Activated receptor tyrosine kinases recruit many signaling proteins to activate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K) consisting of a p85 regulatory protein and a p110 catalytic protein. We have recently shown the p85alpha protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a GAP-defective mutant, p85R274A, resulted in sustained levels of activated platelet-derived growth factor receptors (PDGFRs) and enhanced downstream signaling. In this report we have characterized Rab5- and Rab4-mediated PDGFR trafficking in cells expressing wild type p85 and GAP-defective mutant p85R274A. Wild type p85 overexpressing cells had slower PDGFR trafficking consistent with enhanced GAP activity deactivating Rab5 and Rab4 to block their vesicle trafficking functions. Mutant p85R274A expression increased the internalization rate of PDGFRs, a Rab5-dependent process, without preventing PDGFR ubiquitination. Immunofluorescence studies further demonstrated that p85R274A-expressing cells showed Rab5 accumulation at intracellular locations. Pull-down and FRAP (fluorescence recovery after photobleaching) experiments indicate this is likely membrane-associated Rab5-GTP, sustained due to decreased p85 GAP activity for the p85R274A mutant. These cells also had substantial amounts of activated PDGFRs in Rab4-positive recycling endosomes, a compartment that usually contains primarily deactivated/dephosphorylated receptors. Our results suggest that the PDGFR-associated GAP activity of p85 regulates both Rab5 and Rab4 functions in cells to influence the movement of activated PDGFR through endosomal compartments. Disruption of this regulation by p85R274A expression impacts PDGFR phosphorylation/dephosphorylation, degradation kinetics and downstream signaling by altering the time receptors spend in specific intracellular endosomal compartments. These results demonstrate that the p85alpha protein is an important regulator of Rab-mediated PDGFR trafficking, which significantly impacts receptor signaling and degradation.

Loss of ALS2/alsin function accounts for several recessive motor neuron diseases. ALS2 is a Rab5 activator and its endosomal localization is regulated by Rac1 via macropinocytosis. Here, we show that the pathogenic missense ALS2 mutants fail to be localized to Rac1-induced macropinosomes as well as endosomes, which leads to loss of the ALS2 function as a Rab5 activator on endosomes. Further, these mutants lose the competence to enhance the formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes. Thus, Rac1-induced relocalization of ALS2 might be crucial to exert the ALS2 function associated with the autophagy-endolysosomal degradative pathway.

Rab21, a member of the Rab GTPase family, is known to be involved in membrane trafficking, but its implication in macropinocytosis is unclear. We analyzed the spatiotemporal localization of Rab21 in M-CSF-stimulated RAW264 macrophages by the live-cell imaging of fluorescent protein-fused Rab21. It was demonstrated that wild-type Rab21 was transiently associated with macropinosomes. Rab21 was recruited to the macropinosomes after a decrease in PI(4,5)P(2) and PI(3,4,5)P(3) levels. Although Rab21 was largely colocalized with Rab5, the recruitment of Rab21 to the macropinosomes lagged a minute behind that of Rab5, and preceded that of Rab7. Then, Rab21 was dissociated from the macropinosomes prior to the accumulation of Lamp1, a late endosomal/lysosomal marker. Our analysis of Rab21 mutants revealed that the GTP-bound mutant, Rab21-Q78L, was recruited to the macropinosomes, similarly to wild-type Rab21. However, the GDP-bound mutant, Rab21-T33N, did not localize on the formed macropinosomes, suggesting that the binding of GTP to Rab21 is required for the proper recruitment of Rab21 onto the macropinosomes. However, neither mutation of Rab21 significantly affected the rate of macropinosome formation. These data indicate that Rab21 is a transient component of early and intermediate stages of macropinocytosis, and probably functions in macropinosome maturation before fusing with lysosomal compartments.

Live imaging of subcellular structures is indispensible to advance our understanding of cellular processes. The blurred digital images acquired in light microscopy are, however, complex to analyze, and identification and reconstruction of subcellular structures from such images remains a major challenge. We present a novel, model-based image analysis algorithm to reconstruct outlines of subcellular structures using a sub-pixel representation. The algorithm explicitly accounts for the optical properties of the microscope. We validate the reconstruction performance on synthetic data and apply the new method to fluorescence microscopy images of endosomes identified by the GTPase EGFP-Rab5. The benefits of the new algorithm are outlined by comparison to standard techniques. We demonstrate that the new algorithm leads to better discrimination between different endosomal virus entry pathways and to more robust, accurate, and self-consistent quantification of endosome shape features. This allows establishing a set of features that quantify endosome morphology and robustly capture the dynamics of endosome fusion.

Dorsoventral cell fate in the Drosophila embryo is specified by activation of the Toll receptor, leading to a ventral-to-dorsal gradient across nuclei of the NF-κB transcription factor Dorsal. Toll receptor has been investigated genetically, molecularly, and immunohistologically, but much less is known about its dynamics in living embryos. Using live imaging of fluorescent protein chimeras, we find that Toll is recruited from the plasma membrane to Rab5(+) early endosomes. The distribution of a constitutively active form of Toll, Toll(10b), is shifted from the plasma membrane to early endosomes. Inhibition of endocytosis on the ventral side of the embryo attenuates Toll signaling ventrally and causes Dorsal to accumulate on the dorsal side of the embryo, essentially inverting the dorsal/ventral axis. Conversely, enhancing endocytosis laterally greatly potentiates Toll signaling locally, altering the shape of the Dorsal gradient. Photoactivation and fluorescence recovery after photobleaching studies reveal that Toll exhibits extremely limited lateral diffusion within the plasma membrane, whereas Toll is highly compartmentalized in endosomes. When endocytosis is blocked ventrally, creating an ectopic dorsal signaling center, Toll is preferentially endocytosed at the ectopic signaling center. We propose that Toll signals from an endocytic compartment rather than the plasma membrane. Our studies reveal that endocytosis plays a pivotal role in the spatial regulation of Toll receptor activation and signaling and in the correct shaping of the nuclear Dorsal concentration gradient.

The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling.

Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention.

OBJECTIVE

Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.

Phosphoinositides have emerged as key regulators of membrane traffic through their control of the localization and activity of several effector proteins. Both Rab5 and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] are involved in the early steps of the clathrin-dependent endocytic pathway, but little is known about how their functions are coordinated. We have studied the role of PtdIns(4,5)P(2) and Rab5 in the Drosophila germline during oogenesis. We found that Rab5 is required for the maturation of early endocytic vesicles. We show that PtdIns(4,5)P(2) is required for endocytic-vesicle formation, for Rab5 recruitment to endosomes and, consistently, for endocytosis. Furthermore, we reveal a previously undescribed role of Rab5 in releasing PtdIns(4,5)P(2), PtdIns(4,5)P(2)-binding budding factors and F-actin from early endocytic vesicles. Finally, we show that overexpressing the PtdIns(4,5)P(2)-synthesizing enzyme Skittles leads to an endocytic defect that is similar to that seen in rab5 loss-of-function mutants. Hence, our results argue strongly in favor of the hypothesis that the Rab5-dependant release of PtdIns(4,5)P(2) from endosomes that we discovered in this study is crucial for endocytosis to proceed.

The Nef protein of HIV-1 removes the immune costimulatory proteins CD80 and CD86 from the cell surface by a unique clathrin- and dynamin-independent, actin-based endocytic pathway that deploys coupled activation of c-src and Rac. In this study, we show that, similar to major histocompatibility complex class I (MHCI), Nef subsequently reroutes CD80 and CD86 to the Golgi region. However, not only are CD80/CD86 internalized by a different mechanism from MHCI but also the vesicular pathway of Golgi delivery for CD80/CD86 is distinct from that employed for MHCI. While MHCI passes through early endosomal and sorting compartments marked by Rab5/early embryonic antigen 1 and ADP ribosylation factor 6, respectively, CD80 and CD86 enter endocytic vesicles that do not acquire conventional early endosomal markers but remain accessible to fluid probes. Rather than being delivered to preexisting Rab11-positive recycling compartments, these vesicles recruit Rab11 de novo. Rab11 activity is also necessary for the delivery of CD80/CD86 in these transitional vesicles to the Golgi region. These data reveal an unusual pathway of endocytic vesicular traffic to the Golgi and its recruitment in a viral immune evasion strategy.

OBJECTIVE

Thioredoxin-interacting protein (TXNIP) is an α-arrestin protein whose function is important for the regulation of vascular endothelial growth factor receptor 2 (VEGFR2) signaling and endothelial cell survival. Because VEGFR2 is critical for angiogenesis, we explored the role of TXNIP in VEGF-induced angiogenesis.

RESULTS

TXNIP knockdown inhibited VEGF-induced endothelial cell tube formation and proliferation in cultured human umbilical vein endothelial cell. To elucidate the mechanism by which TXNIP altered VEGFR2 signaling in human umbilical vein endothelial cell, we studied phosphorylation of VEGFR2, phospholipase C gamma-1 (PLCγ1), endothelial NO synthase, and Akt (known as protein kinase B). TXNIP knockdown significantly decreased phosphorylation of VEGFR2 and PLCγ1 at times >5 minutes, but phosphorylation was unchanged at 2 minutes, as was Akt and endothelial NO synthase phosphorylation. Cell-surface biotinylation assay showed that TXNIP knockdown significantly attenuated VEGFR2 internalization. These results suggested that TXNIP was required for sustained VEGFR2 signaling, which is mediated largely by internalized VEGFR2. Rab5 knockdown to inhibit the trafficking and fusion of early endosomes significantly blocked VEGF-induced VEGFR2 internalization and phosphorylation of VEGFR2 and PLCγ1. Immunofluorescence and coimmunoprecipitation showed that TXNIP was part of a complex that included Rab5 and VEGFR2. Finally, TXNIP knockdown prevented the association of VEGFR2 and Rab5.

CONCLUSIONS

Our results show that TXNIP is essential for VEGFR2 internalization in Rab5 positive endosomes, which is required for endothelial cell growth and angiogenesis.

Sorting nexins (SNXs) are key regulators of the endosomal network. In designing an RNAi-mediated loss-of-function screen, we establish that of 30 human SNXs only SNX3, SNX5, SNX9, SNX15 and SNX21 appear to regulate EGF receptor degradative sorting. Suppression of SNX15 results in a delay in receptor degradation arising from a defect in movement of newly internalised EGF-receptor-labelled vesicles into early endosomes. Besides a phosphatidylinositol 3-phosphate- and PX-domain-dependent association to early endosomes, SNX15 also associates with clathrin-coated pits and clathrin-coated vesicles by direct binding to clathrin through a non-canonical clathrin-binding box. From live-cell imaging, it was identified that the activated EGF receptor enters distinct sub-populations of SNX15- and APPL1-labelled peripheral endocytic vesicles, which do not undergo heterotypic fusion. The SNX15-decorated receptor-containing sub-population does, however, undergo direct fusion with the Rab5-labelled early endosome. Our data are consistent with a model in which the EGF receptor enters the early endosome following clathrin-mediated endocytosis through at least two parallel pathways: maturation through an APPL1-intermediate compartment and an alternative more direct fusion between SNX15-decorated endocytic vesicles and the Rab5-positive early endosome.

Integrin recycling is critical for cell migration. Protein kinase D (PKD) mediates signals from the platelet-derived growth factor receptor (PDGF-R) to control αvβ3 integrin recycling. We now show that Rabaptin-5, a Rab5 effector in endosomal membrane fusion, is a PKD substrate. PKD phosphorylates Rabaptin-5 at Ser407, and this is both necessary and sufficient for PDGF-dependent short-loop recycling of αvβ3, which in turn inhibits α5β1 integrin recycling. Rab4, but not Rab5, interacts with phosphorylated Rabaptin-5 toward the front of migrating cells to promote delivery of αvβ3 to the leading edge, thereby driving persistent cell motility and invasion that is dependent on this integrin. Consistently, disruption of Rabaptin-5 Ser407 phosphorylation reduces persistent cell migration in 2D and αvβ3-dependent invasion. Conversely, invasive migration that is dependent on α5β1 integrin is promoted by disrupting Rabaptin phosphorylation. These findings demonstrate that the PKD pathway couples receptor tyrosine kinase signaling to an integrin switch via Rabaptin-5 phosphorylation.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

EHD1 regulates the trafficking of multiple receptors from the endocytic recycling compartment (ERC) to the plasma membrane. However, the potential role of EHD1 in regulating the family of glycosylphosphatidylinositol-anchored proteins (GPI-APs) has not been determined. Here we demonstrate a novel role for EHD1 in regulating the trafficking of CD59, an endogenous GPI-AP, at early stages of trafficking through the endocytic pathway. EHD1 displays significant colocalization with newly internalized CD59. Upon EHD1 depletion, there is a rapid Rab5-independent coalescence of CD59 in the ERC region. However, expression of an active Arf6 mutant (Q67L), which traps internalized pre-sorting endosomal cargo in phosphatidylinositol(4,5)-bisphosphate enriched vacuoles, prevents this coalescence. It is of interest that sustained PKC activation leads to a similar coalescence of CD59 at the ERC, and treatment of EHD1-depleted cells with a PKC inhibitor (Go6976) blocked this rapid relocation of CD59. However, unlike sustained PKC activation, EHD1 depletion does not induce the translocation of PKCα to ERC. The results presented herein provide evidence that EHD1 is involved in the control of CD59 transport from pre-sorting endosomes to the ERC in a PKC-dependent manner. However, the mechanisms of EHD1-induced coalescence of CD59 at the ERC differ from those induced by sustained PKC activation.

Membrane transport is known to be regulated by protein phosphorylation and by small GTPases of the rab family. Using specific antibodies, we have identified a 55 kDa phosphorylated protein which co-immunoprecipitated with the cytosolic forms of rab5 and other rab proteins. We demonstrate, on the basis of its mobility in two-dimensional electrophoresis gels and its immunological properties, that this protein is rab GDI (p55/GDI). We also found that, a minor fraction of p55/GDI is membrane associated, but, whilst also complexed with rab proteins, it is not phosphorylated. On the basis of these data we suggest that the cycling of rab proteins between membranes and cytosol is regulated by phosphorylation of p55/GDI.

Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin that possesses Rho GTPase-activating protein (RhoGAP) and ADP-ribosyltransferase (ADPr) activities. In the current study, the RhoGAP and ADPr activities of ExoS were tested for the ability to disrupt mammalian epithelial cell physiology. RhoGAP, but not ADPr, inhibited internalization/phagocytosis of bacteria, while ADPr, but not RhoGAP, inhibited vesicle trafficking, both general fluid-phase uptake and EGF-activated EGF receptor (EGFR) degradation. In ADPr-intoxicated cells, upon EGF activation, EGFR co-localized with clathrin-coated vesicles (CCV), which did not mature into Rab5-positive early endosomes. Constitutively, active Rab5 recruited EGFR from CCV to early endosomes. Consistent with the inhibition of Rab5 function by ADPr, several Rab proteins including Rab5 and 9, but not Rab4, were ADP ribosylated by ExoS. Thus, the two enzymatic activities of ExoS have different effects on epithelial cells with RhoGAP inhibiting bacterial internalization and ADPr interfering with CCV maturation. The ability ADPr to inhibit mammalian vesicle trafficking provides a new mechanism for bacterial toxin-mediated virulence.

The bidirectional communication between integrin alphavbeta3 and vascular endothelial growth factor (VEGF) receptors acts to integrate and coordinate endothelial cell (EC) activity during angiogenesis. However, the molecular mechanisms involved in this signaling crosstalk are only partially revealed. We have found that protein kinase D1 (PKD1) was activated by VEGF-A, but not by other angiogenic factors, and associated with alphavbeta3 integrin. Moreover, knockdown of PKD1 increased endocytosis of alphavbeta3 and reduced its return from endosomes to the plasma membrane leading to accumulation of the integrin in Rab5- and Rab4-positive endosomes. Consistent with this, PKD1 knockdown caused defects in focal complex formation and reduced EC migration in response to VEGF-A. Moreover, knockdown of PKD1 reduced EC motility on vitronectin, whereas migration on collagen I was not PKD1 dependent. These results suggest that PKD1-regulated alphavbeta3 trafficking contributes to the angiogenesis process by integrating VEGF-A signaling with extracellular matrix interactions.

Members of the syntaxin family play a fundamental role in vesicle docking and fusion of diverse transport events. We have molecularly characterized syntaxin 8, a novel member of the syntaxin family. The nucleotide sequence of cloned rat cDNA predicts a polypeptide of 236 residues with a carboxyl-terminal 18-residue hydrophobic domain that may function as a membrane anchor. Characteristic of syntaxins, syntaxin 8 also contain regions that have the potential to form coiled-coil structures. Among the known syntaxins, syntaxin 8 is most homologous to syntaxin 6 which is predominantly associated with the trans-Golgi network (TGN). The syntaxin 8 transcript is detected in all rat tissues examined by northern blot. Antibodies against recombinant syntaxin 8 recognize a 27 kDa protein that is enriched in membrane fractions containing the Golgi apparatus and the endosomal/lysosomal compartments. Syntaxin 8 in membrane extract could be incorporated into a 20S protein complex in a way that is dependent on the soluble N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein ((alpha)-SNAP), suggesting that syntaxin 8 is indeed a SNAP receptor (SNARE). Indirect immunofluorescence microscopy reveals that the majority of syntaxin 8 is localized to the early endosome marked by Rab5. This is corroborated by immunogold labeling experiments showing enrichment of syntaxin 8 in the early endosome and its co-labeling with Rab5.

During development tissue deformations are essential for the generation of organs and to provide the final form of an organism. These deformations rely on the coordination of individual cell behaviours which have their origin in the modulation of subcellular activities. Here we explore the role endocytosis and recycling on tissue deformations that occur during dorsal closure of the Drosophila embryo. During this process the AS contracts and the epidermis elongates in a coordinated fashion, leading to the closure of a discontinuity in the dorsal epidermis of the Drosophila embryo. We used dominant negative forms of Rab5 and Rab11 to monitor the impact on tissue morphogenesis of altering endocytosis and recycling at the level of single cells. We found different requirements for endocytosis (Rab5) and recycling (Rab11) in dorsal closure, furthermore we found that the two processes are differentially used in the two tissues. Endocytosis is required in the AS to remove membrane during apical constriction, but is not essential in the epidermis. Recycling is required in the AS at early stages and in the epidermis for cell elongation, suggesting a role in membrane addition during these processes. We propose that the modulation of the balance between endocytosis and recycling can regulate cellular morphology and tissue deformations during morphogenesis.

The small GTPase rab5 has been shown to play key roles in the function of both endocytic and phagocytic organelles. Although these organelles share several additional common features, different processes have been proposed to explain their biogenesis. In the present study, we provide evidence that lysosome biogenesis involves mechanisms similar to those previously described for the formation of phagolysosomes. Transient interactions ('kiss and run') between endocytic organelles are shown to occur during lysosome biogenesis. These interactions are regulated initially by the GTPase activity of rab5, as demonstrated by the loss of size-selective fusion between endosomes in cells expressing a GTPase-deficient mutant of rab5. Endocytic compartments in these cells sequentially display properties of early and late endosomes. However, the formation of lysosomes and the sorting of endocytic solute materials to small electron dense vacuoles are not affected by the rab5 mutation. Together, our results indicate that endosome maturation occurs during the early part of lysosome biogenesis. This process involves transient fusion events regulated, in part, by the small GTPase rab5.

The TGFbeta (transforming growth factor beta) pathway is an essential cell signalling pathway that is implicated in both normal developmental processes, such as organogenesis, and pathological disorders, such as cancer and fibrosis. There are three prototypical TbetaRs (TGFbeta receptors): TbetaRI (TbetaR type I), TGbetaRII (TbetaR type II) and TGFbetaRIII (TbetaR type III, also known as betaglycan). Whereas the role of TbetaRII and TbetaRI in TGFbeta signal propagation has been established, the contribution of TbetaRIII to TGFbeta signalling is less well understood. At the cell surface, TbetaRI and TbetaRII receptors can be internalized by clathrin-mediated endocytosis and clathrin-independent membrane-raft-dependent endocytosis. Interestingly, the endocytic route of the receptors plays a direct role in TGFbeta-dependent Smad signal transduction; receptors endocytosed via clathrin-mediated endocytosis activate Smad signalling, whereas receptors endocytosed via membrane rafts are targeted for degradation. The objective of the present study was to evaluate the contribution of TbetaRIII to TbetaRII and TbetaRI membrane partitioning, receptor half-life and signalling. Using sucrose-density ultracentrifugation to isolate membrane-raft fractions, we show that TbetaRIII recruits both TbetaRII and TbetaRI to non-raft membrane fractions. Immunofluorescence microscopy analysis demonstrated that overexpression of TbetaRIII affects intracellular trafficking of TbetaRII by recruiting TbetaRII to EEA1 (early endosome antigen 1)- and Rab5-positive early endosomes. Using 125I-labelled TGFbeta1 to follow cell-surface receptor degradation we show that overexpression of TbetaRIII also extends the receptor half-life of the TbetaRII-TbetaRI complex. Interestingly, we also show, using a luciferase reporter assay, that TbetaRIII increases basal TGFbeta signalling. As numerous pathologies show aberrant activation of TGFbeta signalling, the present study illustrates that TbetaRIII may represent a novel therapeutic target.

The ypt/rab proteins are a family of small GTP-binding proteins thought to be required for different stages of membrane traffic. From the fission yeast Schizosaccharomyces pombe we have isolated and characterized ypt5, a gene encoding a homologue of rab5, a mammalian protein apparently involved in regulating fusion of early endosomes. Recombinant ypt5 protein bound GTP. The ypt5 gene was found to be essential for viability on minimal media, but ypt5-disrupted cells grew slowly on some rich media and accumulated a population of small vesicles not observed in wild-type cells. Canine rab5 cDNA could replace the ypt5 gene in S. pombe and restore normal growth and viability. Ypt5 protein expressed in mammalian cells colocalized with the transferrin receptor to early endosomes. Thus, molecular aspects of the early endocytic pathway may be conserved between mammalian cells and S. pombe and hence may be amenable to genetic analysis.

ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPβ. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.

The neuropathology of Huntington's disease includes nuclear and cytoplasmic inclusions, striatal neuronal loss, and gliosis. Previous work put forward a tantalizing proposal that disruption of axonal transport within long, narrow-caliber axons caused accumulations that could elicit cell death, ultimately resulting in neuronal dysfunction. Although a role for the Huntington's disease protein huntingtin (HTT) has been reported in axonal transport, it is unclear whether HTT affects the transport of all vesicles or influences only a specific class of vesicles. As an interaction between HTT and Rab5 was previously shown to mediate transport on actin filaments, here we tested the hypothesis that a HTT-Rab5 complex also exists for transport on microtubules during axonal transport. Surprisingly, we found that HTT influences Rab11 vesicles, not Rab5 vesicles. Reduction of HTT perturbed the transport of Rab11 vesicles. Reductions in kinesin and dynein motors also perturbed Rab11 vesicle transport indicating that these motors are required for bidirectional transport of Rab11. These results suggest that HTT plays a key role in the movement of Rab11 vesicles within axons. Thus, disruption of transport mediated by mutant HTT could contribute to early neuropathology observed in Huntington's diseases.