Hypoxia Modulates Cellular Endocytic Pathways and Organelles with Enhanced Cell Migration and 3D Cell Invasion.

Hypoxia, a decrease in cellular or tissue level oxygen content, is characteristic of most tumors and has been shown to drive cancer progression by altering multiple subcellular processes. We hypothesized that the cancer cells in a hypoxic environment might have slower proliferation rates and increased invasion and migration rates with altered endocytosis compared to the cancer cells in the periphery of the tumor mass that experience normoxic conditions. We induced cellular hypoxia by exposing cells to cobalt chloride, a chemical hypoxic mimicking agent. This study measured the effect of hypoxia on cell proliferation, migration, and invasion. Uptake of fluorescently labeled transferrin, galectin3, and dextran that undergo endocytosis through major endocytic pathways (Clathrin-mediated pathway (CME), Clathrin-independent pathway (CIE), Fluid phase endocytosis (FPE)) were analyzed during hypoxia. Also, the organelle changes associated with hypoxia were studied with organelle trackers. We found that the proliferation rate decreased, and the migration and invasion rate increased in cancer cells in hypoxic conditions compared to normoxic cancer cells. A short hypoxic exposure increased galectin3 uptake in hypoxic cancer cells, but a prolonged hypoxic exposure decreased clathrin-independent endocytic uptake of galectin 3. Subcellular organelles, such as mitochondria, increased to withstand the hypoxic stress, while other organelles, such as Endoplasmic reticulum (ER), were significantly decreased. These data suggest that hypoxia modulates cellular endocytic pathways with reduced proliferation and enhanced cell migration and invasion.

An Alternative Mechanism of Subcellular Iron Uptake Deficiency in Cardiomyocytes.

BACKGROUND: Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake mechanisms that are independent of systemic absorption are incompletely understood. The main intracellular route for iron uptake in cardiomyocytes is clathrin-mediated endocytosis. METHODS: We investigated subcellular iron uptake mechanisms in patient-derived and CRISPR/Cas-edited induced pluripotent stem cell-derived cardiomyocytes as well as patient-derived heart tissue. We used an integrated platform of DIA-MA (mass spectrometry data-independent acquisition)-based proteomics and signaling pathway interrogation. We employed a genetic induced pluripotent stem cell model of 2 inherited mutations (TnT [troponin T]-R141W and TPM1 [tropomyosin 1]-L185F) that lead to dilated cardiomyopathy (DCM), a frequent cause of heart failure, to study the underlying molecular dysfunctions of DCM mutations. RESULTS: We identified a druggable molecular pathomechanism of impaired subcellular iron deficiency that is independent of systemic iron metabolism. Clathrin-mediated endocytosis defects as well as impaired endosome distribution and cargo transfer were identified as a basis for subcellular iron deficiency in DCM-induced pluripotent stem cell-derived cardiomyocytes. The clathrin-mediated endocytosis defects were also confirmed in the hearts of patients with DCM with end-stage heart failure. Correction of the TPM1-L185F mutation in DCM patient-derived induced pluripotent stem cells, treatment with a peptide, Rho activator II, or iron supplementation rescued the molecular disease pathway and recovered contractility. Phenocopying the effects of the TPM1-L185F mutation into WT induced pluripotent stem cell-derived cardiomyocytes could be ameliorated by iron supplementation. CONCLUSIONS: Our findings suggest that impaired endocytosis and cargo transport resulting in subcellular iron deficiency could be a relevant pathomechanism for patients with DCM carrying inherited mutations. Insight into this molecular mechanism may contribute to the development of treatment strategies and risk management in heart failure.

Ursolic acid-piperazine-dithiocarbamate ruthenium(II) polypyridyl complexes induced necroptosis in MGC-803 cells.

Three ursolic acid-piperazine-dithiocarbamate ruthenium(II) polypyridyl complexes Ru1-Ru3 were designed and synthesized for evaluating antitumor activity. All the complexes exhibited high in vitro cytotoxicity against MGC-803, T24, HepG2, CNE2, MDA-MB-231, MCF-7, A549, and A549/DDP cell lines. Ru1, Ru2, and Ru3 were 11, 8 and 10 times, respectively, more active than cisplatin against A549/DDP. An in vivo study on MGC-803 xenograft mouse models demonstrated that representative Ru2 exhibited an effective inhibitory effect on tumor growth, showing stronger antitumor activity than cisplatin. Biological investigations suggested that Ru2 entered MGC-803 cells by a clathrin-mediated endocytic pathway, initially localizing in the lysosomes and subsequently escaping and localizing in the mitochondria. Mitochondrial swelling resulted in vacuolization, which induced vacuolation-associated cell death and necroptosis with the formation of necrosomes (RIP1-RIP3) and the uptake of propidium iodide. These results demonstrate that the potential of Ru2 as a chemotherapeutic agent to kill cancer cells via a dual mechanism represents an alternative way to eradicate apoptosis-resistant forms of cancer.

In vivo and in vitro toxicity of a stainless-steel aerosol generated during thermal spray coating.

Thermal spray coating is an industrial process in which molten metal is sprayed at high velocity onto a surface as a protective coating. An automated electric arc wire thermal spray coating aerosol generator and inhalation exposure system was developed to simulate an occupational exposure and, using this system, male Sprague-Dawley rats were exposed to stainless steel PMET720 aerosols at 25 mg/m3 × 4 h/day × 9 day. Lung injury, inflammation, and cytokine alteration were determined. Resolution was assessed by evaluating these parameters at 1, 7, 14 and 28 d after exposure. The aerosols generated were also collected and characterized. Macrophages were exposed in vitro over a wide dose range (0-200 µg/ml) to determine cytotoxicity and to screen for known mechanisms of toxicity. Welding fumes were used as comparative particulate controls. In vivo lung damage, inflammation and alteration in cytokines were observed 1 day post exposure and this response resolved by day 7. Alveolar macrophages retained the particulates even after 28 day post-exposure. In line with the pulmonary toxicity findings, in vitro cytotoxicity and membrane damage in macrophages were observed only at the higher doses. Electron paramagnetic resonance showed in an acellular environment the particulate generated free radicals and a dose-dependent increase in intracellular oxidative stress and NF-kB/AP-1 activity was observed. PMET720 particles were internalized via clathrin and caveolar mediated endocytosis as well as actin-dependent pinocytosis/phagocytosis. The results suggest that compared to stainless steel welding fumes, the PMET 720 aerosols were not as overtly toxic, and the animals recovered from the acute pulmonary injury by 7 days.

Nanotherapeutics with suitable properties for advanced anticancer therapy based on HPMA copolymer-bound ritonavir via pH-sensitive spacers.

Ritonavir (RIT) is a widely used antiviral drug that acts as an HIV protease inhibitor with emerging potential in anticancer therapies. RIT causes inhibition of P-glycoprotein, which plays an important role in multidrug resistance (MDR) in cancer cells when overexpressed. Moreover, RIT causes mitochondrial dysfunction, leading to decreased ATP production and reduction of caveolin I expression, which can affect cell migration and tumor progression. To increase its direct antitumor activity, decrease severe side effects induced by the use of free RIT and improve its pharmacokinetics, ritonavir 5-methyl-4-oxohexanoate (RTV) was synthesized and conjugated to a tumor-targeted polymer carrier based on a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. Here we demonstrated that polymer-bound RTV enhanced the internalization of polymer-RTV conjugates, differing in RTV content from 4 to 15 wt%, in HeLa cancer cells compared with polymer without RTV. The most efficient influx and internalization properties were determined for the polymer conjugate bearing 11 wt% of RTV. This conjugate was internalized by cells using both caveolin- and clathrin-dependent endocytic pathways in contrast to the RTV-free polymer, which was preferentially internalized only by clathrin-mediated endocytosis. Moreover, we found the co-localization of the RTV-conjugate with mitochondria and a significant decrease of ATP production in treated cells. Thus, the impact on mitochondrial mechanism can influence the function of ATP-dependent P-glycoprotein and also the cell viability of MDR cancer cells. Overall, this study demonstrated that the polymer-RTV conjugate is a promising polymer-based nanotherapeutic, suitable for antitumor combination therapy with other anticancer drugs and a potential mitochondrial drug delivery system.

Clathrin-mediated endocytosis is required for ANE 30-100K-induced autophagy.

BACKGROUND: We identified an autophagy-inducing areca nut (AN) ingredient (AIAI) in the 30-100 kDa fraction of AN extract (ANE 30-100K). This study was to analyze the role of endocytosis in ANE 30-100K-induced autophagy. METHODS: We used benzyl alcohol, dynasore, and shRNA of clathrin and dynamin to assess whether ANE 30-100K-induced cytotoxicity and accumulation of microtubule-associated protein 1 light chain 3 (LC3)-II were affected in oral (OECM-1) and esophageal (CE81T/VGH) carcinoma cells. RESULTS: Both benzyl alcohol and dynasore effectively reduced ANE 30-100K-induced cytotoxicity and LC3-II accumulation in OECM-1 and CE81T/VGH cells. Downregulated protein expression of both clathrin and dynamin by their shRNA also significantly attenuated ANE 30-100K-induced elevation of LC3-II levels in CE81T/VGH cells. CONCLUSIONS: These results indicate that AIAI may be engulfed by cells through clathrin-mediated endocytosis, which promotes the execution of the following autophagy program.

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis.

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF-EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications.

Comparative Proteomics Reveals Differences in Host-Pathogen Interaction between Infectious and Commensal Relationship with Campylobacter jejuni.

Campylobacter jejuni is the leading food-borne poisoning in industrialized countries. While the bacteria causes disease in humans, it merely colonizes the gut in poultry or pigs, where seems to establish a commensal relationship. Until now, few studies have been conducted to elucidate the relationship between C. jejuni and its different hosts. In this work, a comparative proteomics approach was used to identify the underlying mechanisms involved in the divergent outcome following C. jejuni infection in human and porcine host. Human (INT-407) and porcine (IPEC-1) intestinal cell lines were infected by C. jejuni for 3 h (T3h) and 24 h (T24h). C. jejuni infection prompted an intense inflammatory response at T3h in human intestinal cells, mainly characterized by expression of proteins involved in cell spreading, cell migration and promotion of reactive oxygen species (ROS). Proteomic analysis evidenced significantly regulated biofunctions in human cells related with engulfment and endocytosis, and supported by canonical pathways associated to infection such as caveolar- and clathrin-mediated endocytosis signaling. In porcine IPEC-1 cells, inflammatory response as well as signaling pathways that control cellular functions such as cell migration, endocytosis and cell cycle progression resulted downregulated. These differences in the host response to infection were supported by the different pattern of adhesion and invasion proteins expressed by C. jejuni in human and porcine cells. No marked differences in expression of virulence factors involved in adaptive response and iron acquisition functions were observed. Therefore, the results of this study suggest that both host and pathogen factors are responsible for commensal or infectious character of C. jejuni in different hosts.

Clathrin-Dependent Uptake of Paraquat into SH-SY5Y Cells and Its Internalization into Different Subcellular Compartments.

The herbicide paraquat (PQ) is an exogenous toxin that allows the selective activation of dopaminergic neurons in the mesencephalon to induce injury and also causes its apoptosis in vitro. However, uptake mechanisms between PQ and neurons remain elusive. To address this issue, we undertook a study of PQ endocytosis in a dopaminergic SH-SY5Y cell line as well as explored the subsequent subcellular location and potential functional analysis of PQ. The PQ was found to bind the SH-SY5Y cell membrane and then became internalized via a clathrin-dependent pathway. PQ was internalized by many subcellular organelles in a time- and dose-dependent manner. Interestingly, the taken up PQ and secretogranin III (SCG3), which became dysregulated with PQ treatment that induced SH-SY5Y apoptosis in our previous study, colocalized in cytoplasmic vesicles. Taken together, our findings indicate that PQ is endocytosed by SH-SY5Y cells and that its multiple, subcellular localizations indicate PQ may potentially be involved in subcellular-level functions. More importantly, PQ distributing preferentially into SCG3-positive vesicles demonstrates its selective targeting which may affect SCG3 and cargoes carried by SCG3-positive vesicles. Therefore, it is reasonable to infer that PQ toxic insults may potentially interfere with neurotransmitter storage and transport associated with secretory granules.

Co-delivery of doxorubicin and P-gp inhibitor by a reduction-sensitive liposome to overcome multidrug resistance, enhance anti-tumor efficiency and reduce toxicity.

To overcome multidrug resistance (MDR) in cancer chemotherapy with high efficiency and safety, a reduction-sensitive liposome (CL-R8-LP), which was co-modified with reduction-sensitive cleavable PEG and octaarginine (R8) to increase the tumor accumulation, cellular uptake and lysosome escape, was applied to co-encapsulate doxorubicin (DOX) and a P-glycoprotein (P-gp) inhibitor of verapamil (VER) in this study. The encapsulation efficiency (EE) of DOX and VER in the binary-drug loaded CL-R8-LP (DOX + VER) was about 95 and 70% (w/w), respectively. The uptake efficiencies, the cytotoxicity, and the apoptosis and necrosis-inducing efficiency of CL-R8-LP (DOX + VER) were much higher than those of DOX and the other control liposomes in MCF-7/ADR cells or tumor spheroids. Besides, CL-R8-LP (DOX + VER) was proven to be uptaken into MCF-7/ADR cells by clathrin-mediated and macropinocytosis-mediated endocytosis, followed by efficient lysosomal escape. In vivo, CL-R8-LP (DOX + VER) effectively inhibited the growth of MCF-7/ADR tumor and reduce the toxicity of DOX and VER, which could be ascribed to increased accumulation of drugs in drug-resistant tumor cells and reduced distribution in normal tissues. In summary, the co-delivery of chemotherapeutics and P-gp inhibitors by our reduction-sensitive liposome was a promising approach to overcome MDR, improve anti-tumor effect and reduce the toxicity of chemotherapy.

Recurrent Fusion Genes in Gastric Cancer: CLDN18-ARHGAP26 Induces Loss of Epithelial Integrity.

Genome rearrangements, a hallmark of cancer, can result in gene fusions with oncogenic properties. Using DNA paired-end-tag (DNA-PET) whole-genome sequencing, we analyzed 15 gastric cancers (GCs) from Southeast Asians. Rearrangements were enriched in open chromatin and shaped by chromatin structure. We identified seven rearrangement hot spots and 136 gene fusions. In three out of 100 GC cases, we found recurrent fusions between CLDN18, a tight junction gene, and ARHGAP26, a gene encoding a RHOA inhibitor. Epithelial cell lines expressing CLDN18-ARHGAP26 displayed a dramatic loss of epithelial phenotype and long protrusions indicative of epithelial-mesenchymal transition (EMT). Fusion-positive cell lines showed impaired barrier properties, reduced cell-cell and cell-extracellular matrix adhesion, retarded wound healing, and inhibition of RHOA. Gain of invasion was seen in cancer cell lines expressing the fusion. Thus, CLDN18-ARHGAP26 mediates epithelial disintegration, possibly leading to stomach H(+) leakage, and the fusion might contribute to invasiveness once a cell is transformed.

Tyrosine phosphorylation regulates the endocytosis and surface expression of GluN3A-containing NMDA receptors.

Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity, and development. GluN3A (formerly NR3A) is a nonconventional member of the NMDA receptor (NMDAR) subunit family, which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared with NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing toward GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL), which is located within the cytoplasmic C-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provides a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment.

Peroxynitrite donor SIN-1 alters high-affinity choline transporter activity by modifying its intracellular trafficking.

Sodium-coupled, high-affinity choline transporters (CHTs) are inhibited by 3-morpholinosydnonimine (SIN-1) [peroxynitrite (ONOO⁻) donor]; ONOO⁻ can be produced from nitric oxide and reactive oxygen species during neurodegeneration. SIN-1 rapidly increases CHT internalization from the cell surface, and this correlates with decreased choline uptake. This study addresses mechanisms by which SIN-1 inhibits CHT function in human neuronal SH-SY5Y cells. Thus, mutant L531A-CHT, which does not constitutively internalize into cells by a clathrin-mediated process, is resistant to SIN-1 effects. This suggests that CHT inhibition is not due to oxidative-nitrosative inactivation of the protein and that decreased levels of cell surface CHT in SIN-1-treated cells is related to alterations in its trafficking and subcellular disposition. Dominant-negative proteins AP180C and dynamin-K44A, which interfere with clathrin-mediated and dynamin-dependent endocytosis, respectively, attenuate CHT inhibition by SIN-1. CHT in both vehicle- and SIN-1-treated cells colocalizes with Rab7, Rab9, and Lamp-1 in late endosomes and lysosomes to a similar extent. Lysosome inhibitors increase choline uptake, suggesting that CHT proteins are normally degraded by lysosomes, and this is not altered by oxidative stress. Unexpectedly, inhibitors of proteasomes, but not lysosomes, attenuate SIN-1-mediated inhibition of choline uptake, indicating that proteasomal degradation plays a role in regulating CHT disposition in SIN-1-treated cells. SIN-1 treatment also enhances CHT ubiquitination. Thus, CHT inhibition in SIN-1-treated cells is mediated by proteasomal degradation, which differs from inhibitory mechanisms for some neurotransmitter transporters under similar conditions. Increased oxidative-nitrosative stress in the microenvironment of cholinergic nerve terminals would diminish cholinergic transmission by reducing choline availability for ACh synthesis.

Dynamin 2 mutations in Charcot-Marie-Tooth neuropathy highlight the importance of clathrin-mediated endocytosis in myelination.

Mutations in dynamin 2 (DNM2) lead to dominant intermediate Charcot-Marie-Tooth neuropathy type B, while a different set of DNM2 mutations cause autosomal dominant centronuclear myopathy. In this study, we aimed to elucidate the disease mechanisms in dominant intermediate Charcot-Marie-Tooth neuropathy type B and to find explanations for the tissue-specific defects that are associated with different DNM2 mutations in dominant intermediate Charcot-Marie-Tooth neuropathy type B versus autosomal dominant centronuclear myopathy. We used tissue derived from Dnm2-deficient mice to establish an appropriate peripheral nerve model and found that dominant intermediate Charcot-Marie-Tooth neuropathy type B-associated dynamin 2 mutants, but not autosomal dominant centronuclear myopathy mutants, impaired myelination. In contrast to autosomal dominant centronuclear myopathy mutants, Schwann cells and neurons from the peripheral nervous system expressing dominant intermediate Charcot-Marie-Tooth neuropathy mutants showed defects in clathrin-mediated endocytosis. We demonstrate that, as a consequence, protein surface levels are altered in Schwann cells. Furthermore, we discovered that myelination is strictly dependent on Dnm2 and clathrin-mediated endocytosis function. Thus, we propose that altered endocytosis is a major contributing factor to the disease mechanisms in dominant intermediate Charcot-Marie-Tooth neuropathy type B.

Clathrin-mediated endocytosis in living host cells visualized through quantum dot labeling of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, we found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked reduction in viral entry. We also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymerization is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compound that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments. This is the first report in which QD labeling is used to visualize the dynamic interactions between viruses and endocytic structures; the results presented demonstrate that IHNV enters host cells via clathrin-mediated endocytic, cytoskeleton-dependent, and low-pH-dependent pathways.

Cell specific peptide-conjugated polysaccharide nanogels for protein delivery.

A cell specific peptide (Arg-Gly-Asp; RGD)-modified nanogel was prepared and evaluated for its potential to act as a protein delivery carrier. A bovine serum albumin (BSA)/RGD-modified nanogel complex was efficiently internalized into cells through integrin-mediated endocytosis. Endosomal escape of the RGD-modified nanogel was observed after 24 h incubation. The nanogel proved useful for targeted protein delivery.

Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand.

Targeting and down-regulation of ErbB2, a member of EGF receptor family, is regarded as one of the key aspect for cancer treatment because it is often overexpressed in breast and ovarian cancer cells. Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand. Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells. In this report, we designed divalent and multivalent forms of EC-1 peptide with the Fc portion of the human IgG and bionanocapsule modified with ZZ-tag on its surface to improve the interaction with ErbB2. These forms showed higher affinity to ErbB2 than that of EC-1 monomer. Furthermore, prominent endosomal accumulation of ErbB2 occurred in SK-BR-3 cells when stimulated with EC-Fc ligand multivalently displayed on the surface of the bionanocapsule, whereas SK-BR-3 cells as themselves displayed stringent mechanism against ErbB2 internalization without stimulation. The multivalent form of EC-1 peptide appeared to internalize ErbB2 more efficiently than divalent form did. This internalization was unaffected by the inhibition of clathrin association, but inhibited when the cholesterol was depleted which explained either caveolar or GPI-AP-early endocytic compartment (GEEC) pathway. Because of the lack of caveolin-1 expression, caveolar machinery may be lost in SK-BR-3 cell line. Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.

Regulation of EGF-stimulated EGF receptor endocytosis during M phase.

It has been generally accepted that endocytosis is inhibited during mitotic phase (M phase) as a means to insulate the cell from outside influences. Many endocytic/trafficking proteins are present during M phase, but are associated with partners that are distinct from those involved in trafficking pathways. These findings have led to the 'moonlighting' hypothesis. However, all these findings are based on the study of fluid-phase and constitutive endocytosis. Here, we used epidermal growth factor receptor (EGFR) as a model system to study ligand-induced receptor endocytosis in M phase. We found that EGF-induced EGFR endocytosis still occurs during M phase, but follows different kinetics. EGF-induced EGFR endocytosis is delayed/inhibited for a few minutes and is slower in M phase, especially at metaphase. However, consistent with previous reports, transferrin endocytosis is inhibited under the same conditions. We further showed that EGFR endocytosis is differentially regulated during the cell cycle: dependent on EGFR kinase activation in M phase, but independent of EGFR kinase activation in interphase. We conclude that cells have adopted a system for selective endocytosis in M phase.

Critical role of lipid rafts in virus entry and activation of phosphoinositide 3' kinase/Akt signaling during early stages of Japanese encephalitis virus infection in neural stem/progenitor cells.

Japanese encephalitis virus (JEV), the leading cause of acute encephalitis in South-East Asia is a neurotropic virus infecting various CNS cell types. Most Flaviviruses including JEV get internalised into cells by receptor-mediated endocytosis, which involve clathrin and membrane cholesterol. The cholesterol-enriched membrane microdomains referred to as lipid rafts act as portals for virus entry in a number of enveloped viruses, including Flavivirus. However, the precise role played by membrane lipid rafts in JEV internalisation into neural stem cells is still unknown. We have established neural stem/progenitor cells and C17.2 cell line as models of productive JEV infection. Increase in membrane fluidity and clustering of viral envelope proteins in lipid rafts was observed in early time points of infection. Localisation of non-structural proteins to rafts at later infection stages was also observed. Co-localisation of JEV glycoprotein with Cholera toxin B confirmed that JEV internalisation occurs in a lipid-raft dependent manner. Though JEV entry is raft dependent, however, there is requirement of functional clathrin during endocytosis inside the cells. Besides virus entry, the lipid rafts act as signalling platforms for Src tyrosine kinases and result in activation of phosphoinositìde 3'-kinase/Akt signalling during early JEV infection. Disruption of lipid raft formation by cholesterol depletion using Methyl ß-cyclodextrin, reduced JEV RNA levels and production of infectious virus particles as well as impaired phosphoinositìde 3'-kinase/Akt signalling during initial infection. Overall, our results implicate the importance of host membrane lipid rafts in JEV entry and life cycle, besides maintaining survival of neural stem/progenitor cells during early infection.

Peroxisome proliferator-activated receptor gamma enhances the activity of an insulin degrading enzyme-like metalloprotease for amyloid-beta clearance.

Peroxisome proliferator-activated receptor gamma (PPARgamma) activation results in an increased rate of amyloid-beta (Abeta) clearance from the media of diverse cells in culture, including primary neurons and glial cells. Here, we further investigate the mechanism for Abeta clearance and found that PPARgamma activation modulates a cell surface metalloprotease that can be inhibited by metalloprotease inhibitors, like EDTA and phenanthroline, and also by the peptide hormones insulin and glucagon. The metalloprotease profile of the Abeta-degrading mechanism is surprisingly similar to insulin-degrading enzyme (IDE). This mechanism is maintained in hippocampal and glia primary cultures from IDE loss-of-function mice. We conclude that PPARgamma activates an IDE-like Abeta degrading activity. Our work suggests a drugable pathway that can clear Abeta peptide from the brain.