The role of clathrin-dependent endocytosis in bacterial internalization

Internalization of bacteria into mammalian host cells has been studied extensively in the past two decades. These studies have highlighted the amazingly diverse strategies used by bacterial pathogens to induce their entry in non-phagocytic cells. The roles of actin and of the whole cytoskeletal machinery have been investigated in great detail for several invasive organisms, such as Salmonella, Shigella, Yersinia and Listeria. Recent results using Listeria highlight a role for the endocytosis machinery in bacterial entry, suggesting that clathrin-dependent endocytic mechanisms are also involved in internalization of large particles. This contrasts with the generally accepted dogma but agrees with previous studies of bacterial and viral infections and also of phagocytosis.     

Role of HRB in clathrin-dependent endocytosis

Human immunodeficiency virus Rev-binding protein (HRB), also called human Rev-interacting protein (hRIP) or Rev/Rex activation domain binding (RAB) is a partner of the tyrosine kinase substrate EPS15, and it has been recovered in the AP-2 interactome. EPS15 and AP-2 are involved in endocytosis, but the function of HRB in this process is still unknown. Here we identified HRB as a partner of the vesicular SNARE tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) in yeast two-hybrid screens and using biochemical assays. In HeLa cells, HRB localized both in the nucleus and in the cytoplasm. In the cytoplasm, HRB colocalized with clathrin-, AP-2-, EPS15-, and transferrin receptor-containing vesicles. We did not see significant colocalization between HRB and TI-VAMP in HeLa cells, and we saw partial colocalization with green fluorescent protein-TI-VAMP in stably expressing Madin-Darby canine kidney cells. Nevertheless using a pHLuorin-tagged TI-VAMP construct, we found that HRB and TI-VAMP colocalize close to the plasma membrane after 5 min of anti-green fluorescent protein antibody uptake. These results suggest that TI-VAMP and HRB may interact only during the early stages of endocytosis. Furthermore uptake experiments followed by fluorescence-activated cell sorting showed that the endocytosis of fluorescent transferrin and pHLuorin-TI-VAMP is strongly reduced in HRB knockdown cells. Altogether these results suggest that HRB is involved in clathrin-dependent endocytosis and recruits TI-VAMP in this process.     

p120-Catenin regulates clathrin-dependent endocytosis of VE-cadherin

VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin-p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.     

Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins

Cell migration requires the controlled disassembly of focal adhesions, but the underlying mechanisms remain poorly understood. Here, we show that adhesion turnover is mediated through dynamin- and clathrin-dependent endocytosis of activated β1 integrins. Consistent with this, clathrin and the clathrin adaptors AP-2 and disabled-2 (DAB2) distribute along with dynamin 2 to adhesion sites prior to adhesion disassembly. Moreover, knockdown of either dynamin 2 or both clathrin adaptors blocks β1 integrin internalization, leading to impaired focal adhesion disassembly and cell migration. Together, these results provide important insight into the mechanisms underlying adhesion disassembly and identify novel components of the disassembly pathway.     

Enhanced clathrin-dependent endocytosis in the absence of calnexin

Background

Calnexin, together with calreticulin, constitute the calnexin/calreticulin cycle. Calnexin is a type I endoplasmic reticulum integral membrane protein and molecular chaperone responsible for the folding and quality control of newly-synthesized (glyco)proteins. The endoplasmic reticulum luminal domain of calnexin is responsible for lectin-like activity and interaction with nascent polypeptide chains. The role of the C-terminal, cytoplasmic portion of calnexin is not clear.

Methodology/Principal Findings

Using yeast two hybrid screen and immunoprecipitation techniques, we showed that the Src homology 3-domain growth factor receptor-bound 2-like (Endophilin) interacting protein 1 (SGIP1), a neuronal specific regulator of endocytosis, forms complexes with the C-terminal cytoplasmic domain of calnexin. The calnexin cytoplasmic C-tail interacts with SGIP1 C-terminal domains containing the adaptor complexes medium subunit (Adap-Comp-Sub) region. Calnexin-deficient cells have enhanced clathrin-dependent endocytosis in neuronal cells and mouse neuronal system. This is reversed by expression of full length calnexin or calnexin C-tail.

Conclusions/Significance

We show that the effects of SGIP1 and calnexin C-tail on clathrin-dependent endocytosis are due to modulation of the internalization of the receptor-ligand complexes. Enhanced clathrin-dependent endocytosis in the absence of calnexin may contribute to the neurological phenotype of calnexin-deficient mice.     

JC virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis

The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal alpha(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.     

Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors

Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.     

Fungal lectin, XCL, is internalized via clathrin-dependent endocytosis and facilitates uptake of other molecules

The lectin isolated from Xerocomus chrysenteron (XCL) displays a toxic activity towards insects. In order to assess its possible mode of action and to gather useful data for its potential use in insect-resistant transgenic plants, we investigated the effects of XCL at the cellular level. Immunofluorescence microscopy studies revealed that XCL is rapidly internalized into small endocytic vesicles that further coalesce in the perinuclear region. We show that XCL is endocytosed by the clathrin-dependent pathway, and is delivered to late endosome/lysosome compartments. The internalization of XCL seems to be general since it occurs in different cell types such as insect (SF9) or mammalian (NIH-3T3 and Hela) cell lines. In the presence of XCL, the uptake of GFP and BSA is greatly enhanced, demonstrating that XCL facilitates endocytosis. Thus, XCL could serve as a delivery agent to facilitate the endocytosis of proteins that do not enter the cell alone.     

Specific Grb2-mediated interactions regulate clathrin-dependent endocytosis of the cMet-tyrosine kinase

Lysosomal degradation of the receptor-tyrosine kinase cMet requires receptor ubiquitination by the E3 ubiquitin ligase Cbl followed by clathrin-dependent internalization. A role for Cbl as an adaptor for cMet internalization has been previously reported. However, the requirement for Cbl ubiquitin ligase activity in this process and its mode of recruitment to cMet has yet to be determined. Cbl can directly bind cMet at phosphotyrosine 1003 or indirectly via Grb2 to phosphotyrosine 1356 in the multisubstrate binding domain of cMet. The direct binding of Cbl with cMet is critical for receptor degradation and not receptor internalization. Here we show a strict requirement for Grb2 and the ubiquitin ligase activity of Cbl for cMet endocytosis. Receptor internalization was impaired by small interfering RNA depletion of Grb2, overexpression of dominant negative Grb2 mutants, and point mutations in the cMet multisubstrate docking site that inhibits the direct association of Grb2 with cMet. The requirement for Grb2 was specific and did not involve the multiadaptor Gab1. cMet internalization was impaired in cells expressing an ubiquitin ligase-deficient Cbl mutant or conjugation-deficient ubiquitin but was unaffected in cells expressing a Cbl mutant that is unable to bind cMet directly. Expression of a Cbl-Grb2 chimera rescued impaired cMet endocytosis in cells depleted of endogenous Grb2. These results indicate that the ubiquitin ligase activity of Cbl is critical for clathrin-dependent cMet internalization and suggest a role for Grb2 as an intermediary linking Cbl ubiquitin ligase activity to this process.     

The J-domain protein Rme-8 interacts with Hsc70 to control clathrin-dependent endocytosis in Drosophila

By screening for mutants exhibiting interactions with a dominant-negative dynamin, we have identified the Drosophila homologue of receptor-mediated endocytosis (Rme) 8, a J-domain-containing protein previously shown to be required for endocytosis in Caenorhabditis elegans. Analysis of Drosophila Rme-8 mutants showed that internalization of Bride of sevenless and the uptake of tracers were blocked. In addition, endosomal organization and the distribution of clathrin were greatly disrupted in Rme-8 cells, suggesting that Rme-8 participates in a clathrin-dependent process. The phenotypes of Rme-8 mutants bear a strong resemblance to those of Hsc70-4, suggesting that these two genes act in a common pathway. Indeed, biochemical and genetic data demonstrated that Rme-8 interacts specifically with Hsc70-4 via its J-domain. Thus, Rme-8 appears to function as an unexpected but critical cochaperone with Hsc70 in endocytosis. Because Hsc70 is known to act in clathrin uncoating along with auxilin, another J-protein, its interaction with Rme-8 indicates that Hsc70 can act with multiple cofactors, possibly explaining its pleiotropic effects on the endocytic pathway.     

Differentiation between clathrin-dependent and clathrin-independent endocytosis by means of membrane fluidity measurements

The fluorescence probe [1-(4-trimethylammonium]-6-phenyl-1,3,5-hexatriene (TMA-DPH) displays properties relevant for both monitoring endocytosis kinetics and assessing membrane fluidity by fluorescence-anisotropy measurements (1). Thus, it is, possible with this probe to follow the evolution of membrane fluidity during endocytosis, from the very beginning of the process, i.e., the formation of endocytic vesicles. In most cases, endocytosis is known to start with clathrin-coated vesicles. Still, there are more and more arguments in favor of a complementary endocytic pathway without clathrin. In this article we present membrane-fluidity data for very early endocytosis, which allow an upper limit to be determined for the contribution of a putative nonclathrin pathway. We show that this limit is markedly higher for bone marrow-derived macrophages than for mouse fibroblasts of the L929 cell line.     

Enlargeosome, an exocytic vesicle resistant to nonionic detergents, undergoes endocytosis via a nonacidic route

Enlargeosomes, a new type of widely expressed cytoplasmic vesicles, undergo tetanus toxin-insensitive exocytosis in response to cytosolic Ca(2+) concentration ([Ca(2+)](i)) rises. Cell biology of enlargeosomes is still largely unknown. By combining immunocytochemistry (marker desmoyokin-Ahnak, d/A) to capacitance electrophysiology in the enlargeosome-rich, neurosecretion-defective clone PC12-27, we show that 1) the two responses, cell surface enlargement and d/A surface appearance, occur with similar kinetics and in the same low micromolar [Ca(2+)](i) range, no matter whether induced by photolysis of the caged Ca(2+) compound o-nitrophenyl EGTA or by the Ca(2+) ionophore ionomycin. Thus, enlargeosomes seem to account, at least in large part, for the exocytic processes triggered by the two stimulations. 2. The enlargeosome membranes are resistant to nonionic detergents but distinct from other resistant membranes, rich in caveolin, Thy1, and/or flotillin1. 3. Cell cholesterol depletion, which affects many membrane fusions, neither disrupts enlargeosomes nor affects their regulated exocytosis. 4. The postexocytic cell surface decline is [Ca(2+)](i) dependent. 5. Exocytized d/A-rich membranes are endocytized and trafficked along an intracellular pathway by nonacidic organelles, distinct from classical endosomes and lysosomes. Our data define specific aspects of enlargeosomes and suggest their participation, in addition to cell differentiation and repair, for which evidence already exists, to other physiological and pathological processes.     

Uptake of fluorescent nano beads into BY2-cells involves clathrin-dependent and clathrin-independent endocytosis

To follow endocytosis in BY-2 cells we made use of fluorescent nano beads. Beads with 20 nm in diameter were internalised rapidly and accumulated partially in compartments also labelled by the endocytic marker FM4-64. Studies in BY-2 cells and protoplasts revealed that larger beads (100 nm) were excluded from uptake into turgescent and plasmolysed cells while protoplasts were able to internalise beads with a diameter of up to 1000 nm. Endocytosis of beads was only partially inhibited by the clathrin-specific inhibitor Ikarugamycin and strongly blocked by wortmannin. These results imply that uptake of beads involves clathrin-dependent and clathrin-independent endocytic mechanisms and supports the hypothesis that clathrin-independent endocytosis plays a general role in plants.     

Inhibition of clathrin-dependent endocytosis has multiple effects on human rhinovirus serotype 2 cell entry

Minor group human rhinoviruses (exemplified by human rhinovirus serotype 2 (HRV2)) use members of the low density lipoprotein receptor family for cell entry; all these receptors possess clathrin-coated pit localization signals. Viral infection should thus be inhibited under conditions of impaired clathrin-mediated endocytosis. However, Madshus et al. reported an increase in the cytopathic effect of HRV2 infection in HEp-2 cells upon suppression of clathrin-dependent endocytosis by hypotonic shock and potassium depletion (Madshus, I. H., Sandvig, K., Olsnes, S., and van Deurs, B. (1987) J. Cell. Physiol. 131, 14-22.) To resolve this apparent contradiction, we investigated the binding, internalization, conformational changes, and productive uncoating of HRV2 in HeLa cells subjected to hypotonic shock and potassium depletion. This treatment led to an increase in HRV2 binding, with internalization being barely affected. The generation of C-antigenic particles requiring pH 相似文献   

Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors

Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATPand temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolin-dependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.     

Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process

The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.     

Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis

The lack of a suitable in vitro hepatitis B virus (HBV) infectivity model has limited examination of the early stages of the virus-cell interaction. In this study, we used an immortalized cell line derived from human primary hepatocytes, HuS-E/2, to study the mechanism of HBV infection. HBV infection efficiency was markedly increased after dimethyl sulfoxide (DMSO)-induced differentiation of the cells. Transmission electron microscopy demonstrated the presence of intact HBV particles in DMSO-treated HBV-infected HuS-E/2 cells, which could be infected with HBV for up to at least 50 passages. The pre-S1 domain of the large HBsAg (LHBsAg) protein specifically interacted with clathrin heavy chain (CHC) and clathrin adaptor protein AP-2. Short hairpin RNA knockdown of CHC or AP-2 in HuS-E/2 cells significantly reduced their susceptibility to HBV, indicating that both are necessary for HBV infection. Furthermore, HBV entry was inhibited by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. LHBsAg also interfered with the clathrin-mediated endocytosis of transferrin by human hepatocytes. This infection system using an immortalized human primary hepatocyte cell line will facilitate investigations into HBV entry and in devising therapeutic strategies for manipulating HBV-associated liver disorders.     

Activation of MAPK by TRH requires clathrin-dependent endocytosis and PKC but not receptor interaction with beta-arrestin or receptor endocytosis

To determine whether the interaction of the TRH receptor with beta-arrestin is necessary for TRH activation of MAPK, cells expressing either intact or truncated, internalization-defective TRH receptors were transfected with a beta-arrestin-green fluorescent protein conjugate. In cells expressing the wild-type pituitary TRH receptor, TRH caused translocation of the beta-arrestin-green fluorescent protein conjugate from the cytosol to the plasma membrane within 30 sec. After 5 min, the beta-arrestin-green fluorescent protein conjugate was visible in vesicles, where it colocalized with rhodamine-labeled TRH. In hypertonic sucrose, the beta-arrestin-green fluorescent protein conjugate translocated to the plasma membrane after TRH addition but did not internalize. In cells expressing the truncated TRH receptor, TRH did not cause translocation of the beta-arrestin-green fluorescent protein conjugate. TRH activated MAPK strongly in cells expressing intact or truncated TRH receptors, indicating that the receptor does not need to bind beta-arrestin or internalize. MAPK activation by TRH, epidermal growth factor, and phorbol ester was strongly inhibited by hypertonic sucrose and concanavalin A, which block movement of proteins into coated pits and coated pit assembly. Hypertonic sucrose did not affect MAPK activation in cells overexpressing MAPK kinase 1. Dominant negative dynamin, which blocks conversion of coated pits to vesicles, also reduced receptor internalization and TRH activation of MAPK. TRH activation of MAPK required PKC but was insensitive to pertussis toxin and did not require ras, epidermal growth factor receptor kinase, or PI3K. These results show that the TRH receptor itself does not need to bind beta-arrestin or undergo sequestration to activate MAPK but that the endocytic pathway must be intact.     

Phosphoinositides regulate clathrin-dependent endocytosis at the tip of pollen tubes in Arabidopsis and tobacco

Using the tip-growing pollen tube of Arabidopsis thaliana and Nicotiana tabacum as a model to investigate endocytosis mechanisms, we show that phosphatidylinositol-4-phosphate 5-kinase 6 (PIP5K6) regulates clathrin-dependent endocytosis in pollen tubes. Green fluorescent protein-tagged PIP5K6 was preferentially localized to the subapical plasma membrane (PM) in pollen tubes where it apparently converts phosphatidylinositol 4-phosphate (PI4P) to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. RNA interference-induced suppression of PIP5K6 expression impaired tip growth and inhibited clathrin-dependent endocytosis in pollen tubes. By contrast, PIP5K6 overexpression induced massive aggregation of the PM in pollen tube tips. This PM abnormality was apparently due to excessive clathrin-dependent membrane invagination because this defect was suppressed by the expression of a dominant-negative mutant of clathrin heavy chain. These results support a role for PI(4,5)P(2) in promoting early stages of clathrin-dependent endocytosis (i.e., membrane invagination). Interestingly, the PIP5K6 overexpression-induced PM abnormality was partially suppressed not only by the overexpression of PLC2, which breaks down PI(4,5)P(2), but also by that of PI4Kβ1, which increases the pool of PI4P. Based on these observations, we propose that a proper balance between PI4P and PI(4,5)P(2) is required for clathrin-dependent endocytosis in the tip of pollen tubes.