulate membrane recycling.

Phosphatidylinositol 3-phosphate [PI(3)P] is a phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P2, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling.

The p75 neurotrophin receptor (p75(NTR)) plays multiple roles in neuronal physiology through interactions with many ligands and coreceptors. However, its intracellular neuronal trafficking prior to and after neurotrophin activation is still poorly characterized. We have previously shown that in response to nerve growth factor (NGF), p75(NTR) is retrogradely transported along the axons of motor neurons (MNs) in carriers shared with NGF, brain-derived neurotrophic factor and the tyrosine kinase receptor TrkB. Here, we report that NGF does not enhance the internalization or degradation of p75(NTR), which undergoes a rapid dynamin-dependent and clathrin-independent recycling process in MNs. Instead, incubation of cells with NGF leads to the redirection of a pool of plasma membrane p75(NTR) into clathrin-coated pits. The subsequent internalization of p75(NTR) via clathrin-mediated endocytosis, as well as the activity of Rab5, are essential for the sorting of the p75(NTR)-containing endosomes to the axonal retrograde transport pathway and for the delivery of p75(NTR) to the soma. Our findings suggest that the spatial regulation of p75(NTR) signalling is controlled by these ligand-driven routes of endocytosis.

Rab11-FIP2 is a recently described member of the Rip11/Rab11-FIP/Rab coupling protein family of Rab11 interacting proteins. Rab11-FIP2 interacts with both Rab11 and myosin Vb and co-localizes with Rab11 in both HeLa and Madin-Darby canine kidney cells (Hales, C. M., Griner, R., Hobdy-Henderson, K. C., Dorn, M. C., Hardy, D., Kumar, R., Navarre, J., Chan, E. K., Lapierre, L. A., and Goldenring, J. R. (2001) J. Biol. Chem. 276, 39067-390751). Here, we characterized the specificity of the interaction between Rab11-FIP2 and Rab11 and report that it does not interact with Rab4, Rab3, Rab5, Rab6, or Rab7. We demonstrate that the COOH-terminal region of Rab11-FIP2, which contains the Rab11 binding domain (RBD), is necessary and sufficient for its early endosomal membrane association. In contrast, the amino-terminal region, which contains a phospholipid binding C2-domain, by itself was insufficient for membrane binding. Expression of a deletion mutant of Rab11-FIP2, containing the RBD, caused tubulation of a transferrin receptor-positive early endosomal compartment in HeLa cells. Endogenous Rab11 was also associated with this compartment. This phenotype cannot be reversed by excess wild-type Rab11, or dominant-positive Rab11 (Rab11Q70L), suggesting that Rab11-FIP2 functions downstream of Rab11 in endosomal trafficking.

Sealing of phagosomes is accompanied by the disappearance of phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) from their cytoplasmic leaflet. Elimination of PtdIns(4,5)P(2), which is required for actin remodeling during phagosome formation, has been attributed to hydrolysis by phospholipase C and phosphorylation by phosphatidylinositol 3-kinase. We found that two inositol 5-phosphatases, OCRL and Inpp5B, become associated with nascent phagosomes. Both phosphatases, which are Rab5 effectors, associate with the adaptor protein APPL1, which is recruited to the phagosomes by active Rab5. Knockdown of APPL1 or inhibition of Rab5 impairs association of OCRL and Inpp5B with phagosomes and prolongs the presence of PtdIns(4,5)P(2) and actin on their membranes. Even though APPL1 can serve as an anchor for Akt, its depletion accentuated the activation of the kinase, likely by increasing the amount of PtdIns(4,5)P(2) available to generate phosphatidylinositol (3,4,5)-trisphosphate. Thus, inositol 5-phosphatases are important contributors to the phosphoinositide remodeling and signaling that are pivotal for phagocytosis.

T cell activation requires sustained signaling at the immune synapse, a specialized interface with the antigen-presenting cell (APC) that assembles following T cell antigen receptor (TCR) engagement by major histocompatibility complex (MHC)-bound peptide. Central to sustained signaling is the continuous recruitment of TCRs to the immune synapse. These TCRs are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the immune synapse. Here, we have investigated the interplay of IFT20 with the Rab GTPase network that controls recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown (IFT20KD) resulted in a block in the recycling pathway, leading to a build-up of recycling TCRs in Rab5(+) endosomes. Recycling of the transferrin receptor (TfR), but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components IFT52 and IFT57 were found to act together with IFT20 to regulate TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and immune synapse assembly, and underscore the trafficking-related function of the IFT system beyond ciliogenesis.

Japanese encephalitis virus (JEV) is a mosquito-borne pathogenic flavivirus responsible for acute viral encephalitis in humans. The cellular entry of JEV is poorly characterized in terms of molecular requirements and pathways. Here we present a systematic study of the internalization mechanism of JEV in fibroblasts and neuroblastoma cells. To verify the roles of distinct pathways of cell entry, we used fluorescently labeled virus particles, a combination of pharmacological inhibitors, RNA interference (RNAi), and dominant-negative (DN) mutants of regulatory proteins involved in endocytosis. Our study demonstrates that JEV infects fibroblasts in a clathrin-dependent manner, but it deploys a clathrin-independent mechanism to infect neuronal cells. The clathrin-independent pathway requires dynamin and plasma membrane cholesterol. Virus binding to neuronal cells leads to rapid actin rearrangements and an intact and dynamic actin cytoskeleton, and the small GTPase RhoA plays an important role in viral entry. Immunofluorescence analysis of viral colocalization with endocytic markers showed that JEV traffics through Rab5-positive early endosomes and that release of the viral nucleocapsid occurs at the level of the early and not the late endosomes.

Previous studies have demonstrated that the internalization of the angiotensin II type 1A receptor (AT(1A)R) may be mediated by both beta-arrestin-sensitive and -insensitive mechanisms. Therefore, we have used the AT(1A)R carboxyl-terminal tail to screen a rat brain yeast two-hybrid expression library for novel AT(1A)R-interacting proteins that might contribute to the regulation of AT(1A)R internalization. We have identified Rab5a as an AT(1A)R-binding protein that selectively associates with the AT(1A)R and not with the beta2-adrenergic receptor. A Rab5a-S34N mutant defective in GTP binding does not prevent the internalization of the AT(1A)R but does prevent the trafficking of the AT(1A)R into larger hollow cored vesicular structures. Agonist activation of the AT(1A)R promotes both the formation of Rab5a.AT(1A)R protein complexes and Rab5a GTP binding. Rab5a interactions with the AT(1A)R are mediated in part by the last 10 amino acid residues of the AT(1A)R carboxyl-terminal tail, and although a mutant receptor lacking these residues internalizes normally, it does not redistribute into larger hollow vesicles. Our data suggest that AT(1A)R activation modulates Rab5a activity leading to the homotypic fusion of endocytic vesicles. These observations suggest that vesicular cargo proteins, such as the AT(1A)R, may control their targeting between intracellular compartments by directly regulating the activity of components of the intracellular trafficking machinery such as Rab5a.

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

Survival or destruction of a pathogen following phagocytosis depends, in part, on fusion events between the phagosome and the endosomal or lysosomal compartments. Here we use an in vitro assay to show that phagosome-endosome fusion is regulated by the small GTPase rab5 and that fusion events are influenced by an internalized live organism, Listeria monocytogenes (LM). We compare the in vitro fusion of phagosomes containing heat-killed organisms (dead LM) with that of phagosomes containing a live nonhemolytic mutant (live LMhly-). Unlike the wild-type organism, LMhly- remains trapped inside the phagosome. Phagosome-endosome fusion was reconstituted using biotinylated organisms and endosomes containing horseradish peroxidase conjugated with avidin. With both live LMhly- and dead LM preparations, in vitro phagosome-endosome fusion was time-, temperature-, and cytosol-dependent. Live LMhly- phagosomes exhibited a faster rate of fusion. Fusion in both preparations was regulated by rab5 and possibly by other GTPases. Anti-rab5 antibodies and immunodepletion of cytosolic rab5 inhibited fusion. Addition of glutatione S-transferase-rab5 in the GTP form stimulated phagosome-endosome fusion, whereas addition of a dominant negative mutant of rab5 blocked fusion. Purified live LMhly- phagosomal membranes were enriched in rab5 as revealed by Western blotting, compared with dead LM phagosomes. Fusion of endosomes with dead LM containing phagosomes required ATP and was inhibited by ATP depletion and by N-ethylmaleimide (NEM) and anti-NEM-sensitive factor (NSF) antibodies. Unexpectedly, phagosome-endosome fusion with live LMhly--containing phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Western blot analysis revealed that live LMhly--containing phagosomes were enriched for membrane-bound NSF, while dead LM containing phagosomes contained low or undetectable quantities. Washing live LMhly--containing phagosomes with 0.5 M KCl removed NSF associated with the membranes and rendered them NEM, ATP, anti-NSF antibody sensitive for fusion. We conclude that rab5 regulates phagosome-endosome fusion and that live microorganisms can up-regulate this process by recruiting rab5 to the membrane.

An apical brush border is a characteristic of many mature epithelia. This dynamic structure consists of dense microvilli supported by F-actin bundles that protrude into the apical cytoplasm, where they are crosslinked by spectrin and myosin II to form the terminal web. Little is known about the terminal web, through which vesicles transit to and from the apical membrane. Analysis of mutations in beta(Heavy)-spectrin, the Drosophila brush border spectrin, reveals that this protein is necessary for the maintenance of Rab5 endosomes in the midgut. As a consequence, an apical H+ V-ATPase that is probably responsible for lumenal acidification is lost both from the brush border and Rab5 endosomes. Epistasis tests indicate that beta(Heavy)-spectrin is required during endocytosis after Dynamin and before Rab5-mediated endosome activities. These data are consistent with the location of spectrin in the terminal web, and suggest that this molecule is required for correct sorting decisions at the early endosome.

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-alpha results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both alpha-adaptin and clathrin. Upon EGF stimulation, Eps15 and alpha-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.

The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination.