[Show abstract][Hide abstract] ABSTRACT: The cellular mechanisms by which hepatitis B virus (HBV) is assembled and exported are largely undefined. Recently, it has been suggested that these steps require the multivesicular body (MVB) and the autophagic machinery. However, the mechanisms by which HBV might regulate these compartments are unclear. In this study we have found that by activating Rab7, HBV alters its own secretion by inducing dramatic changes in the morphology of MVB and autophagic compartments. These changes are characterized by the formation of numerous tubules that are dependent upon the increase in Rab7 activity observed in the HBV-expressing HepG2.2.15 cells compared to HepG2 cells. Interestingly, a transfection-based expression of the five individual viral proteins indicated that the precore protein, which is a precursor of HBeAg, was largely responsible for the increased Rab7 activity. Finally, siRNA-mediated depletion of Rab7 significantly increased the secretion of virions, suggesting that reduced delivery of the virus to the lysosome facilitates viral secretion. These findings provide novel evidence indicating that HBV can regulate its own secretion through an activation of the endo-lysosomal and autophagic pathway via Rab7 activation.
[Show abstract][Hide abstract] ABSTRACT: Clathrin-mediated endocytosis in mammalian epithelial cells is believed to require the synergistic action of structural coat proteins and mechanochemical enzymes to deform and sever the plasma membrane (PM) into discreet vesicles. It is generally believed that the formation of clathrin-coated pits in epithelial cells occurs randomly along the apical and basolateral plasma membranes. In this study we visualized the endocytic machinery in living hepatocytes using green fluorescent protein (GFP)-tagged dynamin, a large mechanochemical guanosine triphosphate (GTP)ase implicated in the liberation of nascent vesicles from the plasma membrane and a variety of internal membrane compartments. Confocal microscopy of living cells expressing the epithelial isoform of GFP-tagged dynamin [Dyn2-GFP] revealed a distribution along the ventral PM in discrete vesicle-like puncta or in large (2-10 μm) tubuloreticular plaques. Remarkably, these large structures are dynamic as they form and then disappear, while generating large numbers of motile endocytic vesicles with which dynamin associates. Inhibiting dynamin function by microinjection of purified dynamin antibodies increases the number and size of the tubuloreticular plaques. Importantly, these "hot spots" sequester specific trophic receptors and cognate ligands such as transferrin receptor 1 (TfR1), but not TfR2. CONCLUSION: These findings suggest that hepatocytes sequester or prerecruit both structural and enzymatic components of the clathrin-based endocytic machinery to functional hot spots, from which large numbers of coated pits form and vesicles are generated. This process may mimic the endocytic organization found at the synapse in neuronal cells.
[Show abstract][Hide abstract] ABSTRACT: Pancreatic ductal tumors invade local parenchyma and metastasize to distant organs. Src-mediated tyrosine kinase signaling pathways promote pancreatic ductal adenocarcinoma (PDAC) metastasis, though the molecular mechanisms supporting this invasive process are poorly understood and represent important and novel therapeutic targets. The large GTPase Dynamin 2 (Dyn2), a Src-kinase substrate, regulates membrane-cytoskeletal dynamics although it is yet to be defined if it contributes to tumor cell migration and invasion. Therefore, the goal of this study was to test if Dyn2 is upregulated in human pancreatic tumors and to define its role in cell migration and metastatic invasion using in vitro assays and nude mouse models. Histological analysis showed that 81% of 85 patients had elevated Dyn2 in PDAC. To test if Dyn2 overexpression alters metastatic properties of human pancreatic tumor cells, stable clones of BxPC-3 cells overexpressing either wild-type Dyn2 or a phosphorylation-deficient mutant Dyn2Y(231/597)F known to attenuate Dyn2 function, were generated and analyzed. Importantly, tumor cells overexpressing Dyn2 protruded lamellipodia at twice the rate, migrated faster (180%) and farther (2.5-fold greater distance) on glass and through transwell chambers (2-3-fold more cells through the filter) compared with cells expressing Dyn2Y(231/597)F or vector alone. Further, depletion of Dyn2 and dynamin inhibitors Myristyl trimethyl ammonium bromides and Dynasore significantly reduced cell migration, wound healing and invasion in transwell assays compared with controls. To test the metastatic potential conferred by increased Dyn2 expression, the BxPC-3 cell lines were implanted orthotopically into the pancreas of nude mice. Cells expressing Dyn2-green fluorescent protein exhibited a threefold increase in large distal tumors compared with cells expressing Dyn2Y(231/597)F or vector alone. Finally, histological analysis revealed that Dyn2 is upregulated in 60% of human metastatic pancreatic tumors. These findings are the first to implicate dynamin in any neoplastic condition and to directly demonstrate a role for this mechanoenzyme in invasive cell migration.
[Show abstract][Hide abstract] ABSTRACT: Levels of the epidermal growth factor receptor (EGFR) at the cell surface are tightly regulated by a complex endocytic machinery. Following internalization, EGFR is either recycled back to the cell surface or transported to the late endosome/lysosome for degradation. Currently, the molecular machinery that regulates this sorting pathway is only partially defined. Eps15 (EGFR pathway substrate 15) is an endocytic adaptor protein that is well known to support clathrin-mediated internalization of EGFR at the plasma membrane. Using RT-PCR, we have identified a novel short form of Eps15 (Eps15S) from rat liver that lacks the 111 C-terminal amino acids present in the traditional Eps15 form. The goal of this study was to define the functional role of the novel Eps15S form in EGFR trafficking. Overexpression of a mutant form of Eps15S (Eps15S ΔEH2/EH3) did not block EGFR internalization but reduced its recycling to the cell surface. After knockdown of all Eps15 forms, re-expression of Eps15S significantly reduced EGFR degradation while promoting recycling back to the cell surface. In contrast, re-expression of Eps15 did not potentiate receptor recycling. Furthermore, overexpression of the mutant Eps15S substantially reduced cell proliferation, linking EGFR recycling to downstream mitogenic effects. Finally, we found that Eps15S is localized to the Rab11-positive recycling endosome that is disrupted in cells expressing the Eps15S mutant, leading to an accumulation of the EGFR in early endosomes. These findings suggest that distinct forms of Eps15 direct EGFR to either the late endosome/lysosome for degradation (Eps15) or to the recycling endosome for transit back to the cell surface (Eps15S).
[Show abstract][Hide abstract] ABSTRACT: Tumor cell migration is supported in part by the cyclic formation and disassembly of focal adhesions (FAs); however, the mechanisms that regulate this process are not fully defined. The large guanosine 5'-triphosphatase dynamin (Dyn) plays an important role in FA dynamics and is activated by tyrosine phosphorylation. Using a novel antibody specific to phospho-dynamin (pDyn-Tyr-231), we found that Dyn2 is phosphorylated at FAs by Src kinase and is recruited to FAs by a direct interaction with the 4.1/ezrin/radizin/moesin domain of focal adhesion kinase (FAK), which functions as an adaptor between Src and Dyn2 to facilitate Dyn2 phosphorylation. This Src-FAK-Dyn2 trimeric complex is essential for FA turnover, as mutants disrupting the formation of this complex inhibit FA disassembly. Importantly, phosphoactivated Dyn2 promotes FA turnover by mediating the endocytosis of integrins in a clathrin-dependent manner. This study defines a novel mechanism of how Dyn2 functions as a downstream effector of FAK-Src signaling in turning over FAs.
Molecular biology of the cell 03/2011; 22(9):1529-38. DOI:10.1091/mbc.E10-09-0785 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The size and integrity of the Golgi apparatus is maintained via a tightly controlled regulation of membrane traffic using a variety of different signaling and cytoskeletal proteins. We have recently observed that activation of c-Src has profound effects on Golgi structure, leading to dramatically vesiculated cisternae in a variety of cell types. As the large GTPase dynamin (Dyn2) has been implicated in Golgi vesiculation during secretion, we tested whether inhibiting Dyn2 activity by expression of a Dyn2K44A mutant or siRNA knockdown could attenuate active Src-induced Golgi fragmentation. Indeed, these perturbations attenuated fragmentation, and expression of a Dyn2Y(231/597)F mutant protein that cannot be phosphorylated by Src kinase had a similar effect . Finally, we find that Dyn2 is markedly phosphorylated during the transit of VSV-G protein through the TGN whereas expression of the Dyn2Y(231/597)F mutant significantly reduces exit of the nascent protein from this compartment. These findings demonstrate that activation of Dyn2 by Src kinase regulates Golgi integrity and vesiculation during the secretory process.
Proceedings of the National Academy of Sciences 03/2010; 107(13):5863-8. DOI:10.1073/pnas.0915123107 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanisms by which epithelial cells regulate clathrin-mediated endocytosis (CME) of transferrin are poorly defined and generally viewed as a constitutive process that occurs continuously without regulatory constraints. In this study, we demonstrate for the first time that endocytosis of the transferrin receptor is a regulated process that requires activated Src kinase and, subsequently, phosphorylation of two important components of the endocytic machinery, namely, the large GTPase dynamin 2 (Dyn2) and its associated actin-binding protein, cortactin (Cort). To our knowledge these findings are among the first to implicate an Src-mediated endocytic cascade in what was previously presumed to be a nonregulated internalization process.
[Show abstract][Hide abstract] ABSTRACT: Remodeling of cell-cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell-cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell-cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.
Molecular biology of the cell 08/2009; 20(19):4140-52. DOI:10.1091/mbc.E08-10-1043 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eps15 (EGFR pathway substrate clone 15) is well known for its role in clathrin-coated vesicle formation at the plasma membrane through interactions with other clathrin adaptor proteins such as AP-2. Interestingly, we observed that in addition to its plasma membrane localization, Eps15 is also present at the trans-Golgi network (TGN). Therefore, we predicted that Eps15 might associate with clathrin adaptor proteins at the TGN and thereby mediate the formation of Golgi-derived vesicles. Indeed, we have found that Eps15 and the TGN clathrin adaptor AP-1 coimmunoprecipitate from rat liver Golgi fractions. Furthermore, we have identified a 14-amino acid motif near the AP-2-binding domain of Eps15 that is required for binding to AP-1, but not AP-2. Disruption of the Eps15-AP-1 interaction via siRNA knockdown of AP-1 or expression of mutant Eps15 protein, which lacks a 14-amino acid motif representing the AP-1 binding site of Eps15, significantly reduced the exit of secretory proteins from the TGN. Together, these findings indicate that Eps15 plays an important role in clathrin-coated vesicle formation not only at the plasma membrane but also at the TGN during the secretory process.
Molecular biology of the cell 08/2008; 19(8):3564-75. DOI:10.1091/mbc.E07-10-0997 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is well-known that dynamin 2 (Dyn2) participates in clathrin- and caveolae-mediated endocytosis; however, the role of Dyn2 in coat-independent endocytic processes remains controversial. Here we demonstrate a role for specific spliced variants of Dyn2 in the micropinocytosis of fluid in epithelial cells, independent of coat-mediated endocytic pathways. A general inhibition of Dyn2 was first performed using either microinjection of anti-dynamin antibodies or Dyn2-siRNA treatment. Both of these methods resulted in reduced uptake of transferrin, a marker for clathrin-mediated endocytosis, and, under unstimulated conditions, reduced the uptake of the fluid-phase markers dextran and horseradish peroxidase (HRP). By contrast, cells treated similarly but stimulated with serum or EGF internalized substantial amounts of dextran or HRP, indicating that Dyn2 is not required for stimulated fluid uptake via macropinocytosis. We next tested whether a specific spliced variant might selectively affect fluid-phase endocytosis. Mutation of specific Dyn2 spliced variants resulted in a differential attenuation of transferrin and dextran internalization. Furthermore, the reduction in fluid uptake in Dyn2-siRNA-treated cells was only rescued upon re-expression of select spliced variants. These findings suggest that Dyn2 function is required for the coat-independent internalization of fluid through endocytic pathways distinct from macropinocytosis and, in addition, implicate different Dyn2 spliced variants in specific endocytic functions.
[Show abstract][Hide abstract] ABSTRACT: Cortactin is an actin-binding protein that has recently been implicated in endocytosis. It binds directly to dynamin-2 (Dyn2), a large GTPase that mediates the formation of vesicles from the plasma membrane and the Golgi. Here we show that cortactin associates with the Golgi to regulate the actin- and Dyn2-dependent transport of cargo. Cortactin antibodies stain the Golgi apparatus, labelling peripheral buds and vesicles that are associated with the cisternae. Notably, in vitro or intact-cell experiments show that activation of Arf1 mediates the recruitment of actin, cortactin and Dyn2 to Golgi membranes. Furthermore, selective disruption of the cortactin-Dyn2 interaction significantly reduces the levels of Dyn2 at the Golgi and blocks the transit of nascent proteins from the trans-Golgi network, resulting in swollen and distended cisternae. These findings support the idea of an Arf1-activated recruitment of an actin, cortactin and Dyn2 complex that is essential for Golgi function.
[Show abstract][Hide abstract] ABSTRACT: Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2DeltaPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.
[Show abstract][Hide abstract] ABSTRACT: Dynamin 2 (Dyn2) is a large GTPase involved in vesicle formation and actin reorganization. In this study, we report a novel role for Dyn2 as a component of the centrosome that is involved in centrosome cohesion. By light microscopy, Dyn2 localized aside centrin and colocalized with gamma-tubulin at the centrosome; by immunoelectron microscopy, however, Dyn2 was detected in the pericentriolar material as well as on centrioles. Exogenously expressed green fluorescent protein (GFP)-tagged Dyn2 also localized to the centrosome, whereas glutathione S-transferase (GST)-tagged Dyn2 pulled down a protein complex(es) containing actin, alpha-tubulin and gamma-tubulin from liver homogenate. Furthermore, gel overlay and immunoprecipitation indicated a direct interaction between gamma-tubulin and a 219-amino-acid middle domain of Dyn2. Reduction of Dyn2 protein levels with small-interfering RNA (siRNA) resulted in centrosome splitting, whereas microtubule nucleation from centrosomes was not affected, suggesting a role for Dyn2 in centrosome cohesion. Finally, fluorescence recovery after photobleaching (FRAP) analysis of a GFP-tagged Dyn2 middle domain indicated that Dyn2 is a dynamic exchangeable component of the centrosome. These findings suggest a novel function for Dyn2 as a participant in centrosome cohesion.
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium parvum invasion of epithelia requires polymerization of host cell actin at the attachment site. We analyzed the role of host cell c-Src, a cytoskeleton-associated protein tyrosine kinase, in C. parvum invasion of biliary epithelia.
In vitro models of biliary cryptosporidiosis using a human biliary epithelial cell line were used to assay the role of c-Src signaling pathway in C. parvum invasion.
c-Src and cortactin, an actin-binding protein and a substrate for c-Src, were recruited to the parasite-host cell interface during C. parvum invasion. Tyrosine phosphorylation of cortactin in infected cells was also detected. Inhibition of host cell c-Src significantly blocked C. parvum -induced accumulation and tyrosine phosphorylation of cortactin and actin polymerization at the attachment sites, thereby inhibiting C. parvum invasion of biliary epithelial cells. A triple mutation of tyrosine of cortactin in the epithelia also diminished C. parvum invasion. In addition, proteins originating from the parasite were detected within infected cells at the parasite-host cell interface. Antiserum against C. parvum membrane proteins blocked accumulation of c-Src and cortactin and significantly decreased C. parvum invasion. No accumulation of the endocytosis-related proteins, dynamin 2 and clathrin, was found at the parasite-host cell interface; also, inhibition of dynamin 2 did not block C. parvum invasion.
C. parvum invasion of biliary epithelial cells requires host cell tyrosine phosphorylation of cortactin by a c-Src-mediated signaling pathway to induce actin polymerization at the attachment site, a process associated with microbial secretion but independent of host cell endocytosis.