H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.     

Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.     

Human RME-8 is involved in membrane trafficking through early endosomes

RME-8 is a DnaJ-domain-containing protein that was first identified in Caenorhabditis elegans as being required for uptake of yolk proteins. RME-8 has also been identified in other species, including flies and mammals, and the phenotypes of their RME-8 mutants suggest the importance of this protein in endocytosis. In the present study, we cloned human RME-8 (hRME-8) and characterized its biochemical properties and functions in endocytic pathways. hRME-8 was found to be a peripheral protein that was tightly associated with the membrane via its N-terminal region. It partially colocalized with several early endosomal markers, but not with late endosomal markers, consistent with observations by immunoelectron microscopy. When cells were transfected with a panel of dominant-active Rab proteins, hRME-8 was confined to large vacuoles induced by expression of Rab5aQ79L, but not by Rab7Q67L. Expression of C-terminally-truncated hRME-8 mutants led to the formation of large puncta and vacuoles, and compromised endocytic pathways through early endosomes, i.e., recycling of transferrin and degradation of epidermal growth factor. Taken together, these results indicate that hRME is primarily involved in membrane trafficking through early endosomes, but not through degradative organelles, such as multivesicular bodies and late endosomes.     

Rab15 mediates an early endocytic event in Chinese hamster ovary cells.

Rab GTPases comprise a large family of monomeric proteins that regulate a diverse number of membrane trafficking events, including endocytosis. In this paper, we examine the subcellular distribution and function of the GTPase Rab15. Our biochemical and confocal immunofluorescence studies demonstrate that Rab15 associates with the transferrin receptor, a marker for the early endocytic pathway, but not with Rab7 or the cation-independent mannose 6-phosphate receptor, markers for late endosomal membranes. Furthermore, Rab15 colocalizes with Rab4 and -5 on early/sorting endosomes, as well as Rab11 on pericentriolar recycling endosomes. Consistent with its localization to early endosomal membranes, overexpression of the constitutively active mutant HArab15Q67L reduces receptor-mediated and fluid phase endocytosis. Therefore, our functional studies suggest that Rab15 may function as an inhibitory GTPase in early endocytic trafficking.     

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

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

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses .     

A Highlights from MBoC Selection: The Late Endosome is Essential for mTORC1 Signaling

The multisubunit mTORC1 complex integrates signals from growth factors and nutrients to regulate protein synthesis, cell growth, and autophagy. To examine how endocytic trafficking might be involved in nutrient regulation of mTORC1, we perturbed specific endocytic trafficking pathways and measured mTORC1 activity using S6K1 as a readout. When early/late endosomal conversion was blocked by either overexpression of constitutively active Rab5 (Rab5CA) or knockdown of the Rab7 GEF hVps39, insulin- and amino acid–stimulated mTORC1/S6K1 activation were inhibited, and mTOR localized to hybrid early/late endosomes. Inhibition of other stages of endocytic trafficking had no effect on mTORC1. Overexpression of Rheb, which activates mTOR independently of mTOR localization, rescued mTORC1 signaling in cells expressing Rab5CA, whereas hyperactivation of endogenous Rheb in TSC2−/− MEFs did not. These data suggest that integrity of late endosomes is essential for amino acid– and insulin-stimulated mTORC1 signaling and that blocking the early/late endosomal conversion prevents mTOR from interacting with Rheb in the late endosomal compartment.     

The proteasome alpha-subunit XAPC7 interacts specifically with Rab7 and late endosomes

Rab7 is a key regulatory protein governing early to late endocytic membrane transport. In this study the proteasome alpha-subunit XAPC7 (also known as PSMA7, RC6-1, and HSPC in mammals) was identified to interact specifically with Rab7 and was recruited to multivesicular late endosomes through this interaction. The protein interaction domains were localized to the C terminus of XAPC7 and the N terminus of Rab7. XAPC7 was not found on early or recycling endosomes, but could be recruited to recycling endosomes by expression of a Rab7-(1-174)Rab11-(160-202) chimera, establishing a central role for Rab7 in the membrane recruitment of XAPC7. Although XAPC7 could be shown to associate with membranes bearing ubiquitinated cargo, overexpression had no impact on steady-state ubiquitinated protein levels. Most notably, overexpression of XAPC7 was found to impair late endocytic transport of two different membrane proteins, including EGFR known to be highly dependent on ubiquitination and proteasome activity for proper endocytic sorting and lysosomal transport. Decreased late endocytic transport caused by XAPC7 overexpression was partially rescued by coexpression of wild-type Rab7, suggesting a negative regulatory role for XAPC7. Nevertheless, Rab7 itself was not subject to XAPC7-dependent proteasomal degradation. Together the data establish the first direct molecular link between the endocytic trafficking and cytosolic degradative machineries.     

Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis

BACKGROUND: Embryonic cleavage leads to the formation of an epithelial layer during development. In Drosophila, the process is specialized and called cellularization. The trafficking pathways that underlie this process and that are responsible for the mobilization of membrane pools, however, remain poorly understood. RESULTS: We provide functional evidence for the role of endocytic trafficking through Rab11 endosomes in remobilizing vesicular membrane pools to ensure lateral membrane growth. Part of the membrane stems from endocytosed apical material. Mutants in the endocytic regulators rab5 and shibire/dynamin inhibit basal-lateral membrane growth, and apical endocytosis is blocked in shibire mutants. In addition, shibire controls vesicular trafficking through Rab11-positive endosomes. In shibire mutants, the transmembrane protein Neurotactin follows the secretory pathway normally but is not properly inserted in the plasma membrane and accumulates instead in Rab11 subapical endosomes. Consistent with a direct role of shibire in vesicular trafficking through Rab11 endosomes, Shibire is enriched in this compartment. Moreover, we show by electron microscopy the large accumulation of intracellular coated pits on subapical endocytic structures in shibire mutants. Finally, we show that Rab11 is essential for membrane growth and invagination during cellularization. CONCLUSION: Together, the data show that endocytic trafficking is required for basal-lateral membrane growth during cellularization. We identify Rab11 endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. This pathway may be required in other morphogenetic processes characterized by the growth of a membrane domain.     

beta 2-adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by rab GTPases

Rab GTPases are recognized as critical regulatory factors involved in vesicular membrane transport and endosomal fusion. For example, Rab5 directs the transport and fusion of endocytic vesicles to and with early endosomes, whereas Rab4 is thought to control protein trafficking from early endosomes back to the plasma membrane. In the present study, we investigated the role of Rab5 and Rab4 GTPases in regulating the endocytosis, intracellular sorting, and the plasma membrane recycling of the beta(2)AR. In cells expressing the dominant-negative Rab5-S34N mutant, beta(2)AR internalization was impaired, and beta(2)AR-bearing endocytic vesicles remained in either close juxtaposition or physically attached to the plasma membrane. In contrast, a constitutively active Rab5-Q79L mutant redirected internalized beta(2)AR to enlarged endosomes but did not prevent beta(2)AR dephosphorylation and recycling. The expression of either wild-type Rab4 or a Rab4-N121I mutant did not prevent beta(2)AR dephosphorylation. However, the dominant-negative Rab4-N121I mutant blocked beta(2)AR resensitization by blocking receptor recycling from endosomes back to the cell surface. Our data indicate that, in addition to regulating the intracellular trafficking and fusion of beta(2)AR-bearing endocytic vesicles, Rab5 also contributes to the formation and/or budding of clathrin-coated vesicles. Furthermore, beta(2)AR dephosphorylation occurs as the receptor transits between Rab5- and Rab4-positive compartments.     

Endocytic trafficking routes of wild type and DeltaF508 cystic fibrosis transmembrane conductance regulator

Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. DeltaF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and DeltaF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16 degrees C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations DeltaF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis.     

Rab5 and Rab7, but not ARF6, govern the early events of HIV-1 infection in polarized human placental cells

Trophoblasts, the structural cells of the placenta, are thought to play a determinant role in in utero HIV type 1 (HIV-1) transmission. We have accumulated evidence suggesting that HIV-1 infection of these cells is associated with uptake by an unusual clathrin/caveolae-independent endocytic pathway and that endocytosis is followed by trafficking through multiple organelles. Furthermore, part of this trafficking involves the transit of HIV-1 from transferrin-negative to EEA1 and transferrin-positive endosomes, suggesting a merger from nonclassical to classical endocytic pathways in these cells. In the present article, the relationship between the presence of HIV-1 within specific endosomes and infection was studied. We demonstrate that viral infection is virtually lost when endosome inhibitors are added shortly after exposure to HIV-1. Thus, contrary to what is seen in CD4+ T lymphocytes, the initial presence of HIV-1 within the endosomes is mandatory for infection to take place. Importantly, this process is independent of the viral envelope proteins gp120 and gp41. The Rab family of small GTPases coordinates the vesicular transport between the different endocytic organelles. Experiments performed with various expression vectors indicated that HIV-1 infection in polarized trophoblasts relies on Rab5 and Rab7 without the contribution of Arf6 or Rab11. Furthermore, we conclude that Rab5 drives movements from raft-rich region to early endosomes, and this transit is required for subsequently reaching late endosomes via Rab7. This complex trafficking is mandatory for HIV-1 infection to proceed in human polarized trophoblasts.     

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

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

Regulation of endocytosis by the small GTP-ase Rab5

Rab5 is a small GTPase associated with the plasma membrane and the early endosomes. Expression of wild type rab5 and a mutant protein defective in GTP-binding in BHK cells led to alterations in the rate of endocytosis and in the morphology of endocytic organelles. The results obtained suggest that rab5 is a rate limiting GTPase that regulate the kinetics of both lateral fusion of early endosomes and fusion of plasma membrane-derived endocytic vesicles with early endosomes.     

Regulation of endocytosis by the small GTP-ase Rab5

Rab5 is a small GTPase associated with the plasma membrane and the early endosomes. Expression of wild type rab5 and a mutant protein defective in GTP-binding in BHK cells led to alterations in the rate of endocytosis and in the morphology of endocytic organelles. The results obtained suggest that rab5 is a rate limiting GTPase that regulate the kinetics of both lateral fusion of early endosomes and fusion of plasma membrane-derived endocytic vesicles with early endosomes.     

Late endocytic dysfunction as a putative cause of amyloid fibril formation in Alzheimer's disease

The assembly of amyloid β-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid β-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid β-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid β-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.     

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural–functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.     

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN).     

Infectious adenovirus type 2 transport through early but not late endosomes

Receptor-mediated endocytosis is a major gate for pathogens into cells. In this study, we analyzed the trafficking of human adenovirus type 2 and 5 (Ad2/5) and the escape-defective temperature-sensitive Ad2-ts1 mutant in epithelial cancer cells. Ad2/5 and Ad2-ts1 uptake into endosomes containing transferrin, major histocompatibility antigen 1 and the Rab5 effector early endosome antigen 1 (EEA1) involved dynamin, amphiphysin, clathrin and Eps15. Cointernalization experiments showed that most of the Ad2/5 and Ad2-ts1 visited the same EEA1-positive endosomes. In contrast to Ad2/5, Ad2-ts1 required functional Rab5 for endocytosis and lysosomal transport and was sensitive to the phosphatidyl-inositol-3 (PI3)-kinase inhibitor wortmannin or the ubiquitin-binding protein Hrs for sorting from early to late endosomes. Endosomal escape of Ad2 was not affected by incubation at 19 degrees C, which blocked membrane sorting in early endosomes and inhibited Ad2-ts1 transport to lysosomes. Unlike Semliki Forest Virus (SFV), sorting of Ad2-ts1 to late endosomes was independent of Rab7 and Ad2/5 infection independent of EEA1. The data indicate that Ad2/5 and Ad2-ts1 use an invariant machinery for clathrin-mediated uptake to early endosomes. We suggest that the infectious Ad2 particles are either directly released from early endosomes to the cytosol or sorted by a temperature-insensitive and PI3-kinase-independent mechanism to an escape compartment different from late endosomes or lysosomes.