[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: 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. · 5.98 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. · 9.74 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.
Molecular and cellular biology 12/2009; 30(3):781-92. · 6.06 Impact Factor
[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. · 5.98 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. · 5.98 Impact Factor
[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.
Journal of Molecular Biology 05/2005; 348(2):491-501. · 3.91 Impact Factor
[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.
[show abstract][hide abstract] ABSTRACT: The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. In this study, we demonstrate that Dyn2 and cortactin function to mediate dynamic remodeling of the actin cytoskeleton in response to stimulation with the motogenic growth factor platelet-derived growth factor. On stimulation, Dyn2 and cortactin coassemble into large, circular structures on the dorsal cell surface. These "waves" promote an active reorganization of actin filaments in the anterior cytoplasm and function to disassemble actin stress fibers. Importantly, inhibition of Dyn2 and cortactin function potently blocked the formation of waves and subsequent actin reorganization. These findings demonstrate that cortactin and Dyn2 function together in a supramolecular complex that assembles in response to growth factor stimulation and mediates the remodeling of actin to facilitate lamellipodial protrusion at the leading edge of migrating cells.
Molecular Biology of the Cell 04/2003; 14(3):1085-96. · 4.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria is a member of a small but important class of proteins that exhibit strong visible fluorescence without the requirement of
cofactors or other enzymes. In vivo, the photoprotein aequorin binds dimers of GFP allowing the blue light emitted by aequorin
to be converted to green light (1). This visible fluorescence emitted by GFP is made possible by an internal p-hydroxybenzylideneimidazolinone chromophore formed post-translationally by cyclization of Ser65, Tyr66, and Gly67 and 1,2-dehydrogenation
of the tyrosine. A number of mutations have been made in GFP, most of which result in a partial or complete loss of fluorescence
without significant change in relative absorption or emission peaks. These mutations probably cause misfolding of the protein,
failure of chromophore formation, or quenching of the fluorescence by insufficient shielding. A few mutations, however, have
resulted in enhanced GFP spectra, increased brightness, a slower rate of photobleaching, or a spectral shift resulting in
the emission of a different color, such as blue, yellow, or cyan. These GFP variants emitting new colors are thus called blue
fluorescent protein (BFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP) (2).
[show abstract][hide abstract] ABSTRACT: The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory
pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton
has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been
postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed
that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration
with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the
plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated
with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate
an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety
of biochemical methods we demonstrate that the cortactin–SH3 domain associates with the proline-rich domain (PRD) of dynamin.
Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro
observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or
expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortΔSH3)
significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD
(Dyn 2(aa)ΔPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable,
but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in
the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin
cytoskeleton to regulate actin reorganization and subsequently cell shape.
The Journal of Cell Biology 10/2000; 151(1):187-198. · 10.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion
plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed
cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular
to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those
generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities
for dynamin 2 and for the dynamin 2–binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and
osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching,
GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aaG273D abolished podosomes while GFP-dynaminK44A was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin
family and actin at attachment sites between cells and the substratum.
The Journal of Cell Biology 07/2000; 150(2):377-390. · 10.82 Impact Factor