Clathrin Inhibitor Research Articles

The endocytic adaptor protein Disabled-2 is required for cellular uptake of fibrinogen

Endocytosis is pivotal for uptake of fibrinogen from plasma into megakaryocytes and platelet α-granules. Due to the complex adaptor and cargo contents in endocytic vehicles, the underlying mechanism of fibrinogen uptake is not yet completely elucidated. In this study, we investigated whether the endocytic adaptor protein Disabled-2 (DAB2) mediates fibrinogen uptake in an adaptor-specific manner. By employing primary megakaryocytes and megakaryocytic differentiating human leukemic K562 cells as the study models, we found that fibrinogen uptake is associated with the expression of integrin αIIbβ3 and DAB2 and is mediated through clathrin-dependent manner. Accordingly, constitutive and inducible knockdown of DAB2 by small interfering RNA reduced fibrinogen uptake for 53.2±9.8% and 59.0±10.7%, respectively. Culturing the cells in hypertonic solution or in the presence of clathrin inhibitor chlorpromazine abrogated clathrin-dependent endocytosis and diminished the uptake of fibrinogen. Consistent with these findings, 72.2±0.2% of cellular DAB2 was colocalized with clathrin, whereas 56.4±4.1% and 54.6±2.0% of the internalized fibrinogen were colocalized with clathrin and DAB2, respectively. To delineate whether DAB2 mediates fibrinogen uptake in an adaptor-specific manner, K562 stable cell lines with knockdown of the adaptor protein-2 (AP-2) or double knockdown of AP-2/DAB2 were established. The AP-2 knockdown cells elicited normal fibrinogen uptake activity but the uptake of collagen was diminished. In addition, collagen uptake was further reduced in DAB2/AP-2 knockdown cells. These findings thereby define an adaptor-specific mechanism in the control of fibrinogen uptake and implicate that DAB2 is the key adaptor in the clathrin-associated endocytic complexes to mediate fibrinogen internalization.

The Mechanism of Lipofectamine 2000 Mediated Transmembrane Gene Delivery

In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway were investigated. Clathrin and caveolae-mediated endocytic pathway contributions to transfection efficiency were studied. The inhibitors of endocytosis were used to treat HEp-2 cells before lipofectamine 2000/pGFP-N2 transfection. Transfection efficiency was evaluated with green fluorescence protein (GFP) expression assays. Cell viability and cytotoxicity were evaluated with MTT method. The results indicated that inhibitors of clathrin (chlorpromazine or wortmannin) and caveolin (genistein) could reduce the cell transfection efficiency observably. Both clathrin and caveolae-mediated endocytic pathways play important roles in transmembrane gene delivery.

Inhibition of clathrin/dynamin-dependent internalization interferes with LPS-mediated TRAM–TRIF-dependent signaling pathway

Recognition of lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) activates two district proinflammatory signaling pathway and initiates LPS internalization. To investigate roles of LPS internalization, a traditionally regarded metabolic pathway for LPS, in regulation of these two pathways, three internalization inhibitors, monodansylcadaverine (MDC, a clathrin inhibitor), dynasore (DS, a dynamin inhibitor) and chloroquine (CQ, an endosome acidifying maturation inhibitor) were applied to induce internalization dysfunction in macrophages. Results showed MDC and DS affected LPS internalization but did not interfere with their colocalization. Additionally, they decreased cytokines and chemokines release and inhibited signaling molecules activation mediated by TRAM–TRIF-dependent pathway as determined by protein array. In contrast, CQ did not inhibit LPS internalization but affected the colocalization. It also suppressed macrophage activation mediated by both MyD88-dependent and TRAM–TRIF-dependent pathways. The above data indicated that LPS internalization was clathrin/dynamin dependent and it was essential for activation of TRAM–TRIF-dependent signaling pathway.

Infectious dengue-1 virus entry into mosquito C6/36 cells

The entry of dengue virus-1 (DENV-1) strain Hawaii into mosquito C6/36 cells was analyzed using a variety of biochemical inhibitors together with electron microscopy. The treatment with ammonium chloride, chlorpromazine, dansylcadaverine and dynasore inhibited virus yields, determined by infectivity titrations, whereas nystatin and methyl-β-cyclodextrin did not have any effect. The effect of the clathrin and dynamin inhibitors on DENV-1 entry was corroborated by detection of internalized virions using immunofluorescence staining. Furthermore, electron micrographs showed the incoming virions attached to electron-dense invaginations of the plasma membrane and within coated vesicles that resembled clathrin-coated pits and vesicles, respectively. The susceptibility to clathrin and dynamin inhibitors of clinical isolates from recent outbreaks was comparable to that shown by the cell culture-adapted reference strain. Similarly, DENV-3 strain H87 and DENV-4 strain 8124 were also inhibited in the presence of ammonium chloride, chlorpromazine and dynasore, allowing conclude that the infectious entry of DENV serotypes to mosquito cells occurs by low pH-dependent clathrin-mediated endocytosis.

Mechanistic studies on the uptake and intracellular trafficking of novel cyclodextrin transfection complexes by intestinal epithelial cells

Oral delivery of gene therapeutics would facilitate treatment of local intestinal disease, including colon cancer and inflammatory bowel disease, thus avoiding invasive surgery. The aims of this study were to investigate; if the orientation of the lipid tail on the cyclodextrin (CD) influenced the efficacy of a novel poly-6-cationic amphiphilic CD to transfect intestinal enterocytes; the endocytotic uptake pathway(s), and, the intracellular trafficking of the CD·DNA complexes. Inhibitors of clathrin- and caveolae-mediated endocytosis and macropinocytosis were used to determine the mechanism(s) of CD·DNA uptake by both undifferentiated and differentiated Caco-2 cells. Cell surface heparan sulphate proteoglycans were involved in the association of CD·DNA complexes with undifferentiated Caco-2 cells. Complexation of pDNA with CD facilitated significant levels of pDNA uptake and gene expression (comparable to PEI) in both undifferentiated and differentiated Caco-2 cells. Disruption of intracellular vesicular trafficking reduced transfection activity. CD was also capable of transfecting the more physiologically relevant differentiated Caco-2 model. Macropinocytosis was responsible for the uptake of CD·DNA transfection complexes by both undifferentiated and differentiated Caco-2 cells. The ability of this novel CD to transfect differentiated intestinal cells indicates the potential of this vector for oral gene delivery.

Internalization of Sambucus nigra agglutinins I and II in insect midgut CF‐203 cells

In this project, the uptake mechanisms and localization of two lectins from Sambucus nigra, further referred to as S. nigra agglutinin (SNA)-I and SNA-II, into insect midgut CF-203 cells were studied. SNA-I is a chimeric lectin belonging to the class of ribosome-inactivating proteins, whereas SNA-II is a hololectin devoid of enzymatic activity. Internalization of the fluorescein isothiocyanate-labeled lectin was investigated using confocal microscopy. Both lectins were internalized into the cytoplasm of CF-203 cells at similar rates. Preexposure of the insect midgut cells to specific inhibitors of clathrin- and caveolae-mediated endocytosis resulted in an inhibition of lectin uptake in CF-203 cells and caspase-induced cytotoxicity caused by SNA-I and SNA-II, confirming the involvement of both endocytosis pathways. Further studies demonstrated that the uptake mechanism(s) for both lectins required phosphoinositide 3-kinases, but did not depend on the actin cytoskeleton. Since the hololectin SNA-II apparently uses a similar endocytosis pathway as the chimerolectin SNA-I, it can be concluded that the endocytosis process mainly relies on the carbohydrate-binding activity of the lectins under investigation. © 2011 Wiley Periodicals, Inc.

Nanomaterials can dynamically steer cell responses to biological ligands.

Traditional tissue regeneration approaches to activate cell behaviors on biomaterials rely on the use of extracellular-matrix-based or soluble growth-factor cues. In this article, a novel approach is highlighted to dynamically steer cellular phenomena such as cell motility based on nanoscale substratum features of biological ligands. Albumin-derived nanocarriers (ANCs) with variable nanoscale-size features are functionalized with fibronectin III9-10 matrix ligands, and their effects on primary human keratinocyte activation are investigated. The presentation of fibronectin fragments from ANCs significantly enhances cell migration as compared to free ligands at equivalent concentrations. Notably, cell migration is influenced by the size of the underlying ANCs even for variably sized ANCs covered in comparable levels of fibronectin fragment. For equivalent ligand concentrations, cell migration on the smaller-sized ANCs (30 and 50 nm) is significantly enhanced as compared to that on larger-sized ANCs (75 and 100 nm). In contrast, the enhancement of cell migration on nanocarriers is abolished by the use of immobilized, biofunctionalized ANCs, indicating that "dynamic" nanocarrier internalization events underlie the role of nanocarrier geometry on the differential regulation of cell migration kinetics. Uptake studies using fluorescent ANCs indicate that larger-sized ANCs cause delayed endocytic kinetics and hence could present barriers for internalization during the cell adhesion and motility processes. Motile cells exhibit diminished migration upon exposure to clathrin inhibitors, but not caveolin inhibitors, suggesting the role of clathrin-mediated endocytosis in facilitating cell migratory responsiveness to the nanocarriers. Overall, a monotonic relationship is found between the nanocarrier cytointernalization rate and the cell migration rate, suggesting the possibility of designing biointerfacial features for the dynamic control of cell migration. Thus, the functionalization of a mobile nanocarrier by a biorelevant ligand can be used to sensitize cellular motility activation to the adhesion ligands, and such nanocarrier interfaces can dynamically attune cell migration kinetics by modulating the uptake of the ligand-nanocarrier complex via nanocarrier size.

Cellular Entry of G3.5 Poly (amido amine) Dendrimers by Clathrin- and Dynamin-Dependent Endocytosis Promotes Tight Junctional Opening in Intestinal Epithelia

This study investigates the mechanisms of G3.5 poly (amido amine) dendrimer cellular uptake, intracellular trafficking, transepithelial transport and tight junction modulation in Caco-2 cells in the context of oral drug delivery. Chemical inhibitors blocking clathrin-, caveolin- and dynamin-dependent endocytosis pathways were used to investigate the mechanisms of dendrimer cellular uptake and transport across Caco-2 cells using flow cytometry and confocal microscopy. Dendrimer cellular uptake was found to be dynamin-dependent and was reduced by both clathrin and caveolin endocytosis inhibitors, while transepithelial transport was only dependent on dynamin- and clathrin-mediated endocytosis. Dendrimers were quickly trafficked to the lysosomes after 15 min of incubation and showed increased endosomal accumulation at later time points, suggesting saturation of this pathway. Dendrimers were unable to open tight junctions in cell monolayers treated with dynasore, a selective inhibitor of dynamin, confirming that dendrimer internalization promotes tight junction modulation. G3.5 PAMAM dendrimers take advantage of several receptor-mediated endocytosis pathways for cellular entry in Caco-2 cells. Dendrimer internalization by dynamin-dependent mechanisms promotes tight junction opening, suggesting that dendrimers act on intracellular cytoskeletal proteins to modulate tight junctions, thus catalyzing their own transport via the paracellular route.

Unique leptin trafficking by a tailless receptor

Impairment in blood-to-brain transport of leptin is a major cause as well as consequence of obesity. Leptin crosses the blood-brain barrier by transcytosis rather than undergoing intracellular degradation. Results from previous studies have indicated that the membrane juxtapositional cytoplasmic sequence of the leptin receptor ObR is responsible for leptin transport. To identify the specific structural domains, we generated a series of ObR truncates with different lengths of the intracellular sequence, overexpressed them in 3 types of mammalian cells including cerebral endothelia, and quantified leptin binding and endocytosis. All mutant ObRs were able to bind and mediate the internalization of leptin. Surprisingly, ObR860, a construct with no cytoplasmic sequence, could act like the classical ObRa transporter in internalizing leptin. There were some cell type-dependent variations in the intracellular trafficking of Alexa-labeled leptin when mediated by ObR860 or ObRa because of differential involvement of membrane microdomains, as shown by use of the clathrin inhibitor chlorpromazine and the dynamin inhibitor Dynasore. The clathrin- and dynamin-mediated endocytosis of leptin contrasts with the lack of effect of the caveolae inhibitors nystatin and filipin. Thus, leptin-induced internalization of the ligand-receptor complex can occur without specific sorting signals in the cytoplasmic region of ObR. This novel finding may have significant implications for leptin transport.

Ebola virus uses clathrin-mediated endocytosis as an entry pathway

Ebola virus (EBOV) infects several cell types and while viral entry is known to be pH-dependent, the exact entry pathway(s) remains unknown. To gain insights into EBOV entry, the role of several inhibitors of clathrin-mediated endocytosis in blocking infection mediated by HIV pseudotyped with the EBOV envelope glycoprotein (EbGP) was examined. Wild type HIV and envelope-minus HIV pseudotyped with Vesicular Stomatitis Virus glycoprotein (VSVg) were used as controls to assess cell viability after inhibiting clathrin pathway. Inhibition of clathrin pathway using dominant-negative Eps15, siRNA-mediated knockdown of clathrin heavy chain, chlorpromazine and sucrose blocked EbGP pseudotyped HIV infection. Also, both chlorpromazine and Bafilomycin A1 inhibited entry of infectious EBOV. Sensitivity of EbGP pseudotyped HIV as well as infectious EBOV to inhibitors of clathrin suggests that EBOV uses clathrin-mediated endocytosis as an entry pathway. Furthermore, since chlorpromazine inhibits EBOV infection, novel therapeutic modalities could be designed based on this lead compound.

On the Mechanism and Dynamics of Uptake and Permeation of Polyether-Copolyester Dendrimers Across an In Vitro Blood–Brain Barrier Model

Dendrimers have emerged as a promising drug delivery system due to their well defined size, tailorability, and multifunctional nature. However, their application in brain delivery is relatively a new area of research. The present study was aimed at evaluating the uptake and permeation of polyether-copolyester (PEPE) dendrimers across the blood–brain barrier model and exploring the underlying mechanisms. Saturation was observed in the uptake of rhodamine B labeled PEPE dendrimers by brain vascular endothelial (bEnd.3) cells at high concentrations. Clathrin and caveolin inhibitors produced partial inhibition of the dendrimer uptake, signifying contribution of both pathways in the uptake process. PEPE dendrimers were able to cross in vitro BBB model in high amounts with Papp of 19.7±1.9×10−6 cm/s and 38.6±4.1×10−6 cm/s for den-1-(G2)-400 and den-2-(G2)-400, respectively; and only 11–14% reduction in transendothelial electrical resistance during initial 4 h. The results of this study suggest that architecture of dendrimers plays a major role not only in influencing the extent and mechanism of uptake by bEnd.3 cells but also permeation across the BBB model. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3748–3760, 2009

Arsonoliposomes for the Potential Treatment of Medulloblastoma

To investigate the arsonoliposome effect on medulloblastoma cells (VC312Rs) related to uptake, endocytotic mechanism and cell viability. VC312R viability in presence of either arsonoliposomes or stealth liposomes was studied using MTT assay for 1-4 days. Fibroblasts (3T3) were used as control. Apoptosis was studied for 2 h, 5 h and 24 h. Bodipy-labelled arsonoliposome uptake (time- and dose-dependent) was estimated using FACS analysis. The endocytotic mechanism was investigated using inhibitors of clathrin- (chlorpromazine) and caveolae-mediated endocytosis (filipin). Arsonoliposomes affected significantly the VC312R viability compared to 3T3 cells and induced apoptosis to VC312Rs after 2 h of incubation. Apoptosis was not observed for 3T3 cells. Liposome uptake versus time showed a bimodal pattern. Clathrin-mediated endocytosis was the main endocytotic mechanism at low lipid concentrations and caveolae at higher ones; thus, dose-dependent uptake did not show a plateau at increased lipid concentrations. Arsonoliposomes showed "selective" toxicity towards medulloblastoma cells inducing apoptosis after 2 hs of incubation. Therefore, arsonoliposomes are promising anticancer vehicles for brain tumour treatment.

Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

The role of actin dynamics in clathrin-mediated endocytosis in mammalian cells is unclear. In this study, we define the role of actin cytoskeleton in Kaposi's sarcoma-associated herpesvirus (KSHV) entry and trafficking in endothelial cells using an immunofluorescence-based assay to visualize viral capsids and the associated cellular components. In contrast to infectivity or reporter assays, this method does not rely on the expression of any viral and reporter genes, but instead directly tracks the accumulation of individual viral particles at the nuclear membrane as an indicator of successful viral entry and trafficking in cells. Inhibitors of endosomal acidification reduced both the percentage of nuclei with viral particles and the total number of viral particles docking at the perinuclear region, indicating endocytosis, rather than plasma membrane fusion, as the primary route for KSHV entry into endothelial cells. Accordingly, a viral envelope protein was only detected on internalized KSHV particles at the early but not late stage of infection. Inhibitors of clathrin- but not caveolae/lipid raft-mediated endocytosis blocked KSHV entry, indicating that clathrin-mediated endocytosis is the major route of KSHV entry into endothelial cells. KSHV particles were colocalized not only with markers of early and recycling endosomes, and lysosomes, but also with actin filaments at the early time points of infection. Consistent with these observations, transferrin, which enters cells by clathrin-mediated endocytosis, was found to be associated with actin filaments together with early and recycling endosomes, and to a lesser degree, with late endosomes and lysosomes. KSHV infection induced dynamic actin cytoskeleton rearrangements. Disruption of the actin cytoskeleton and inhibition of regulators of actin nucleation such as Rho GTPases and Arp2/3 complex profoundly blocked KSHV entry and trafficking. Together, these results indicate an important role for actin dynamics in the internalization and endosomal sorting/trafficking of KSHV and clathrin-mediated endocytosis in endothelial cells.

551 Shiga Toxin 1 Uptake By Intestinal Epithelial Cells Is CDC-42 Mediated and Clathrin-Independent

Shiga toxins (Stx) 1 and 2 are major contributors to foodborne disease caused by enterohemorrhagic E.coli (EHEC). Human intestinal epithelial cells (IEC) do not normally express Stx receptor (the glycosphingolipid Gb3), leaving the mechanism of Stx uptake poorly understood. Inhibition of clathrin-mediated endocytosis significantly increases Stx1 uptake in Gb3-free IEC. We thus now test the hypothesis that cell division cycle 42 (Cdc-42) dependent fluid phase endocytosis might be involved in clathrin-independent Stx1 uptake by IEC In Vitro and In Vivo. Methods: In Vivo: We compared pathologies in New Zealand white rabbits fed with either a) an experimental RDEC-H19A strain engineered to express Stx1, since it produces intestinal pathology quite similar to that noted in human EHEC or b) a control RDEC-1 E. coli strain that adheres to rabbit mucosa to provide an attaching and effacing lesion or c) PBS, to provide a negative control for effects of bacterial infection. Immunofluorescence of frozen sections from cecal tissue of rabbits studied at times up to 7 days after infection assessed a) uptake of Stx1 and b) presence of activated Cdc-42 in IEC. In Vitro: The apical uptake of the recombinant B-subunit of Stx1 labeled with Alexa 680 fluorescent dye (Stx1B) was assessed in T84 IEC that were: a) treated with agents known to inhibit clathrin-mediated endocytosis, b) depleted of Cdc42 by shRNA or c) untreated controls using biochemical and immunofluorescence methods. Results: 1) Cecal tissues from rabbits treated with either RDEC-1 or RDEC-H19A bacteria, but not those from PBS-treated animals, displayed Cdc42 activation in subsets of epithelial cells at sites near and those distant from attached bacteria. 2) T84 cells incubated with the inhibitors of clathrin mediated endocytosis chlorpromazine or low potassium increased Stx1B uptake in a time-dependent fashion. This stimulation was virtually eliminated, however, in T84 cells in which Cdc-42 was downregulated using shRNA. 3) The specific blocker of Cdc-42 function and nucleotide exchange, Pirl-1, also prevented stimulation of Stx1B uptake in concentration-dependent way in cells treated with inhibitors of clathrin-mediated endocytosis. 4) This clathrin independent Cdc-42 mediated Stx1B uptake was also dynamin dependent. Dynasore, a specific inhibitor of dynamin GTPase activity, inhibited the Stx1B uptake in cells with inactivated clathrin induced endocytic pathway. Conclusions: 1) Basal Stx1uptake by Gb3 negative T84 cells is regulated by Cdc-42. 2) Cdc-42 dependent clathrin independent pathway is likely to be involved in IEC uptake of Stx1 during EHEC infections.

Mechanism of Insulin Uptake in Rat Alveolar Type II and Type I-Like Epithelial Cells

The mechanism underlying the handling of protein and peptide drugs such as insulin in alveolar epithelial cells is still unclear. We therefore examined fluorescein isothiocyanate-labeled (FITC)-insulin uptake in rat primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells. FITC-insulin uptake in these cells was much higher than those of FITC-immunoglobulin (IgG), transferrin, and dextran. FITC-insulin uptake was time- and concentration-dependent, and was almost completely inhibited by metabolic inhibitors in both cells, while bafilomycin A(1) inhibited the uptake only in type II cells. Inhibitors of clathrin- and caveolae-mediated endocytosis did not affect FITC-insulin uptake in either type of cell. Dynasore, a dynamin GTPase inhibitor, potently inhibited FITC-insulin uptake in type II cells. These results suggest that the characteristics of insulin uptake in type II and type I cells are different, and dynamin-dependent endocytosis that utilizes neither clathrin nor caveolae is involved in type II cells, while a dynamin-independent pathway is mainly involved in type I cells.

Pemphigus Vulgaris IgG-induced Desmoglein-3 Endocytosis and Desmosomal Disassembly Are Mediated by a Clathrin- and Dynamin-independent Mechanism

Pemphigus vulgaris (PV) is a life-threatening autoimmune disease characterized by oral mucosal erosions and epidermal blistering. The autoantibodies generated target the desmosomal cadherin desmoglein-3 (Dsg3). Previous studies demonstrate that upon PV IgG binding, Dsg3 is internalized and enters an endo-lysosomal pathway where it is degraded. To define the endocytic machinery involved in PV IgG-induced Dsg3 internalization, human keratinocytes were incubated with PV IgG, and various tools were used to perturb distinct endocytic pathways. The PV IgG.Dsg3 complex failed to colocalize with clathrin, and inhibitors of clathrin- and dynamin-dependent pathways had little or no effect on Dsg3 internalization. In contrast, cholesterol binding agents such as filipin and nystatin and the tyrosine kinase inhibitor genistein dramatically inhibited Dsg3 internalization. Furthermore, the Dsg3 cytoplasmic tail specified sensitivity to these inhibitors. Moreover, inhibition of Dsg3 endocytosis with genistein prevented disruption of desmosomes and loss of adhesion in the presence of PV IgG. Altogether, these results suggest that PV IgG-induced Dsg3 internalization is mediated through a clathrin- and dynamin-independent pathway and that Dsg3 endocytosis is tightly coupled to the pathogenic activity of PV IgG.

Overexpression of functional TrkA receptors after internalisation in human airway smooth muscle cells

Trafficking of the TrkA receptor after stimulation by NGF is of emerging importance in structural cells in the context of airway inflammatory diseases. We have recently reported the expression of functional TrkA receptors in human airway smooth muscle cells (HASMC). We have here studied the TrkA trafficking mechanisms in these cells. TrkA disappearance from the cell membrane was induced within 5 min of NGF (3pM) stimulation. Co-immunoprecipitation of clathrin-TrkA was revealed, and TrkA internalisation inhibited either by clathrin inhibitors or by siRNA inducing downregulation of endogenous clathrin. TrkA internalised receptors were totally degraded in lysosomes, with no recycling phenomenon. Newly synthesized TrkA receptors were thereafter re-expressed at the cell membrane within 10 h. TrkA re-synthesis was inhibited by blockade of clathrin-dependent internalisation, but not of TrkA receptors lysosomal degradation. Finally, we observed that NGF multiple stimulations progressively increased TrkA expression in HASMC, which was associated with an increase in NGF/TrkA-dependent proliferation. In conclusion, we show here the occurrence of clathrin-dependent TrkA internalisation and lysosomal degradation in the airway smooth muscle, followed by upregulated re-synthesis of functional TrkA receptors and increased proliferative effect in the human airway smooth muscle. This may have pathophysiological consequences in airway inflammatory diseases.

Clathrin-dependent Endocytosis Is Required for TrkB-dependent Akt-mediated Neuronal Protection and Dendritic Growth

Endocytosis of Trk (tropomyosin-related kinase) receptors is critical for neurotrophin signal transduction and biological functions. However, the mechanism governing endocytosis of TrkB (tropomyosin-related kinase B) and the specific contributions of TrkB endocytosis to downstream signaling are unknown. In this study, we report that blocking clathrin, dynamin, or AP2 in cultured neurons of the central nervous system inhibited brain-derived neurotrophic factor (BDNF)-induced activation of Akt but not ERK. Treating neurons with the clathrin inhibitor monodansylcadaverine or a peptide that blocks dynamin function specifically abrogated Akt pathway activation in response to BDNF but did not affect the response of other downstream effectors or the up-regulation of immediate early genes neuropeptide Y and activity-regulated cytoskeleton-associated protein. Similar effects were found in neurons expressing small interfering RNA to silence AP2 or a dominant negative form of dynamin that inhibits clathrin-mediated endocytosis. In PC12 cells, ERK but not Akt activation required TrkA endocytosis following stimulation with nerve growth factor, whereas the opposite was true when TrkA-expressing neurons were stimulated with nerve growth factor in the central nervous system. Thus, the specific effects of internalized Trk receptors probably depend on the presence of cell type-specific modulators of neurotrophin signaling and not on differences inherent to Trk receptors themselves. Endocytosis-dependent activation of Akt in neurons was found to be critical for BDNF-supported survival and dendrite outgrowth. Together, these results demonstrate the functional requirement of clathrin- and dynamin-dependent endocytosis in generating the full intracellular response of neurons to BDNF in the central nervous system.

Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB5toxin that targets the endoplasmic reticulum chaperone BiP

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the endoplasmic reticulum (ER) chaperone BiP. However, its trafficking within target cells has not been investigated previously. In Vero cells, fluorescence colocalization with subcellular markers established that SubAB is trafficked from the cell surface to the ER via a retrograde pathway similar, but not identical, to those of Shiga toxin (Stx) and cholera toxin (Ctx), with their pathways converging at the Golgi. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalization is exclusively clathrin-dependent.

Transforming Growth Factor β Activation of c-Abl Is Independent of Receptor Internalization and Regulated by Phosphatidylinositol 3-Kinase and PAK2 in Mesenchymal Cultures

Transforming growth factor beta (TGF-beta) modulates a number of cellular phenotypes as divergent as growth stimulation and growth inhibition. Although the Smad pathway is critical for many of these responses, recent evidence indicates that Smad-independent pathways may also have a critical role. One such protein previously shown to regulate TGF-beta action independent of the Smad proteins is the c-Abl nonreceptor tyrosine kinase. In the current study we determined that TGF-beta receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures. This cell type-specific response occurs in a membrane-proximal locale independent of receptor internalization and upstream of dynamin action. Although c-Abl activation by TGF-beta is independent of Smad2 or Smad3, it is prevented by inhibitors of phosphatidylinositol 3-kinase or PAK2. Thus, c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines and integrates serine/threonine receptor kinases with tyrosine kinase pathways.