Aggregation of amyloid-β (Aβ) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aβ clearance pathway in the brain is cellular uptake and degradation. However, how Aβ traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aβ in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aβ endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aβ traffics through Rab11-positive recycling vesicles. A blockage of this Aβ recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aβ accumulation. Inhibition of lysosomal enzymes results in Aβ accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aβ uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aβ trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aβ endocytic trafficking to lysosomes for degradation is a major Aβ clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aβ capable of causing neurotoxicity and seeding amyloid.

Rab proteins comprise a family of small GTPases that serve a regulatory role in vesicular membrane traffic. Geranylgeranylation of these proteins on C-terminal cysteine motifs is crucial for their membrane association and function. This post-translational modification is catalysed by rab geranylgeranyl transferase (Rab-GGTase), a multisubunit enzyme consisting of a catalytic heterodimer and an accessory component, named rab escort protein (REP)-1. Previous in vitro studies have suggested that REP-1 presents newly synthesized rab proteins to the catalytic component of the enzyme, and forms a stable complex with the prenylated proteins following the transfer reaction. According to this model, a cellular factor would be required to dissociate the rab protein from REP-1 and to allow it to recycle in the prenylation reaction. RabGDP dissociation inhibitor (RabGDI) was considered an ideal candidate for this role, given its established function in mediating membrane association of prenylated rab proteins. Here we demonstrate that dissociation from REP-1 and binding of rab proteins to the membrane do not require RabGDI or other cytosolic factors. The mechanism of REP-1-mediated membrane association of rab5 appears to be very similar to that mediated by RabGDI. Furthermore, REP-1 and RabGDI share several other functional properties, the ability to inhibit the release of GDP and to remove rab proteins from membranes; however, RabGDI cannot assist in the prenylation reaction. These data suggest that REP-1 is per se sufficient to chaperone newly prenylated rab proteins to their target membranes.

The biological function of receptors is determined by their appropriate trafficking through the endosomal pathway. Following internalization, the transferrin (Tf) receptor quantitatively recycles to the plasma membrane, whereas the epidermal growth factor (EGF) receptor undergoes degradation. To determine how Tf and EGF engage these two different pathways we imaged their binding and early endocytic pathway in live cells using total internal reflection fluorescence microscopy (TIRF-M). We find that EGF and Tf bind to distinct plasma membrane regions and are incorporated into different endocytic vesicles. After internalization, both EGF-enriched and Tf-enriched vesicles interact with endosomes containing early endosome antigen 1 (EEA1). EGF is incorporated and retained in these endosomes, while Tf-containing vesicles rapidly dissociate and move to a juxtanuclear compartment. Endocytic vesicles carrying EGF recruit more Rab5 GTPase than those carrying Tf, which, by strengthening their association with EEA1-enriched endosomes, may provide a mechanism for the observed cargo-specific sorting. These results reveal pre-endocytic sorting of Tf and EGF, a specialized role for EEA1-enriched endosomes in EGF trafficking, and a potential mechanism for cargo-specified sorting of endocytic vesicles by these endosomes.

Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce "membranous web" formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.

PI 3-kinases are important regulators of endocytic trafficking. We have previously proposed a model in which the Rab5 GTPase recruits EEA1 to the early endosome both directly, by binding to EEA1, and indirectly, through the recruitment of the p150/hVps34 PI 3-kinase and the production of PI[3]P in the endosomal membrane. In this study we have examined this model in vivo. We find that both endogenous hVps34 and p150 are targeted to enlarged endosomal structures in cells expressing constitutively activated Rab5, where they are significantly colocalized with EEA1. Recombinant fragments of p150 disrupt the endosomal localization of EEA1, showing that p150 is required for EEA1 targeting. We further analyzed the mechanism of GTP-dependent Rab5-p150 binding, and showed the p150 HEAT and WD40 domains are required for binding, whereas deletion of the protein kinase domain increases binding to Rab5. Overexpression of constitutively active Rab5 caused a redistribution of epitope-tagged hVps34 and p150 to Rab5-positive endosomes. However, subcellular fractionation showed that this was not due to a significant recruitment of hVps34 or p150 from the cytosolic to the particulate fraction. These data suggest that the binding of Rab5 to the HEAT/WD40 domains of p150 is important in regulating the localization of hVps34/p150. However, Rab5 does not appear to act by directly recruiting p150/hVps34 complexes from the cytosol to the endosomal membrane.

We have characterized the intracellular fate of Toxoplasma in bone marrow-derived macrophages following two disparate modes of uptake: phagocytosis vs active invasion. The fate of parasite-containing vacuoles was followed by immunofluorescence localization of endogenous endocytic markers to track phagocytic processing in pulse-infected cells. During uptake of both opsonized and dead parasites, host cell plasma membrane MHC class II molecules and FcR were internalized into the phagosome and then gradually lost. Maturation of phagosomes containing Toxoplasma was a rapid, dynamic process of sequential fusion with early endosomes, late endosomes, and lysosomes that was complete within 15 min. Toxoplasma-containing phagosomes were transiently positive for transferrin receptor between 0 and 2.5 min, then contained the cation-independent mannose 6-phosphate receptor between 2.5 and 7.5 min, and finally matured to lysosome-like compartments containing lysosomal membrane glycoprotein 1 and the proton pump, but lacking cation-independent mannose 6-phosphate receptor. Toxoplasma-containing phagosomes also sequentially acquired host proteins that regulate endocytic fusion including rab5, N-ethylmaleimide-sensitive factor, and rab7. In marked contrast, MHC class II molecules and FcR were excluded from the parasitophorous vacuole formed by active parasite invasion. The parasitophorous vacuole also failed to acquire any host compartmental markers or fusion proteins analyzed. Our results indicate that Toxoplasma evades endocytic processing due to an absence of host regulatory proteins necessary to drive endocytic fusion, and that this divergence from normal maturation occurs during the formation of the primary vacuole.

In fungal hyphal cells, intracellular membrane trafficking is constrained by the relatively long intracellular distances and the mode of growth, exclusively by apical extension. Endocytosis plays a key role in hyphal tip growth, which involves the coupling of secretory membrane delivery to the apical region with subapical compensatory endocytosis. However, the identity, dynamics and function of filamentous fungal endosomal compartments remain largely unexplored. Aspergillus nidulans RabA(Rab5) localizes to a population of endosomes that show long range bidirectional movement on microtubule (MT) tracks and are labelled with FM4-64 shortly after dye internalization. RabA(Rab5) membranes do not overlap with largely static mature endosomes/vacuoles. Impaired delivery of dynein to the MT plus ends or downregulation of cytoplasmic dynein using the dynein heavy chain nudA1(ts)mutation results in accumulation of RabA(Rab5) endosomal membranes in an abnormal NudA1 compartment at the tip, strongly supporting the existence in A. nidulans hyphal tips of a dynein loading region. We show that the SynA synaptobrevin endocytic recycling cargo traffics through this region, which strongly supports the contention that polarized hyphal growth involves the association of endocytic recycling with the plus ends of MTs located at the tip, near the endocytic internalization collar.

Cargo transfer from trans-Golgi network (TGN)-derived transport carriers to endosomes involves a still undefined set of tethering/fusion events. Here we analyze a molecular interaction that may play a role in this process. We demonstrate that the GGAs, a family of Arf-dependent clathrin adaptors involved in selection of TGN cargo, interact with the Rabaptin-5-Rabex-5 complex, a Rab4/Rab5 effector regulating endosome fusion. These interactions are bipartite: GGA-GAE domains recognize an FGPLV sequence (residues 439-443) in a predicted random coil of Rabaptin-5 (a sequence also recognized by the gamma1- and gamma2-adaptin ears), while GGA-GAT domains bind to the C-terminal coiled-coils of Rabaptin-5. The GGA-Rabaptin-5 interaction decreases binding of clathrin to the GGA-hinge domain, and expression of green fluorescent protein (GFP)-Rabaptin-5 shifts the localization of endogenous GGA1 and associated cargo to enlarged early endosomes. These observations thus identify a binding sequence for GAE/gamma-adaptin ear domains and reveal a functional link between proteins regulating TGN cargo export and endosomal tethering/fusion events.

Endosomal sorting is essential for cell homeostasis. Proteins targeted for degradation are retained in the maturing endosome vacuole while others are recycled to the cell surface or sorted to the biosynthetic pathway via tubular transport carriers. Sorting nexin (SNX) proteins containing a BAR (for Bin-Amphiphysin-Rvs) domain are key regulators of phosphoinositide-mediated, tubular-based endosomal sorting, but how such sorting is co-ordinated with endosomal maturation is not known. Here, using well-defined Rab GTPases as endosomal compartment markers, we have analyzed the localization of SNX1 [endosome-to-trans-Golgi network (TGN) transport as part of the SNX-BAR-retromer complex], SNX4 (cargo-recycling from endosomes to the plasma membrane) and SNX8 (endosomes-to-TGN trafficking in a retromer-independent manner). We show that these SNX-BARs are primarily localized to early endosomes, but display the highest frequency of tubule formation at the moment of early-to-late endosome transition: the Rab5-to-Rab7 switch. Perturbing this switch shifts SNX-BAR tubulation to early endosomes, resulting in SNX1-decorated tubules that lack retromer components VPS26 and VPS35, suggesting that both early and late endosomal characteristics of the endosome are important for SNX-BAR-retromer-tubule formation. We also establish that SNX4, but not SNX1 and SNX8, is associated with the Rab11-recycling endosomes and that a high frequency of SNX4-mediated tubule formation is observed as endosomes undergo Rab4-to-Rab11 transition. Our study therefore provides evidence for fine-tuning between the processes of endosomal maturation and the formation of endosomal tubules. As tubulation is required for SNX1-, SNX4- and SNX8-mediated sorting, these data reveal a previously unrecognized co-ordination between maturation and tubular-based sorting.

Rab22a is a small GTPase that is expressed ubiquitously in mammalian tissues and displays the highest sequence homology to Rab5. In BHK-21 cells, overexpression of the wild-type Rab22a caused formation of abnormally large vacuole-like structures containing the early-endosomal antigen EEA1 but not Rab11, a marker of recycling endosomes or the late-endosomal/lysosomal markers LAMP-1 and lyso-bis-phosphatidic acid. In HeLa cells, overexpressed Rab22a was found on smaller EEA1-positive endosomes, but a portion of the protein was also found in the Golgi complex. Using the yeast two-hybrid system and a biochemical pull-down assay, the GTP-bound form of Rab22a was found to interact with the N-terminus of EEA1. In HeLa cells overexpressing Rab22a or its mutants affected in the GTPase cycle, no significant changes were observed in the uptake of Alexa-transferrin. However, the GTPase-deficient Rab22a Q64L mutant caused a redistribution of transferrin-positive endosomes to the leading edges of cells and a fragmentation of the Golgi complex. In BHK cells, the Q64L mutant caused the accumulation of a fluid phase marker, TRITC-dextran, and a lysosomal hydrolase, aspartylglucosaminidase, in abnormal vacuole-like structures that contained both early and late endosome markers. Both the wild-type Rab22a and the Q64L mutant were found to interfere with the degradation of EGF. These results suggest that Rab22a may regulate the dynamic interactions of endosomal compartments and it may be involved in the communication between the biosynthetic and early endocytic pathways.

Invasion of glial cells by the human polyomavirus, JC virus (JCV), leads to a rapidly progressing and uniformly fatal demyelinating disease known as progressive multifocal leukoencephalopathy. The endocytic trafficking steps used by JCV to invade cells and initiate infection are not known. We demonstrated that JCV infection was inhibited by dominant defective and constitutively active Rab5-GTPase mutants that acted at distinct steps in endosomal sorting. We also found that labeled JCV colocalized with labeled cholera toxin B and with caveolin-1 (cav-1) on early endosomes following internalization by clathrin-dependent endocytosis. JCV entry and infection were both inhibited by dominant defective mutants of eps15 and Rab5-GTPase. Expression of a dominant-negative scaffolding mutant of cav-1 did not inhibit entry or infection by JCV. A single-cell knockdown experiment using cav-1 shRNA did not inhibit JCV entry but interfered with a downstream trafficking event important for infection. These data show that JCV enters cells by clathrin-dependent endocytosis, is transported immediately to early endosomes, and is then sorted to a caveolin-1-positive endosomal compartment. This latter step is dependent on Rab5-GTPase, cholesterol, caveolin-1, and pH. This is the first example of a ligand that enters cells by clathrin-dependent endocytosis and is then sorted from early endosomes to caveosomes, indicating that caveolae-derived vesicles play a more important role than previously realized in sorting cargo from early endosomes.

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

Rab proteins comprise a family of monomeric GTPases that control cellular membrane traffic. Rab21 is a poorly characterised member with no known function. Human Rab21 cDNA from K562 cells was subcloned into GFP expression vectors to generate Rab21 and Rab21 mutants defective in either GTP hydrolysis (Rab21 Q78L) or binding (Rab21 T33N) for transfection studies in HeLa cells. Confocal fluorescence microscopy and ultrastructural studies revealed Rab21 to be predominantly localised to the early endocytic pathway, on vesicles containing earlyendosomal antigen 1 EEA1, transferrin receptor and internalised ligands. EEA1 was localised to enlarged endosomes in Rab21 wild-type expressing cells but the GTP hydrolysis and GDP binding mutants had unique phenotypes labelling tubular reticular structures and the trans-Golgi network, respectively. Early endosome localisation for Rab21 was confirmed in a hepatoma cell line that allowed analysis of the subcellular distribution of the endogenous protein. Comparison of the localisation of Rab21 with other Rabs revealed extensive colocalisation with early endocytic variants Rab4, Rab5, Rab17 and Rab22 but much less overlap with those associated with late endosomes, recycling endosomes and the early secretory pathway. Cells expressing Rab21 T33N had defects in endocytosis of transferrin and epidermal growth factor and failed to effectively deliver the latter ligand to late endosomes and lysosomes for degradation. Collectively, our data provide the first characterisation of Rab21 function in early endosome dynamics.

The efficient engulfment of apoptotic cells by professional or nonprofessional phagocytes is critical to maintain mammalian homeostasis. To identify molecules involved in the engulfment of apoptotic cells, we established a retrovirus-based expression cloning system coupled with the engulfment assay. By screening a cDNA library of a mouse macrophage cell line, we identified two small GTPase family members (RhoG and Rab5) that enhanced the engulfment of apoptotic cells. By examining other small GTPase family members, we found that Rac1 enhanced the engulfment of apoptotic cells, whereas RhoA inhibited the process. Accordingly, the expression of a dominant-negative form of RhoG or Rac1 in primary macrophage cultures severely reduced the ability of the macrophages to engulf apoptotic cells, and a dominant-negative form of RhoA enhanced the process. These results indicated that the efficient engulfment of apoptotic cells requires the concerted action of small GTPase family members. We demonstrated previously that NIH3T3 cells expressing the alphav beta3 integrin efficiently engulf apoptotic cells in the presence of milk fat globule epidermal growth factor 8 via a phosphatidylserine-dependent mechanism. The dominant-negative form of RhoG or Rac1 inhibited this process, which suggested RhoG and Rac1 are also involved in the integrin-mediated engulfment.

RABEX-5 and other exchange factors with VPS9 domains regulate endocytic trafficking through activation of the Rab family GTPases RAB5, RAB21 and RAB22. Here we report the crystal structure of the RABEX-5 catalytic core in complex with nucleotide-free RAB21, a key intermediate in the exchange reaction pathway. The structure reveals how VPS9 domain exchange factors recognize Rab GTPase substrates, accelerate GDP release and stabilize the nucleotide-free conformation. We further identify an autoinhibitory element in a predicted amphipathic helix located near the C terminus of the VPS9 domain. The autoinhibitory element overlaps with the binding site for the multivalent effector RABAPTIN-5 and potently suppresses the exchange activity of RABEX-5. Autoinhibition can be partially reversed by mutation of conserved residues on the nonpolar face of the predicted amphipathic helix or by assembly of the complex with RABAPTIN-5.

High-risk human papillomaviruses (HPVs) are small nonenveloped DNA viruses with a strict tropism for squamous epithelium. The viruses are causative agents of cervical cancer and some head and neck cancers, but their differentiation-dependent life cycles have made them difficult to study in simple cell culture. Thus, many aspects of early HPV infection remain mysterious. We recently showed the high-risk HPV type 31 (HPV31) enters its natural host cell type via caveola-dependent endocytosis, a distinct mechanism from that of the closely related HPV16 (Smith et al., J. Virol. 81:9922-9931, 2007). Here, we determined the downstream trafficking events after caveolar entry of HPV31 into human keratinocytes. After initial plasma membrane binding, HPV31 associates with caveolin-1 and transiently localizes to the caveosome before trafficking to the early endosome and proceeding through the endosomal pathway. Caveosome-to-endosome transport was found to be Rab5 GTPase dependent. Although HPV31 capsids were observed in the lysosome, Rab7 GTPase was dispensable for HPV31 infection, suggesting that viral genomes escape from the endosomal pathway prior to Rab7-mediated capsid transport. Consistent with this, the acidic pH encountered by HPV31 within the early endosomal pathway induces a conformational change in the capsid resulting in increased DNase susceptibility of the viral genome, which likely aids in uncoating and/or endosomal escape. The entry and trafficking route of HPV31 into human keratinocytes represents a unique viral pathway by which the virions use caveolar entry to eventually access a low-pH site that appears to facilitate endosomal escape of genomes.

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.

The human granulocytic ehrlichiosis (HGE) agent resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. Double immunofluorescence labeling was used to characterize the nature of the HGE agent replicative inclusions and to compare them with inclusions containing the human monocytic ehrlichia, Ehrlichia chaffeensis, in HL-60 cells. Although both Ehrlichia spp. can coinfect HL-60 cells, they resided in separate inclusions. Inclusions of both Ehrlichia spp. were not labeled with either anti-lysosome-associated membrane protein 1 or anti-CD63. Accumulation of myeloperoxidase-positive granules were seen around HGE agent inclusions but not around E. chaffeensis inclusions. 3-(2, 4-Dinitroanilino)-3'-amino-N-methyldipropylamine and acridine orange were not localized to either inclusion type. Vacuolar-type H+-ATPase was not colocalized with HGE agent inclusions but was weakly colocalized with E. chaffeensis inclusions. E. chaffeensis inclusions were labeled with the transferrin receptor, early endosomal antigen 1, and rab5, but HGE agent inclusions were not. Some HGE agent and E. chaffeensis inclusions colocalized with major histocompatibility complex class I and II antigens. These two inclusions were not labeled for annexins I, II, IV, and VI; alpha-adaptin; clathrin heavy chain; or beta-coatomer protein. Vesicle-associated membrane protein 2 colocalized to both inclusions. The cation-independent mannose 6-phosphate receptor was not colocalized with either inclusion type. Endogenously synthesized sphingomyelin, from C6-NBD-ceramide, was not incorporated into either inclusion type. Brefeldin A did not affect the growth of either Ehrlichia sp. in HL-60 cells. These results suggest that the HGE agent resides in inclusions which are neither early nor late endosomes and does not fuse with lysosomes or Golgi-derived vesicles, while E. chaffeensis resides in an early endosomal compartment which accumulates the transferrin receptor.

Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response. Rab5 is known as a key regulator of the early endocytic pathway and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominant-negative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.

BACKGROUND

Trypanosomatids utilise polycistronic transcription for production of the vast majority of protein-coding mRNAs, which operates in the absence of gene-specific promoters. Resolution of nascent transcripts by polyadenylation and trans-splicing, together with specific rates of mRNA turnover, serve to generate steady state transcript levels that can differ in abundance across several orders of magnitude and can be developmentally regulated. We used a targeted oligonucleotide microarray, representing the strongly developmentally-regulated T. brucei membrane trafficking system and approximately 10% of the Trypanosoma brucei genome, to investigate both between-stage, or differentiation-dependent, transcriptome changes and within-stage flexibility in response to various challenges.

RESULTS

6% of the gene cohort are developmentally regulated, including several small GTPases, SNAREs, vesicle coat factors and protein kinases both consistent with and extending previous data. Therefore substantial differentiation-dependent remodeling of the trypanosome transcriptome is associated with membrane transport. Both the microarray and qRT-PCR were then used to analyse transcriptome changes resulting from specific gene over-expression, knockdown, altered culture conditions and chemical stress. Firstly, manipulation of Rab5 expression results in co-ordinate changes to clathrin protein expression levels and endocytotic activity, but no detectable changes to steady-state mRNA levels, which indicates that the effect is mediated post-transcriptionally. Secondly, knockdown of clathrin or the variant surface glycoprotein failed to perturb transcription. Thirdly, exposure to dithiothreitol or tunicamycin revealed no evidence for a classical unfolded protein response, mediated in higher eukaryotes by transcriptional changes. Finally, altered serum levels invoked little transcriptome alteration beyond changes to expression of ESAG6/7, the transferrin receptor.

CONCLUSIONS

While trypanosomes regulate mRNA abundance to effect the major changes accompanying differentiation, a given differentiated state appears transcriptionally inflexible. The implications of the absence of a transcriptome response in trypanosomes for both virulence and models of life cycle progression are discussed.

Agonist-induced endocytosis and processing of the G protein-coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)- or hemagglutinin epitope-tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5-GFP and Rab4-GFP in early endosomes, and subsequently with Rab11-GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand-receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa(R)-transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine-Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase-dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed.

The Rab family of small GTPases serves as cellular regulators of vesicular transport in all eukaryotes. Higher organisms have a vastly expanded number of Rabs. Some of these would presumably perform cell-type or tissue-specific functions. Roles for Rab proteins in several aspects of cellular physiology that are peculiar to neurons have been revealed. Rabs, in conjunction with their effectors and regulators, participate in central nervous system development (Rab23), polarized neurite growth (Rab8 and Rab11), endocytosis and axonal retrograde transport (Rab5 and Rab7). At chemical synapses, Rabs perform specific functions in synaptic vesicle exocytosis (Rab3) and postsynaptic compartment dynamics of glutamate receptors (Rab8). Much less is known about the role of Rabs in astroglia/oligodendroglia-specific processes, although Rab family members that are glia-enriched (Rab22B, Rab40C) have been reported. We summarized and discussed recent findings on the roles played by Rabs in neurons and glia in the central nervous system, and speculate on future directions.

The small GTPase Rab5, which cycles between active (GTP-bound) and inactive (GDP-bound) states, plays essential roles in membrane budding and trafficking in the early endocytic pathway. However, the molecular mechanisms underlying the Rab5-regulated processes are not fully understood other than the targeting event to early endosomes. Here, we report a novel Rab5-binding protein, RIN3, that contains many functional domains shared with other RIN members and additional Pro-rich domains. RIN3 displays the same biochemical properties as RIN2, the stimulator and stabilizer of GTP-Rab5. In addition, RIN3 exhibits its unique intracellular localization. RIN3 expressed in HeLa cells localized to cytoplasmic vesicles and the RIN3-positive vesicles contained Rab5 but not the early endosomal marker EEA1. Transferrin appeared to be transported partly through the RIN3-positive vesicles to early endosomes. RIN3 was also capable of interacting via its Pro-rich domain with amphiphysin II, which contains SH3 domain and participates in receptor-mediated endocytosis. Interestingly, cytoplasmic amphiphysin II was translocated into the RIN3- and Rab5-positive vesicles when co-expressed with RIN3. These results indicate that RIN3 biochemically characterized as the stimulator and stabilizer for GTP-Rab5 plays an important role in the transport pathway from plasma membrane to early endosomes.