Novel function of a dynein light chain in actin assembly during clathrin-mediated endocytosis.

Clathrin- and actin-mediated endocytosis is essential in eukaryotic cells. In this study, we demonstrate that Tda2 is a novel protein of the endocytic machinery necessary for normal internalization of native cargo in yeast. Tda2 has not been classified in any protein family. Unexpectedly, solving the crystal structure of Tda2 revealed it belongs to the dynein light chain family. However, Tda2 works independently of the dynein motor complex and microtubules. Tda2 forms a tight complex with the polyproline motif-rich protein Aim21, which interacts physically with the SH3 domain of the Arp2/3 complex regulator Bbc1. The Tda2-Aim21 complex localizes to endocytic sites in a Bbc1- and filamentous actin-dependent manner. Importantly, the Tda2-Aim21 complex interacts directly with and facilitates the recruitment of actin-capping protein, revealing barbed-end filament capping at endocytic sites to be a regulated event. Thus, we have uncovered a new layer of regulation of the actin cytoskeleton by a member of a conserved protein family that has not been previously associated with a function in endocytosis.

Phospholipase D is involved in the formation of Golgi associated clathrin coated vesicles in human parotid duct cells.

Phospholipase D (PLD) has been implicated in many cellular functions, such as vesicle trafficking, exocytosis, differentiation, and proliferation. The aim of this study was to characterize the role of PLD in HSY cells, a human cell line originating from the intercalated duct of the parotid gland. As the function and intracellular localization of PLD varies according to cell type, initially, the intracellular localization of PLD1 and PLD2 was determined. By immunofluorescence, PLD1 and PLD2 both showed a punctate cytoplasmic distribution with extensive co-localization with TGN-46. PLD1 was also found in the nucleus, while PLD2 was associated with the plasma membrane. Treatment of cells with the primary alcohol 1-butanol inhibits the hydrolysis of phosphatidylcoline by PLD thereby suppressing phosphatidic acid (PA) production. In untreated HSY cells, there was only a slight co-localization of PLD with the clathrin coated vesicles. When HSY cells were incubated with 1-butanol the total number of clathrin coated vesicles increased, especially in the juxtanuclear region and the co-localization of PLD with the clathrin coated vesicles was augmented. Transmission electron microscopy confirmed that the number of Golgi-associated coated vesicles was greater. Treatment with 1-butanol also affected the Golgi apparatus, increasing the volume of the Golgi saccules. The decrease in PA levels after treatment with 1-butanol likewise resulted in an accumulation of enlarged lysosomes in the perinuclear region. Therefore, in HSY cells PLD appears to be involved in the formation of Golgi associated clathrin coated vesicles as well as in the structural maintenance of the Golgi apparatus.

Fast modulation of µ-opioid receptor (MOR) recycling is mediated by receptor agonists.

The µ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.

Acetaldehyde/alcohol dehydrogenase-2 (EhADH2) and clathrin are involved in internalization of human transferrin by Entamoeba histolytica.

Transferrin (Tf) is a host glycoprotein capable of binding two ferric-iron ions to become holotransferrin (holoTf), which transports iron in to all cells. Entamoeba histolytica is a parasitic protozoan able to use holoTf as a sole iron source in vitro. The mechanism by which this parasite scavenges iron from holoTf is unknown. An E. histolytica holoTf-binding protein (EhTfbp) was purified by using an anti-human transferrin receptor (TfR) monoclonal antibody. EhTfbp was identified by MS/MS analysis and database searches as E. histolytica acetaldehyde/alcohol dehydrogenase-2 (EhADH2), an iron-dependent enzyme. Both EhTfbp and EhADH2 bound holoTf and were recognized by the anti-human TfR antibody, indicating that they correspond to the same protein. It was found that the amoebae internalized holoTf through clathrin-coated pits, suggesting that holoTf endocytosis could be important for the parasite during colonization and invasion of the intestinal mucosa and liver.

Native type IV collagen induces cell migration through a CD9 and DDR1-dependent pathway in MDA-MB-231 breast cancer cells.

CD9 is a member of the tetraspanin family and is widely expressed in the plasma membrane of several cell types as well as malignant cells. CD9 associates with a number of transmembrane proteins, which facilitates biological processes, including cell signaling, adhesion, migration and proliferation. DDR1 is activated by native type IV collagen and overexpressed in human breast cancer. Type IV collagen is the main component of basement membranes, and may interact with cell surface biomolecules, promoting adhesion and motility. However, the role of DDR1 and type IV collagen in the regulation of CD9-cell surface levels and migration in breast cancer cells has not been studied in detail. We demonstrate here that native type IV collagen induces a transient increase of CD9-cell surface levels through a DDR1-dependent pathway in MDA-MB-231 breast cancer cells, as revealed by flow cytometry and Western blotting using specific antibodies that recognize CD9. In contrast, type IV collagen does not induce any increase of CD9-cell surface levels in the mammary non-tumorigenic epithelial cells MCF10A and MCF12A. Transient increase of CD9-cell surface levels is coupled with clathrin-mediated endocytosis and it is dependent of DDR1 expression. In addition, type IV collagen induces cell migration through a DDR1 and CD9-dependent pathway. In summary, our data demonstrate, for the first time, that native type IV collagen induces a transient increase of CD9-cell surface levels and cell migration through a DDR1 and CD9-dependent pathway in MDA-MB-231 breast cancer cells.

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles.

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.

Complex gangliosides are apically sorted in polarized MDCK cells and internalized by clathrin-independent endocytosis.

Gangliosides are glycosphingolipids mainly present at the outer leaflet of the plasma membrane of eukaryotic cells, where they participate in recognition and signalling activities. The synthesis of gangliosides is carried out in the lumen of the Golgi apparatus by a complex system of glycosyltransferases. After synthesis, gangliosides leave the Golgi apparatus via the lumenal surface of transport vesicles destined to the plasma membrane. In this study, we analysed the synthesis and membrane distribution of GD3 and GM1 gangliosides endogenously synthesized by Madin-Darby canine kidney (MDCK) cell lines genetically modified to express appropriate ganglioside glycosyltransferases. Using biochemical techniques and confocal laser scanning microscopy analysis, we demonstrated that GD3 and GM1, after being synthesized at the Golgi apparatus, were transported and accumulated mainly at the plasma membrane of nonpolarized MDCK cell lines. More interestingly, both complex gangliosides were found to be enriched mainly at the apical domain when these cell lines were induced to polarize. In addition, we demonstrated that, after arrival at the plasma membrane, GD3 and GM1 gangliosides were endocytosed using a clathrin-independent pathway. Then, internalized GD3, in association with a specific monoclonal antibody, was accumulated in endosomal compartments and transported back to the plasma membrane. In contrast, endocytosed GM1, in association with cholera toxin, was transported to endosomal compartments en route to the Golgi apparatus. In conclusion, our results demonstrate that complex gangliosides are apically sorted in polarized MDCK cells, and that GD3 and GM1 gangliosides are internalized by clathrin-independent endocytosis to follow different intracellular destinations.

Oropouche virus entry into HeLa cells involves clathrin and requires endosomal acidification.

Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1h.

Intoxication of epithelial cells by plasmid-encoded toxin requires clathrin-mediated endocytosis.

It has been shown that the autotransporter plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli (EAEC) produces cytotoxic and enterotoxic effects. Both effects can be explained by the proteolytic activity of Pet on its intracellular target alpha-fodrin (alphaII spectrin). In addition, Pet cytotoxicity and enterotoxicity depend on Pet serine protease activity, and on its internalization into epithelial cells. However, the mechanisms of Pet uptake by epithelial cells are unknown. Here, we show that Pet interacts with the plasma membrane of epithelial cells, and afterwards is detected inside the cells. Furthermore, Pet was internalized via clathrin-mediated endocytosis, since its internalization was inhibited by monodansylcadaverine and sucrose, but not by filipin or methyl-beta-cyclodextrin, which are drugs that interfere with protein entry via a clathrin-independent pathway. Additionally, Pet was immunoprecipitated by anti-clathrin antibodies, but not by anti-caveolin antibodies. Moreover, small interfering RNA (siRNA), designed to knock out clathrin gene expression in HEp-2 cells, prevented Pet internalization, and thereby the Pet-induced cytotoxic effect. However, the use of siRNA to knock out caveolin expression had no effect on Pet internalization, and the cytotoxic effect was clearly observed. Together, these data indicate that Pet secreted by EAEC binds to the cell surface via an unknown receptor, to be taken up by clathrin-mediated endocytosis, and exert its toxic effect in the cytoplasm.

The antibody to GD3 ganglioside, R24, is rapidly endocytosed and recycled to the plasma membrane via the endocytic recycling compartment. Inhibitory effect of brefeldin A and monensin.

Gangliosides are sialic acid-containing glycosphingolipids present on mammalian plasma membranes, where they participate in cell-surface events such as modulation of growth factor receptors and cell-to-cell and cell-to-matrix interactions. Antibodies to gangliosides have been associated with a wide range of clinically identifiable acute and chronic neuropathy syndromes. In addition, antibodies to tumor-associated gangliosides are being used as therapeutic agents. Their binding to and release from cell membranes and intracellular destinations have not so far been extensively examined. In this study, we characterized in both GD3 ganglioside-expressing Chinese hamster ovary (CHO)-K1 and SK-Mel 28 melanoma cells the intracellular trafficking and subcellular localization of the mouse monoclonal antibody to GD3, R24. By biochemical techniques and detailed confocal microscopic analysis, we demonstrate that the GD3-R24 antibody complex is rapidly and specifically internalized by a dynamin 2-independent pathway and then accumulates in the endocytic recycling compartment. In addition, we show that the R24 antibody exits the recycling compartment en route to the plasma membrane by a dynamin 2-dependent pathway sensitive to brefeldin A and monensin. Taken together, our results indicate that the GD3-R24 complex is endocytosed in GD3-expressing cells, accumulates in the recycling endosome, and is transported back to the plasma membrane via a route that involves clathrin-coated vesicles.

The calcium chelator BAPTA affects the binding of assembly protein AP-2 to membranes.

Clathrin coated vesicles are involved in receptor-mediated transport. The coat of these vesicles is constituted mostly of clathrin and the assembly proteins AP-1 or AP-2. In the present study using an in vitro binding system, we found that the interaction of AP-2 but not AP-1 with membranes diminished when the calcium chelating agent BAPTA was added. The maximal inhibitory effect was observed with 10 mM of the chelating agent. Binding of AP-2 to membranes was recovered by adding calcium in a concentration-dependent fashion. Binding was also affected when the membranes were previously treated with BAPTA and then washed. However, other chelating agents such as EDTA or EGTA, as well as the zinc chelating TPEN, did not have any effect on the binding. From these results we postulate a role for calcium in regulating the assembly-disassembly cycle of adaptors in the formation of clathrin coated vesicles.

Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line.

Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.

The calcium chelator BAPTA affects the binding of assembly protein AP-2 to membranes

Clathrin coated vesicles are involved in receptor-mediated transport. The coat of these vesicles is constituted mostly of clathrin and the assembly proteins AP-1 or AP-2. In the present study using an in vitro binding system, we found that the interaction of AP-2 but not AP-1 with membranes diminished when the calcium chelating agent BAPTA was added. The maximal inhibitory effect was observed with 10 mM of the chelating agent. Binding of AP-2 to membranes was recovered by adding calcium in a concentration-dependent fashion. Binding was also affected when the membranes were previously treated with BAPTA and then washed. However, other chelating agents such as EDTA or EGTA, as well as the zinc chelating TPEN, did not have any effect on the binding. From these results we postulate a role for calcium in regulating the assembly-disassembly cycle of adaptors in the formation of clathrin coated vesicles.(AU)