Recycling of the Ca2+-activated K+ Channel,KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C,and an N-terminal Domain

Regulation of the number of Ca²⁺-activated K⁺ channels at the endothelial cell surface contributes to control of the endothelium-derived hyperpolarizing factor response, although this process is poorly understood. To address the fate of plasma membrane-localized KCa2.3, we utilized an extracellular epitope-tagged channel in combination with fluorescence and biotinylation techniques in both human embryonic kidney cells and the human microvascular endothelial cell line, HMEC-1. KCa2.3 was internalized from the plasma membrane and degraded with a time constant of 18 h. Cell surface biotinylation demonstrated that KCa2.3 was rapidly endocytosed and recycled back to the plasma membrane. Consistent with recycling, expression of a dominant negative (DN) RME-1 or Rab35 as well as wild type EPI64C, the Rab35 GTPase-activating protein, resulted in accumulation of KCa2.3 in an intracellular compartment. Expression of DN RME-1, DN Rab35, or wild type EPI64C resulted in a decrease in steady-state plasma membrane expression. Knockdown of EPI64C increased cell surface expression of KCa2.3. Furthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly²⁰⁶–Pro²¹⁷, as being required for the rapid recycling of KCa2.3. Deletion of Gly²⁰⁶–Pro²¹⁷ had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.     

The large GTPase dynamin2: a new player in connexin 43 gap junction endocytosis, recycling and degradation

Connexins (Cx) are key regulators of cell proliferation, differentiation and apoptosis. Cx trafficking and endocytosis need interactions with a large number of signaling and scaffolding proteins. We demonstrate herein that Cx43-GFP gap junction plaque endocytosis was blocked in cells transfected by the dominant-negative form of dynamin2 (Dyn2K44A) and by dynasore, an inhibitor of dynamin GTPase activity, which reduced the association between dynamin2 and Cx43. Our data also reveal that recruitment of the GTPase at the plasma membrane and its activation by c-Src are key events for Cx43 internalization. In addition they show that dynamin2 participated in internalization and degradation of the gap junction plaque but also in recycling of Cx43 to the plasma membrane through respectively Rab5/Rab7 and Rab11 pathways. These results demonstrate for the first time that dynamin2 is a new Cx partner and report an innovating mechanistic model by which dynamin2 may control Cx43 gap junction plaque invagination, endocytosis, recycling and degradation. These processes are magnified in response to carcinogen exposure underlining their potential importance during carcinogenesis.     

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.     

MUC1 intra-cellular trafficking is clathrin, dynamin, and rab5 dependent

MUC1, a transmembrane glycoprotein, is abnormally over-expressed in most human adenocarcinomas. MUC1 association with cytoplasmic cell signal regulators and nuclear accumulation are important for its tumor related activities. Little is known about how MUC1 translocates from the cell membrane to the cytoplasm. In this study, live cell imaging was used to study MUC1 intracellular trafficking. The interaction between EGFR and MUC1 was mapped by FRET analysis and EGF stimulated MUC1 endocytosis was observed directly through live cell imaging. MUC1-CT endocytosis was clathrin and dynamin dependent. Rab5 over-expression resulted in decreased cell membrane localization of MUC1, with accumulation of MUC1 endocytic vesicles in the peri-nuclear region. Conversely, over-expression of a Rab5 dominant negative mutant (S34N) resulted in redistribution of MUC1 from the peri-nuclear region to the cytoplasm. Collectively, these results indicated that MUC1 intra-cellular trafficking occurs through a regulated process that was stimulated by direct EGFR and MUC1 interaction, mediated by clathrin coated pits that were dynamin dependent and regulated by Rab5.     

HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane

The human immunodeficiency virus (HIV) type-1 viral protein U (Vpu) protein enhances the release of diverse retroviruses from human, but not monkey, cells and is thought to do so by ablating a dominant restriction to particle release. Here, we determined how Vpu expression affects the subcellular distribution of HIV-1 and murine leukemia virus (MLV) Gag proteins in human cells where Vpu is, or is not, required for efficient particle release. In HeLa cells, where Vpu enhances HIV-1 and MLV release approximately 10-fold, concentrations of HIV-1 Gag and MLV Gag fused to cyan fluorescent protein (CFP) were initially detected at the plasma membrane, but then accumulated over time in early and late endosomes. Endosomal accumulation of Gag-CFP was prevented by Vpu expression and, importantly, inhibition of plasma membrane to early endosome transport by dominant negative mutants of Rab5a, dynamin, and EPS-15. Additionally, accumulation of both HIV and MLV Gag in endosomes required a functional late-budding domain. In human HOS cells, where HIV-1 and MLV release was efficient even in the absence of Vpu, Gag proteins were localized predominantly at the plasma membrane, irrespective of Vpu expression or manipulation of endocytic transport. While these data indicated that Vpu inhibits nascent virion endocytosis, Vpu did not affect transferrin endocytosis. Moreover, inhibition of endocytosis did not restore Vpu-defective HIV-1 release in HeLa cells, but instead resulted in accumulation of mature virions that could be released from the cell surface by protease treatment. Thus, these findings suggest that a specific activity that is present in HeLa cells, but not in HOS cells, and is counteracted by Vpu, traps assembled retrovirus particles at the cell surface. This entrapment leads to subsequent endocytosis by a Rab5a- and clathrin-dependent mechanism and intracellular sequestration of virions in endosomes.     

Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.     

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.     

IL4/PGE2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E2 (PGE2) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells.     

Trans-Golgi Network (TGN) as a regulatory node for β1-adrenergic receptor (β1AR) down-modulation and recycling

Receptor down-modulation is the key mechanism by which G protein-coupled receptors (GPCRs) prevent excessive receptor signaling in response to agonist stimulation. Recently, the trans-Golgi network (TGN) has been implicated as a key checkpoint for receptor endocytosis and degradation. Here, we investigated the involvement of the TGN in down-modulation of β1-adrenergic receptor in response to persistent isoprotenerol stimulation. Immunofluorescent staining showed that ~50% of endocytosed β1AR colocalized with TGN-46 at 5 h. Disruption of the TGN by brefeldin A (BFA) led to the robust accumulation of endocytosed β1AR in Rab11(+) recycling endosomes, inhibited β1AR entry into LAMP1(+) lysosomes, and as a result enhanced β1AR recycling to the plasma membrane. The lysosomotropic agent, chloroquine, arrested the majority of endocytosed β1AR in the TGN by 4 h. Immunoblot analysis showed that either disruption of the TGN or blockage of the lysosome prevented β1AR degradation. Co-expression of GFP-arrestin-3 in β1AR cells increased the endocytosis of β1AR and facilitated its entry to the TGN but inhibited recycling to the plasma membrane. Arrestin-3-induced inhibition of β1AR recycling was reversed by BFA treatment, whereas chloroquine induced the accumulation of arrestin-3 with β1AR in the TGN. These results demonstrate for the first time that the TGN acts as a checkpoint for both the recycling and down-regulation of β1AR and that arrestin-3 not only mediates β1AR endocytosis but also its recycling through the TGN.     

Clathrin-dependent APP endocytosis and Aβ secretion are highly sensitive to the level of plasma membrane cholesterol

Several lines of evidence support a strong relationship between cholesterol and Alzheimer's disease pathogenesis. Membrane cholesterol is known to modulate amyloid precursor protein (APP) endocytosis and amyloid-β (Aβ) secretion. Here we show in a human cell line model of endocytosis (HEK293 cells) that cholesterol exerts these effects in a dose-dependent and linear manner, over a wide range of concentrations (-40% to + 40% variations of plasma membrane cholesterol induced by methyl-beta-cyclodextrin (MBCD) and MBCD-cholesterol complex respectively). We found that the gradual effect of cholesterol is inhibited by small interference RNA-mediated downregulation of clathrin. Modulation of clathrin-mediated APP endocytosis by cholesterol was further demonstrated using mutants of proteins involved in the formation of early endosomes (dynamin2, Eps15 and Rab5). Importantly we show that membrane proteins other than APP are not affected by cholesterol to the same extent. Indeed clathrin-dependent endocytosis of transferrin and cannabinoid1 receptors as well as internalization of surface proteins labelled with a biotin derivative (sulfo-NHS-SS-biotin) were not sensitive to variations of plasma membrane cholesterol from -40% to 40%. In conclusion clathrin-dependent APP endocytosis appears to be very sensitive to the levels of membrane cholesterol. These results suggest that cholesterol increase in AD could be responsible for the enhanced internalization of clathrin-, dynamin2-, Eps15- and Rab5-dependent endocytosis of APP and the ensuing overproduction of Aβ.     

Regulation of Trypanosoma cruzi invasion of nonphagocytic cells by the endocytically active GTPases dynamin, Rab5, and Rab7

During invasion of nonphagocytic cells by Trypanosoma cruzi (T. cruzi), host cell lysosomes are recruited to the plasma membrane attachment site followed by lysosomal enzyme secretion. The membrane trafficking events involved in invasion have not been delineated. We demonstrate here that T. cruzi invasion of nonphagocytic cells was completely abolished by overexpression of a dominant negative mutant of dynamin. Likewise, overexpression of a dominant negative mutant of Rab5, the rate-limiting GTPase for endocytosis, resulted in reduced infection rates compared with cells expressing Rab5 wild-type. Moreover, cells expressing the activated mutant of Rab5 experienced higher infection rates. A similar pattern was also observed when Rab7-transfected cells were examined. Confocal microscopy experiments showed that parasites colocalized with green fluorescent protein-Rab5-positive early endosomes after 5 min of invasion. These data clearly indicate that newly forming T. cruzi phagosomes first interact with an early endosomal compartment and subsequently with other late component markers before lysosomal interaction occurs.     

Rab13 mediates the continuous endocytic recycling of occludin to the cell surface

During epithelial morphogenesis, adherens junctions (AJs) and tight junctions (TJs) undergo dynamic reorganization, whereas epithelial polarity is transiently lost and reestablished. Although ARF6-mediated endocytic recycling of E-cadherin has been characterized and implicated in the rapid remodeling of AJs, the molecular basis for the dynamic rearrangement of TJs remains elusive. Occludin and claudins are integral membrane proteins comprising TJ strands and are thought to be responsible for establishing and maintaining epithelial polarity. Here we investigated the intracellular transport of occludin and claudins to and from the cell surface. Using cell surface biotinylation and immunofluorescence, we found that a pool of occludin was continuously endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney cells and epithelial MTD-1A cells. Biochemical endocytosis and recycling assays revealed that a Rab13 dominant active mutant (Rab13 Q67L) inhibited the postendocytic recycling of occludin, but not that of transferrin receptor and polymeric immunoglobulin receptor in MTD-1A cells. Double immunolabelings showed that a fraction of endocytosed occludin was colocalized with Rab13 in MTD-1A cells. These results suggest that Rab13 specifically mediates the continuous endocytic recycling of occludin to the cell surface in both fibroblastic and epithelial cells.     

Regulation of ENaC expression at the cell surface by Rab11

The epithelial Na⁺ channel (ENaC) is an essential channel responsible for Na⁺ reabsorption. Coexpression of Rab11a and Rab3a small G proteins with ENaC results in a significant increase in channel activity. In contrast, coexpression of Rab5, Rab27a, and Arf-1 had no effect or slightly decreased ENaC activity. Inhibition of MEK with PD98059, Rho-kinase with Y27632 or PI3-kinase with LY294002 had no effect on ENaC activity in Rab11a-transfected CHO cells. Fluorescence imaging methods demonstrate that Rab11a colocalized with ENaC. Rab11a increases ENaC activity in an additive manner with dominant-negative dynamin, which is a GTPase responsible for endocytosis. Brefeldin A, an inhibitor of intracellular protein translocation, blocked the stimulatory action of Rab11a on ENaC activity. We conclude that ENaC channels, present on the apical plasma membrane, are being exchanged with channels from the intracellular pool in a Rab11-dependent manner.     

Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes

The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes to insulin, IRAP translocated to the plasma membrane as assessed by either cell fractionation, surface biotinylation, or the plasma membrane sheet assay. The rate of exocytosis closely paralleled that of GLUT4. In the continuous presence of insulin, IRAP was endocytosed with a half-time of about 3-5 min. IRAP endocytosis is inhibited by cytosol acidification, a property of clathrin-mediated endocytosis, but not by the expression of a constitutively active Akt/PKB. Arrival in an LDM fraction derived via subcellular fractionation exhibited a slower time course than disappearance from the cell surface, suggesting additional endocytic intermediates. As assayed by membrane "sheets," GLUT4 and IRAP showed similar internalization rates that are wortmannin-insensitive and occur with a half-time of roughly 5 min. IRAP remaining on the cell surface 10 min following insulin removal was both biotin- and avidin-accessible, implying the absence of thin-necked invaginations. Finally, endocytosed IRAP quickly recycled back to the plasma membrane in a wortmannin-sensitive process. These results demonstrate rapid endocytosis and recycling of IRAP in the presence of insulin and trafficking that matches GLUT4 in rate.     

The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis

The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin-dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin.     

Dynamic partitioning into lipid rafts controls the endo-exocytic cycle of the alphaL/beta2 integrin, LFA-1, during leukocyte chemotaxis

Cell migration entails the dynamic redistribution of adhesion receptors from the cell rear toward the cell front, where they form new protrusions and adhesions. This process may involve regulated endo-exocytosis of integrins. Here we show that in primary neutrophils unengaged alphaL/beta2 integrin (LFA-1) is internalized and rapidly recycled upon chemoattractant stimulation via a clathrin-independent, cholesterol-sensitive pathway involving dynamic partitioning into detergent-resistant membranes (DRM). Persistent DRM association is required for recycling of the internalized receptor because 1) >90% of endocytosed LFA-1 is associated with DRM, and a large fraction of the internalized receptor colocalizes intracellularly with markers of DRM and the recycling endocytic compartment; 2) a recycling-defective mutant (alphaL/beta2Y735A) dissociates rapidly from DRM upon being endocytosed and is subsequently diverted into a late endosomal pathway; and 3) a dominant negative Rab11 mutant (Rab11S25N) induces intracellular accumulation of endocytosed alphaL/beta2 and prevents its enrichment in chemoattractant-induced lamellipodia. Notably, chemokine-induced migration of neutrophils over immobilized ICAM-1 is abrogated by cholesterol-sequestering agents. We propose that DRM-associated endocytosis allows efficient retrieval of integrins, as they detach from their ligands, followed by polarized recycling to areas of the plasma membrane, such as lamellipodia, where they establish new adhesive interactions and promote outside-in signaling events.     

Endocytosis of hERG Is Clathrin-Independent and Involves Arf6

The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.     

Bovine Ephemeral Fever Virus Uses a Clathrin-Mediated and Dynamin 2-Dependent Endocytosis Pathway That Requires Rab5 and Rab7 as Well as Microtubules

The specific cell pathways involved in bovine ephemeral fever virus (BEFV) cell entry have not been determined. In this work, colocalization of the M protein of BEFV with clathrin or dynamin 2 was observed under a fluorescence microscope. To better understand BEFV entry, we carried out internalization studies with a fluorescently labeled BEFV by using a lipophilic dye, 3,30-dilinoleyloxacarbocyanine perchlorate (DiO), further suggesting that BEFV uses a clathrin-mediated endocytosis pathway. Our results suggest that clathrin-mediated and dynamin 2-dependent endocytosis is an important avenue of BEFV entry. Suppression of Rab5 or Rab7a through the use of a Rab5 dominant negative mutant and Rab7a short hairpin RNA (shRNA) demonstrated that BEFV requires both early and late endosomes for endocytosis and subsequent infection in MDBK and Vero cells. Treatment of BEFV-infected cells with nocodazole significantly decreased the M protein synthesis and viral yield, indicating that microtubules play an important role in BEFV productive infection, likely by mediating trafficking of BEFV-containing endosomes. Furthermore, BEFV infection was strongly blocked by different inhibitors of endosomal acidification, suggesting that virus enters host cells by clathrin-mediated and dynamin 2-dependent endocytosis in a pH-dependent manner.     

Differential role of actin,clathrin, and dynamin in Fc gamma receptor-mediated endocytosis and phagocytosis

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

The role of endosomal signaling triggered by metastatic growth factors in tumor progression

Within tumor microenvironment, a lot of growth factors such as hepatocyte growth factor and epidermal growth factor may induce similar signal cascade downstream of receptor tyrosine kinase (RTK) and trigger tumor metastasis synergistically. In the past decades, the intimate relationship of RTK-mediated receptor endocytosis with signal transduction was well established. In general, most RTK undergoes clathrin-dependent endocytosis and/or clathrin-independent endocytosis. The internalized receptors may sustain the signaling within early endosome, recycling to plasma membrane for subsequent ligand engagement or sorting to late endosomes/lysosome for receptor degradation. Moreover, receptor endocytosis influences signal transduction in a temporal and spatial manner for periodical and polarized cellular processes such as cell migration. The endosomal signalings triggered by various metastatic factors are quite similar in some critical points, which are essential for triggering cell migration and tumor progression. There are common regulators for receptor endocytosis including dynamin, Rab4, Rab5, Rab11 and Cbl. Moreover, many critical regulators within the RTK signal pathway such as Grb2, p38, PKC and Src were also modulators of endocytosis. In the future, these may constitute a new category of targets for prevention of tumor metastasis.