Clathrin-dependent Receptor-mediated Endocytosis Research Articles

Influenza A virus exploits transferrin receptor recycling to enter host cells

Influenza A virus (IAV) enters host cells mostly through clathrin-dependent receptor-mediated endocytosis. A single bona fide entry receptor protein supporting this entry mechanism remains elusive. Here we performed proximity ligation of biotin to host cell surface proteins in the vicinity of attached trimeric hemagglutinin-HRP and characterized biotinylated targets using mass spectrometry. This approach identified transferrin receptor 1 (TfR1) as a candidate entry protein. Genetic gain-of-function and loss-of-function experiments, as well as invitro and invivo chemical inhibition, confirmed the functional involvement of TfR1 in IAV entry. Recycling deficient mutants of TfR1 do not support entry, indicating that TfR1 recycling is essential for this function. The binding of virions to TfR1 via sialic acids confirmed its role as a directly acting entry factor, but unexpectedly even headless TfR1 promoted IAV particle uptake in trans. TIRF microscopy localized the entering virus-like particles in the vicinity of TfR1. Our data identify TfR1 recycling as a revolving door mechanism exploited by IAV to enter host cells.

Characterization of the Atl-mediated staphylococcal internalization mechanism

Staphylococcus aureus internalization by non-professional phagocytes is considered a main pathogenicity mechanism leading to chronic infections. The well-established mechanism of Staphylococcus aureus internalization is mediated by fibronectin (Fn)-binding proteins (FnBPs), Fn as a bridging molecule and the host cell α5β1 integrin. We previously identified a novel alternative internalization mechanism in Staphylococcus aureus, which involves the major autolysin Atl and the host cell heat shock cognate protein 70 (Hsc70). Atl-dependent internalization is also employed by the coagulase-negative Staphylococcus epidermidis, where it might represent the major or even sole internalization mechanism, because of the lack of FnBP-homologous proteins. In this study, we aimed to further characterize the Atl-dependent staphylococcal internalization mechanism. We performed biomolecular interaction analysis (BIA) to quantify the adhesive properties of Atl and found multivalent and high affinity interactions of Atl with Fn and Hsc70. Confocal laser scanning microscopy (CLSM) and a flow-cytometric internalization assay in combination with different pharmacological inhibitors suggested an involvement of the α5β1 integrin, Fn and Hsc70 and subsequent signaling events mediated by Src and phosphoinositide 3 (PI3) kinases in the Atl-dependent staphylococcal uptake by EA.hy 926 cells. Further characterization of the endocytic machinery implicated a role for clathrin-dependent receptor-mediated endocytosis involving actin cytoskeletal rearrangements and microtubules. In conclusion, Atl ubiquitous among staphylococcal species may substitute for the FnBPs ensuring low-level internalization via a mechanism that seems to share important features with the FnBP-mediated staphylococcal uptake potentially being the prerequisite for the development of therapy-resistant chronic infections by staphylococcal strains that lack FnBPs.

Engineering of pectin-capped gold nanoparticles for delivery of doxorubicin to hepatocarcinoma cells: an insight into mechanism of cellular uptake

In this study, we have reported the fabrication and evaluation of pectin-capped gold nanoparticles (PEC-AuNPs) for delivery of anticancer drug, doxorubicin (DOX) to cells overexpressing asialoglycoprotein receptor (ASGPR). Pectin was used as a reducing, stabilizing and targeting agent. The pectin-capped gold nanoparticles demonstrated surface plasmon resonance band at 519 nm. The PEC-AuNPs were spherical in shape with a particle size of 14 nm and zeta potential value of −33 mV and were biocompatible and non-cytotoxic. The PEC-AuNPs exhibited a high drug loading efficiency of 78%. The DOX-loaded gold nanoparticles (DOX-PEC-AuNPs) showed excellent stability under varying pH and electrolytic conditions. The cytotoxicity study of the DOX-PEC-AuNPs in human Caucasian hepatocyte cells demonstrated their greater potency in killing these cells as compared to free DOX. The uptake and targeting potential of DOX-PEC-AuNPs was thoroughly investigated. Further, it was found that the PEC-AuNPs were taken up by HepG2 cells via a clathrin-dependent receptor-mediated endocytosis by asialoglycoprotein receptor present of the surface of these cells. Thus, the PEC-capped AuNPs can prove a promising carrier for anticancer drug in the treatment of hepatocellular carcinoma.

Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles.

Lung cancer is the leading cause of cancer mortality worldwide, resulting in 88% deaths of all diagnosed patients. Hence, novel therapeutic modalities are urgently needed. Single-stranded oligonucleotide-based aptamers (APTs) are excellent ligands for tumor cell targeting. However, the molecular mechanisms underlying their internalization into living cells have been poorly studied. Towards the application of APTs for active drug targeting to cancer cells, we herein studied the mechanism underlying S15-APT internalization into human non-small cell lung cancer A549 cells. We thus delineated the mode of entry of a model nanomedical system based on quantum dots (QDs) decorated with S15-APTs as a selective targeting moiety for uptake by A549 cells. These APT-decorated QDs displayed selective binding to, and internalization by target A549 cells, but not by normal human bronchial epithelial BEAS2B, cervical carcinoma (HeLa) and colon adenocarcinoma CaCo-2 cells, hence demonstrating high specificity. Flow cytometric analysis revealed a remarkably low dissociation constant of S15-APTs-decorated QDs to A549 cells (Kd = 13.1 ± 1.6 nM). Through the systematic application of a series of established inhibitors of known mechanisms of endocytosis, we show that the uptake of S15-APTs proceeds via a classical clathrin-dependent receptor-mediated endocytosis. This cancer cell-selective mode of entry could possibly be used in the future to evade plasma membrane-localized multidrug resistance efflux pumps, thereby overcoming an important mechanism of cancer multidrug resistance.

Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells.

During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses.IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.

Comparative study on cellular entry of incinerated ancient gold particles (Swarna Bhasma) and chemically synthesized gold particles

Gold nanoparticles (AuNPs) are used for a number of imaging and therapeutic applications in east and western part of the world. For thousands of years, the traditional Indian Ayurvedic approach to healing involves the use of incinerated gold ash, prepared with a variety of plant extracts and minerals depending on the region. Here, we describe the characterization of incinerated gold particles (IAuPs) in HeLa (human cells derived from cervical cancer) and HFF-1 (human foreskin fibroblast cells) in comparison to synthesized citrate-capped gold nanoparticles (AuNPs). We found that while individual IAuP crystallites are around 60 nm in size, they form large aggregates with a mean diameter of 4711.7 nm, some of which can enter cells. Fewer cells appeared to have IAuPs compared to AuNPs, although neither type of particle was toxic to cells. Imaging studies revealed that IAuPs were in vesicles, cytosol, or in the nucleus. We found that their nuclear accumulation likely occurred after nuclear envelope breakdown during cell division. We also found that larger IAuPs entered cells via macropinocytosis, while smaller particles entered via clathrin-dependent receptor-mediated endocytosis.

Asialoglycoprotein receptor-targeted liposomes loaded with a norcantharimide derivative for hepatocyte-selective targeting

In order to overcome the shortcomings associated with the clinical application of norcantharidin (NCTD), including intense irritation and a short half-life, and to obtain a hepatocyte-selective liposome system with high encapsulation efficiency (EE) and low leakage, we synthesized a C14 alkyl chain norcantharimide derivative of NCTD (2-tetradecylhexahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione, N-14NCTDA). Asialoglycoprotein receptor-targeted, galactosylated liposomes loaded with N-14NCTDA (GAL-Lipo) were prepared by the lipid film hydration method. GAL-Lipo with a satisfactory particle size of approximately 120nm has a higher encapsulation efficiency of more than 98.0%, which is markedly increased compared with NCTD loaded liposomes (EE%=47.6%). In addition, GAL-Lipo remained stable for at least 1 month at 4°C. In cytotoxicity assays, GAL-Lipo demonstrated stronger cytotoxicity effects (IC50=24.58μmolL−1) on Hep G2 cells than free N-14NCTDA (100μmol/L) and conventional liposomes (Con-Lipo, 39.49μmol/L) without the GAL modification. GAL-Lipo can continuously accumulate in Hep G2 cells and be internalized into cells via two pathways, namely caveolin-dependent endocytosis and clathrin-dependent asialoglycoprotein receptors (ASGP-R) mediated endocytosis and produces considerably more significant cellular apoptosis. The results of vivo toxicity studies showed that GAL-Lipo dramatically reduced renal toxicity. In addition, GAL-Lipo has a markedly improved pharmacokinetic profile in vivo and a longer circulation time (AUC=6.700±2.964mgL−1h, t1/2z=1.347±0.519h) than Con-Lipo (AUC=2.319±0.121mgL−1h, t1/2z=0.413±0.238h). In conclusion, N-14NCTDA with an ideal logP is a better alternative for the treatment of primary hepatic carcinoma. GAL-Lipo offers an attractive strategy to specifically target hepatocytes via caveolin-dependent and clathrin-dependent asialoglycoprotein receptor-mediated endocytosis resulting in higher anticancer activity and fewer side-effects.

Receptor-Mediated Enhanced Cellular Delivery of Nanoparticles Using Recombinant Receptor-Binding Domain of Diphtheria Toxin

Antibodies and peptides are often used to home nanoparticles (NPs) to specific cells. Here in this work, we have used recombinant receptor-binding domain of diphtheria toxin (RDT) as a homing molecule for NPs. Diphtheria toxin binds to heparin binding EGF-like growth factor (HB-EGF) through its receptor-binding domain. HB-EGF is often overexpressed as cell surface molecule in various types of cancer. We have prepared monodispersed, spherical PLGA NPs and coated these NPs with RDT. These NPs are characterized by FESEM and FT-IR spectroscopy. Using flow cytometry and fluorescence spectroscopy, we show that coating with RDT increases cellular uptake of PLGA NPs. We further show that RDT-coated nanoparticles are internalized through clathrin-dependent receptor-mediated endocytosis that can be reduced by specific inhibitor. These RDT-coated nanoparticles (RDT-NP) were further used for preferential delivery of Irinotecan, a chemotherapeutic agent, to cells overexpressing HB-EGF. We show that receptor-mediated enhanced uptake of RDT-NPs increases the potency of irinotecan in these cells.

Entry of Classical Swine Fever Virus into PK-15 Cells via a pH-, Dynamin-, and Cholesterol-Dependent, Clathrin-Mediated Endocytic Pathway That Requires Rab5 and Rab7.

Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small, enveloped, positive-strand RNA virus. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. However, the mechanisms of CSFV entry into cells are not well characterized. In this study, we used systematic approaches to dissect CSFV cell entry. We first observed that CSFV infection was inhibited by chloroquine and NH4Cl, suggesting that viral entry required a low-pH environment. By using the specific inhibitor dynasore, or by expressing the dominant negative (DN) K44A mutant, we verified that dynamin is required for CSFV entry. CSFV particles were observed to colocalize with clathrin at 5 min postinternalization, and CSFV infection was significantly reduced by chlorpromazine treatment, overexpression of a dominant negative form of the EPS15 protein, or knockdown of the clathrin heavy chain by RNA interference. These results suggested that CSFV entry depends on clathrin. Additionally, we found that endocytosis of CSFV was dependent on membrane cholesterol, while neither the overexpression of a dominant negative caveolin mutant nor the knockdown of caveolin had an effect. These results further suggested that CSFV entry required cholesterol and not caveolae. Importantly, the effect of DN mutants of three Rab proteins that regulate endosomal traffic on CSFV infection was examined. Expression of DN Rab5 and Rab7 mutants, but not the DN Rab11 mutant, significantly inhibited CSFV replication. These results were confirmed by silencing of Rab5 and Rab7. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab7 during the early phase of infection within 45 min after virus entry. These results indicated that after internalization, CSFV moved to early and late endosomes before releasing its RNA. Taken together, our findings demonstrate for the first time that CSFV enters cells through the endocytic pathway, providing new insights into the life cycle of pestiviruses. Bovine viral diarrhea virus (BVDV), a single-stranded, positive-sense pestivirus within the family Flaviviridae, is internalized by clathrin-dependent receptor-mediated endocytosis. However, the detailed mechanism of cell entry is unknown for other pestiviruses, such as classical swine fever (CSF) virus (CSFV). CSFV is the etiological agent of CSF, a highly contagious disease of swine that causes numerous deaths in pigs and enormous economic losses in China. Understanding the entry pathway of CSFV will not only advance our knowledge of CSFV infection and pathogenesis but also provide novel drug targets for antiviral intervention. Based on this objective, we used systematic approaches to dissect the pathway of entry of CSFV into PK-15 cells. This is the first report to show that the entry of CSFV into PK-15 cells requires a low-pH environment and involves dynamin- and cholesterol-dependent, clathrin-mediated endocytosis that requires Rab5 and Rab7.

Keratinocyte-Derived IL-17E Contributes to Inflammation in Psoriasis

Psoriasis is a chronic inflammatory skin disorder effectively treated by blocking IL-17RA, a receptor chain used by several IL-17 family members, including IL-17E. Although IL-17A is critically involved in the pathogenesis of psoriasis, the contribution of IL-17E remains unknown. Here we show that IL-17E(+) cells are more abundant than IL-17A(+) cells in lesional psoriatic skin. IL-17E synthesis is increased in keratinocytes within psoriatic plaques, and macrophages having a mixed M1/M2 phenotype represent an important proportion of the IL-17E(+) cells infiltrating the dermis. Mechanistically, macrophages do not synthetize but rather take up IL-17E via receptor-mediated clathrin-dependent endocytosis. Furthermore, monocyte-derived macrophages invitro polarized in M2, but not M1, express the IL-17E receptor and respond to IL-17E by preferentially producing inflammatory cytokines and chemokines involved in neutrophil recruitment. Remarkably, IL-17E expression in lesional psoriatic skin correlates with the number of neutrophils while being inversely proportional to the number of infiltrating T cells. These data provide strong evidence for a proinflammatory role of keratinocyte-derived IL-17E in psoriasis, possibly via macrophage activation.

Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria.

The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endo-somes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intra-cellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells.

Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the 70 kDa heat-shock protein (Hsp70). Heat-shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is constitutively expressed; thus, it may be expressed under physiological conditions. In C6/36 cells, Hsc70 is upregulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at the late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C and D; of these, isoform D helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.

The Usp8 deubiquitination enzyme is post-translationally modified by tyrosine and serine phosphorylation

The ERBB1–ERBB4 receptors belong to a family of receptor tyrosine kinases that trigger a network of signaling pathways after ligand binding, thereby regulating cellular growth, differentiation and development. Ligand-induced signaling through ERBB1, also known as EGFR, is attenuated by the clathrin-dependent receptor-mediated endocytosis and RING E3-ligase Cbl-mediated receptor ubiquitination, which is followed by incorporation into multi-vesicular bodies (MVBs) and subsequent degradation in lysosomes. Before incorporation into MVBs, the EGFR is deubiquitinated by Usp8. We previously demonstrated that Usp8 is tyrosine phosphorylated in an EGFR- and SRC-kinase dependent manner. In the present study we show that overexpression of constitutively active SRC enhances constitutive and ligand-induced Usp8 tyrosine phosphorylation. We also show that enhanced endosomal recycling of the EGFR induced by TGFα stimulation is associated with decreased Usp8 tyrosine phosphorylation. We therefore hypothesize that tyrosine phosphorylation of Usp8 could regulate the function of Usp8. To identify Usp8 tyrosine phosphorylation site(s), we used Usp8 deletion constructs, site-directed mutagenesis of nine individual Usp8 tyrosine residues and mass spectrometry (MS) analysis. Our results demonstrate that the MIT-domain is necessary for ligand-induced tyrosine phosphorylation of Usp8 1–504. However, mutation of three MIT domain tyrosine residues did not abolish Usp8 tyrosine phosphorylation. Similar results were obtained upon mutation of six exposed tyrosine residues in the Rhod domain and linker region. Repeated MS analysis of both Usp8 WT and C748A mutants readily detected serine phosphorylation, including the S680 14–3–3 binding site, but did not reveal any phospho-tyrosine residues. Notably, mutation of the tyrosine residue in the Usp8 14–3–3 binding motif (Y679) did not abolish phosphoserine-dependent binding of 14–3–3 to Usp8. Our findings are most consistent with the model that MIT domain-dependent recruitment of Usp8 to endosomal membranes is important for low stoichiometry SRC-mediated tyrosine phosphorylation of multiple Usp8 tyrosines. Our findings demonstrate that Usp8 is a target for the post-translational serine and tyrosine phosphorylation, most likely characterized by low abundant tyrosine phosphorylation on multiple residues, and high abundant serine phosphorylation on several residues.

Endocytic mechanism of transferrin-conjugated nanoparticles and the effects of their size and ligand number on the efficiency of drug delivery

When considering drug delivery, the amount of drug that can be carried at a particular time and how the drug is incorporated efficiently into cells are important parameters. Transferrin (Tf)-conjugated nanocarriers have been used for the targeted delivery of drugs to cancer cells due to the availability of receptor-mediated clathrin-dependent endocytosis. In general, however, endocytosis seems to differ according to the size and shape of carriers. Large substances are generally internalized into cells by phagocytosis. We studied the internalization mechanism of Tf-conjugated nanoparticles (diameter, 522 nm). Tf-conjugated polystyrene particles were incorporated into cells by receptor-mediated endocytosis with large clathrin-coated vesicles even though their diameter was >500 nm and despite that fact that clathrin-coated vesicles have a diameter of ≈100 nm. This finding suggests that signals for internalization generated by stimulated Tf receptors (TfRs) activate clathrin-mediated endocytosis preferentially. Whether these larger particles could deliver drugs more efficiently than smaller particles was then examined. The toxicity of larger Tf-conjugated biodegradable nanoparticles (poly(lactic-co-glycolic acid)) encapsulating doxorubicin (diameter, 216 ± 38 nm) was appreciably dependent on the number of Tf molecules conjugated on a particle and the number of TfRs expressed on the cell membrane. Larger Tf-conjugated particles delivered drugs to cancer cells expressing many TfRs more selectively than their smaller counterparts (diameter, 56 ± 9 nm) if they were decorated with an appropriate number of Tf molecules.

Recycling of EGFR and ErbB2 is associated with impaired Hrs tyrosine phosphorylation and decreased deubiquitination by AMSH

ErbB receptors play an important role in normal cellular growth, differentiation and development, but overexpression or poor downregulation can result in enhanced signaling and cancerous growth. ErbB signaling is terminated by clathrin-dependent receptor-mediated endocytosis, followed by incorporation in multi-vesicular bodies and subsequent degradation in lysosomes. In contrast to EGFR, ErbB2 displays poor ligand-induced downregulation and enhanced recycling, but the molecular mechanisms underlying this difference are poorly understood. Given our previous observation that both EGFR and an EGFR–ErbB2 chimera undergo Cbl-mediated K63-polyubiquitination, we investigated in the present study whether activation of the EGFR and the EGFR–ErbB2 chimera is associated with tyrosine phosphorylation of the ESCRT-0 complex subunit Hrs and AMSH-mediated deubiquitination. EGF stimulation of the EGFR resulted in efficient Hrs tyrosine phosphorylation and deubiquitination by the K63-polyubiquitin chain-specific deubiquitinating enzyme AMSH. In contrast, EGF activation of EGFR–ErbB2 showed significantly decreased Hrs tyrosine phosphorylation and deubiquitination by AMSH. To test whether this phenotype is the result of endosomal recycling, we induced recycling of the EGFR by stimulation with TGFα. Indeed, even though TGFα-stimulation of EGFR is associated with efficient ligand-stimulated K63-polyubiquitination, we observed that Hrs tyrosine phosphorylation as well as AMSH-mediated deubiquitination is significantly reduced under these conditions. Using various EGFR–ErbB2 chimeras, we demonstrate that enhanced recycling, decreased Hrs tyrosine phosphorylation and decreased AMSH mediated deubiquitination of EGFR–ErbB2 chimeras is primarily due to the presence of ErbB2 sequences or the absence of EGFR sequences C-terminal to the Cbl binding site. We conclude that endosomal recycling of the EGFR and ErbB2 receptors is associated with significantly impaired tyrosine phosphorylation of the ESCRT-0 subunit Hrs as well as decreased deubiquitination by AMSH, which is consistent with the finding that recycling receptors are not efficiently incorporated in the MVB pathway.

Decoupling of Activation and Effector Binding Underlies ARF6 Priming of Fast Endocytic Recycling

The small GTP-binding protein ADP-ribosylation factor 6 (ARF6) controls the endocytic recycling pathway of several plasma membrane receptors. We analyzed the localization and GDP/GTP cycle of GFP-tagged ARF6 by total internal reflection fluorescent microscopy. We found that ARF6-GFP associates with clathrin-coated pits (CCPs) at the plasma membrane in a GTP-dependent manner in a mechanism requiring the adaptor protein complex AP-2. In CCP, GTP-ARF6 mediates the recruitment of the ARF-binding domain of downstream effectors including JNK-interacting proteins 3 and 4 (JIP3 and JIP4) after the burst recruitment of the clathrin uncoating component auxilin. ARF6 does not contribute to receptor-mediated clathrin-dependent endocytosis. In contrast, we found that interaction of ARF6 and JIPs on endocytic vesicles is required for trafficking of the transferrin receptor in the fast, microtubule-dependent endocytic recycling pathway. Our findings unravel a novel mechanism of separation of ARF6 activation and effector function, ensuring that fast recycling may be determined at the level of receptor incorporation into CCPs.

Abstract 4510: Similarities in trans-Golgi localization of chlorotoxin in tumor and in normal vascular endothelial cells

Abstract Introduction: Chlorotoxin (TM601), a scorpion derived 36-amino acid peptide is a new experimental drug currently in Phase II clinical trials for adult recurrent glioma. Despite its known specificity for tumor cells, its exact mode of action is not known but anti-angiogenic effects have been observed. Here we provide evidence that TM601 localizes near the Golgi complex in human glioma (U373, U87), lung carcinoma (A549), and in normal human vascular endothelial cells (HUVEC) but not in normal human dermal fibroblasts (HDF) and astrocytes (NHA). TM601 enters glioma cells via clathrin-mediated transport and affects level of several Golgi associated peptides. Methods: Cells were treated with or without 10 µM TM601 or fluorescent TM601 for 1 or 24 hours. We evaluated: 1) TM601 cellular localization in relation to the Golgi (antibodies to TM601 and Golgin97 marker) and lysosomes (fluorescent trackers) using immunocytochemistry or direct immunofluorescence visually and quantitatively by confocal microscopy; 2) TM601 cellular entry in U373 and HDF cells using an inhibitor of clathrin-dependent endocytosis (chlorpromazine 10 µg/ml) or inhibitor of macropinocytosis (amiloride 300 µM). Cell lysates were prepared and proteins evaluated by Western blot; 3) TM601 effects on the U373 proteome in the sucrose fractionated Golgi-enriched fraction using liquid chromatography coupled to tandem mass spectrometry. Results: 1) In all tumor cells and HUVEC cells, TM601 was observed as an intense granular staining at the trans-Golgi. In normal fibroblasts and astrocytes it was found at a low level in the cytoplasm. In U373 glioma, but not in the normal HDF and NHA cells, TM601 showed a statistically significant colocalization with the LysoTracker (p=3.9E-05) that stained Golgi associated lysosomal vesicles. 2) A statistically significant effect on TM601 cellular level in U373 was observed after treatment with chlorpromazine and in HDF (but not U373) with amiloride. 3) Preliminary data (n=2) of Golgi-enriched fractions from TM601 treated U373 cells showed that TM601 treatment significantly affected levels of several peptides. Further evaluation by Western blots is planned. Summary: Our data show similarity between glioma, lung carcinoma and the normal vascular endothelial cells in the cellular uptake/transport of TM601 toward Golgi complexes. These cells differ drastically from other normal cells such as HDF and NHA which present low cytoplasmic level of TM601. It is expected that endothelial cell transport of TM601 may occur via receptor mediated clathrin dependent endocytosis pathway as in the glioma cells. In addition, our preliminary data showed TM601 effect on the level of several Golgi associated proteins in glioma cells. A similar study with endothelial cells is planned. The information obtained from these studies will assist in understanding how TM601 affects angiogenesis and tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4510.

Inequivalent Contribution of the Five Tryptophan Residues in the C-Lobe of Human Serum Transferrin to the Fluorescence Increase when Iron is Released

Human serum transferrin (hTF), with two Fe3+ binding lobes, transports iron into cells. Diferric hTF preferentially binds to a specific receptor (TFR) on the surface of cells, and the complex undergoes clathrin dependent receptor-mediated endocytosis. The clathrin-coated vesicle fuses with an endosome where the pH is lowered, facilitating iron release from hTF. On a biologically relevant time scale (2-3 min), the factors critical to iron release include pH, anions, a chelator, and the interaction of hTF with the TFR. Previous work, in which the increase in the intrinsic fluorescence signal was used to monitor iron release from the hTF/TFR complex, established that the TFR significantly enhances the rate of iron release from the C-lobe of hTF. In the current study, the role of the five C-lobe Trp residues in reporting the fluorescence change has been evaluated (+/-sTFR). Only four of the five recombinant Trp --> Phe mutants produced well. A single slow rate constant for iron release is found for the monoferric C-lobe (FeC hTF) and the four Trp mutants in the FeC hTF background. The three Trp residues equivalent to those in the N-lobe differed from the N-lobe and each other in their contributions to the fluorescent signal. Two rate constants are observed for the FeC hTF control and the four Trp mutants in complex with the TFR: k(obsC1) reports conformational changes in the C-lobe initiated by the TFR, and k(obsC2) is ascribed to iron release. Excitation at 295 nm (Trp only) and at 280 nm (Trp and Tyr) reveals interesting and significant differences in the rate constants for the complex.

Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression

Previous studies have suggested that most papillomaviruses enter the host cell via clathrin-dependent receptor-mediated endocytosis but have not addressed later steps in viral entry. To examine these events, we followed the localization of L2 and packaged DNA after entry of infectious virions or L1/L2 pseudovirions. Confocal microscopic analyses of HeLa cells showed a time-dependent uncoating of capsids in cytoplasmic vesicles and the accumulation of both L2 and viral DNA at distinct nuclear domains identified as nuclear domain 10 (ND10). Both L2 and the pseudogenome had a punctate distribution and localized to ND10 in promyelocytic leukemia protein (PML)-expressing cells, whereas L2 had a diffuse nuclear distribution in PML-/- cells. The number of pseudovirus-infected cells was an order of magnitude higher in the PML+ cells compared with the PML-/- cells, and viral genome transcription after infection with authentic bovine papillomavirus virions was similarly elevated in PML+ cells. The results identify a role for PML in the enhancement of viral infectivity in the early part of the life cycle. We propose a model in which L2 chaperones the viral genome to ND10 to efficiently initiate viral transcription.

A JC virus-induced signal is required for infection of glial cells by a clathrin- and eps15-dependent pathway.

Infectious entry of JC virus (JCV) into human glial cells occurs by receptor-mediated clathrin-dependent endocytosis. In this report we demonstrate that the tyrosine kinase inhibitor genistein blocks virus entry and inhibits infection. Transient expression of dominant-negative eps15 mutants, including a phosphorylation-defective mutant, inhibited both virus entry and infection. We also show that the JCV-induced signal activates the mitogen-activated protein kinases ERK1 and ERK2. These data demonstrate that JC virus binding to human glial cells induces an intracellular signal that is critical for entry and infection by a ligand-inducible clathrin-dependent mechanism.