Clathrin-dependent Process Research Articles

Taking a Raft to the Cell Interior

Abrami et al. investigated the mechanism that leads to internalization of anthrax toxin and discovered that oligomerization-driven anthrax toxin receptor (ATR) clustering leads to its association with lipid rafts, cholesterol- and sphingolipid-rich microdomains of the plasma membrane, and promotes toxin internalization. Anthrax toxin consists of three subunits: edema factor (EF), a calmodulin-dependent adenylate cyclase; lethal factor (LF), which targets mitogen-activated protein kinase kinases (MAPKKs); and protective antigen (PA), which enables EF and LF to gain access to the cell interior. PA is an 83-kD protein (PA83) that binds to the ATR and is then cleaved by cell surface proteases to a 63-kD form (PA63) that forms a ring-shaped heptamer. EF and LF associate with heptamerized PA63, and then the entire toxin-receptor complex is endocytosed. After endocytosis, the PA63 heptamer forms a channel, and EF and LF enter the cytoplasm. The authors used Western analysis to show that PA63, but not PA83 or an unbound epitope-tagged version of the ATR, was associated with a lipid raft fraction from the membranes of CHO cells. Clustering PA83-bound ATR through antibody cross-linking promoted its association with lipid rafts and rapid internalization of a mutant PA that cannot be cleaved and normally remains at the cell surface. Depleting the cells of cholesterol inhibited intracellular accumulation of P63, channel formation, and LF-dependent cleavage of a MAPKK. Internalization did not depend on caveolin, but occurred through a clathrin-mediated pathway. The authors describe how the physiology of the ATR--slow internalization of the unbound or monomerically bound form but rapid internalization of the clustered form--provides an ideal target for anthrax toxin. They suggest that lipid rafts may provide a therapeutic target against anthrax.L. Abrami, S. Liu, P. Cosson, S. H. Leppla, F. G. van der Goot, Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process. J. Cell Biol. 160, 321-328 (2003). [Abstract] [Full Text]T. Kurzchalia, Anthrax toxin rafts into cells. J. Cell Biol. 160, 295-296 (2003). [Abstract] [Full Text]

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.

Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex.

Internalization of β-adrenergic receptors (βARs) occurs by the sequential binding of β-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that βARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from βARK1 and prevents βARK1-mediated translocation of PI3K to activated β2ARs. Furthermore, disruption of the βARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the β2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to βARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

Association of Caveolin with Chlamydia trachomatis Inclusions at Early and Late Stages of Infection

The mechanism by which the intracellular bacterial pathogen Chlamydia trachomatis enters eukaryotic cells is poorly understood. There are conflicting reports of entry occurring by clathrin-dependent and clathrin-independent processes. We report here that C. trachomatis serovar K enters HEp-2 and HeLa 229 epithelial cells and J-774A.1 mouse macrophage/monocyte cells via caveolin-containing sphingolipid and cholesterol-enriched raft microdomains in the host cell plasma membranes. First, filipin and nystatin, drugs that specifically disrupt raft function by cholesterol chelation, each impaired entry of C. trachomatis serovar K. In control experiments, filipin did not impair entry of the same organism by an antibody-mediated opsonic process, nor did it impair entry of BSA-coated microspheres. Second, the chlamydia-containing endocytic vesicles specifically reacted with antisera against the caveolae marker protein caveolin. These vesicles are known to become the inclusions in which parasite replication occurs. They avoid fusion with lysosomes and instead traffic to the Golgi region, where they intercept Golgi-derived vesicles that recycle sphingolipids and cholesterol to the plasma membrane. We also report that late-stage C. trachomatis inclusions continue to display high levels of caveolin, which they likely acquire from the exocytic Golgi vesicles. We suggest that the atypical raft-mediated entry process may have important consequences for the host–pathogen interaction well after entry has occurred. These consequences include enabling the chlamydial vesicle to avoid acidification and fusion with lysosomes, to traffic to the Golgi region, and to intercept sphingolipid-containing vesicles from the Golgi.

Inositol hexakisphosphate‐mediated regulation of glutamate receptors in rat brain sections

D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6), one of the most abundant inositol phosphates within cells, has been proposed to play a key role in vesicle trafficking and receptor compartmentalization. In the present study, we used in vitro receptor autoradiography, subcellular fractionation, and immunoblotting to investigate its effects on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Qualitative and quantitative analysis of 3H-AMPA binding indicated that incubation of frozen-thawed brain sections with InsP6 at 35 degrees C enhanced AMPA receptor binding in several brain regions, with maximal increases in the hippocampus and cerebellum. Moreover, saturation kinetics demonstrated that InsP6-induced augmentation of AMPA binding was due to an increment in the maximal number of AMPA binding sites. At the immunological level, Western blots performed on crude mitochondrial/synaptic (P2) fractions revealed that InsP6 (but not InsP5 and InsP3) treatment increased glutamate receptor (GluR)1 and GluR2 subunits of AMPA receptors, an effect that was associated with concomitant reductions in microsomal (P3) fractions. Interestingly, the InsP6-induced modulation of AMPA receptor binding was blocked at room temperature, and pretreatment with heparin also dampered its action on both AMPA receptor binding and GluR subunits. These effects of InsP6 appear to be specific to AMPA receptors, as neither 3H-glutamate binding to NMDA receptors nor levels of NR1 and NR2A subunits in P2 and P3 fractions were affected. Taken together, our data strongly suggest that InsP6 specifically regulates AMPA receptor distribution, possibly through a clathrin-dependent process.

Uptake by macrophages of triacylglycerol-rich artificial emulsion particles co-incubated with native LDL is a clathrin-dependent process

Uptake by macrophages of triacylglycerol-rich artificial emulsion particles co-incubated with native LDL is a clathrin-dependent process

Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network.

Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone alpha-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor maturation defects and exacerbated alpha-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN.

Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis.

Porcine alveolar macrophages (AMphi) are the dominant cell type that supports the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in vivo and in vitro. In order to determine the characteristics of the virus-receptor interaction, the attachment of PRRSV to cells was examined by using biotinylated virus in a series of flow cytometric assays. PRRSV bound specifically to AMphi in a dose-dependent manner. Binding of PRRSV to AMphi increased gradually and reached a maximum within 60 min at 4 degrees C. By confocal microscopy, it was shown that different degrees of PRRSV binding exist and that entry is by endocytosis. Virus uptake in vesicles is a clathrin-dependent process, as it was blocked by the addition of cytochalasin D and co-localization of PRRSV and clathrin was found. Furthermore, by the use of two weak bases, NH4Cl and chloroquine, it was demonstrated that PRRSV uses a low pH-dependent entry pathway. In the presence of these reagents, input virions accumulated in large vacuoles, indicating that uncoating was prevented. These results indicate that PRRSV entry into AMphi involves attachment to a specific virus receptor(s) followed by a process of endocytosis, by which virions are taken into the cell within vesicles by a clathrin-dependent pathway. A subsequent drop in pH is required for proper virus replication.

Synaptic Vesicle Size and Number Are Regulated by a Clathrin Adaptor Protein Required for Endocytosis

Clathrin-mediated endocytosis is thought to involve the activity of the clathrin adaptor protein AP180. However, the role of this protein in endocytosis in vivo remains unknown. Here, we show that a mutation that eliminates an AP180 homolog (LAP) in Drosophila severely impairs the efficiency of synaptic vesicle endocytosis and alters the normal localization of clathrin in nerve terminals. Most importantly, the size of both synaptic vesicles and quanta is significantly increased in lap mutants. These results provide novel insights into the molecular mechanism of endocytosis and reveal a role for AP180 in regulating vesicle size through a clathrin-dependent reassembly process.