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Razidlo G.L.,Center for Basic Research in Digestive Diseases | Schroeder B.,Center for Basic Research in Digestive Diseases | Chen J.,Center for Basic Research in Digestive Diseases | Billadeau D.D.,Mayo Medical School | And 2 more authors.
Current Biology | Year: 2014

Invadopodia are protrusive structures used by tumor cells for degradation of the extracellular matrix to promote invasion [1]. Invadopodia formation and function are regulated by cytoskeletal-remodeling pathways and the oncogenic kinase Src. The guanine nucleotide exchange factor Vav1, which is an activator of Rho family GTPases, is ectopically expressed in many pancreatic cancers, where it promotes tumor cell survival and migration [2, 3]. We have now determined that Vav1 is also a potent regulator of matrix degradation by pancreatic tumor cells as depletion of Vav1 by siRNA-mediated knockdown inhibits the formation of invadopodia. This requires the exchange function of Vav1 toward the GTPase Cdc42, which is required for invadopodia assembly [4, 5]. In addition, we have determined that Src-mediated phosphorylation and activation of Vav1 are both required for, and, unexpectedly, sufficient for, invadopodia formation. Expression of Vav1 Y174F, which mimics its activated state, is a potent inducer of invadopodia formation through Cdc42, even in the absence of Src activation and phosphorylation of other Src substrates, such as cortactin. Thus, these data identify a novel mechanism by which Vav1 can enhance the tumorigenicity and invasive potential of cancer cells. These data suggest that Vav1 promotes the matrix-degrading processes underlying tumor cell migration and further, under conditions of ectopic Vav1 expression, that Vav1 is a central regulator and major driver of invasive matrix remodeling by pancreatic tumor cells. © 2014 Elsevier Ltd.


Razidlo G.L.,Center for Basic Research in Digestive Diseases | Wang Y.,Mayo Medical School | Chen J.,Center for Basic Research in Digestive Diseases | Krueger E.W.,Center for Basic Research in Digestive Diseases | And 3 more authors.
Developmental Cell | Year: 2013

The large GTPase Dynamin 2 (Dyn2) is markedly upregulated in pancreatic cancer, is a potent activator of metastatic migration, and is required for Rac1-mediated formation of lamellipodia. Here we demonstrate an unexpected mechanism of Dyn2 action in these contexts via direct binding to the Rac1 guanine nucleotide exchange factor (GEF) Vav1. Surprisingly, disruption of the Dyn2-Vav1 interaction targets Vav1 to the lysosome for degradation via an interaction with the cytoplasmic chaperone Hsc70, resulting in a dramatic reduction of Vav1 protein stability. Importantly, a specific mutation in Vav1 near its Dyn2-binding C-terminal Src homology 3 (SH3) domain prevents Hsc70 binding, resulting in a stabilization of Vav1 levels. Dyn2 binding regulates the interaction of Vav1 with Hsc70 to control the stability and subsequent activity of this oncogenic GEF. These findings elucidate how Dyn2 activates Rac1, lamellipod protrusion, and invasive cellular migration and provide insight into how this specific Vav is ectopically expressed in pancreatic tumors. © 2013 Elsevier Inc.


Feng H.,University of Pittsburgh | Liu K.W.,University of Pittsburgh | Guo P.,University of Pittsburgh | Guo P.,Childrens Hospital | And 7 more authors.
Oncogene | Year: 2012

Dynamin 2 (Dyn2), a large GTPase, is involved in receptor tyrosine kinase (RTK)-promoted cell migration. However, the molecular mechanisms by which Dyn2 regulates RTK-induced cell migration have not been established. Recently, we reported that tyrosine-protein phosphatase non-receptor type 11 (SHP-2) and phosphatidylinositol 3-kinase (PI3K) mediate platelet-derived growth factor receptor-α (PDGFRα)-promoted glioma tumor growth and invasion. Here, we show that Dyn2 is an effector downstream of the PDGFRα-PI3K/SHP-2 signaling in glioma cells. Depletion of endogenous Dyn2 by short hairpin RNAs (shRNAs) inhibited PDGFRα-stimulated phosphorylation of Akt, Erk1/2, Rac1 and Cdc42 activities, glioma cell migration and survival in vitro and tumor growth and invasion in the brains of mice. Dyn2 binds to SHP-2 and PI3K and colocalizes with PDGFRα at the invasive fronts in PDGF-A-stimulated glioma cells. Inhibition of SHP-2 by siRNA knockdown abrogated Dyn2 association with activated PDGFRα and PDGFRα activation of Rac1 and Cdc42, and glioma cell migration, thereby establishing a link between SHP-2 interaction with Dyn2 and the PDGFRα signaling. Furthermore, a dominant-negative SHP-2 C459S mutant inhibited PDGF-A-stimulated glioma cell migration, phosphorylation of Dyn2 and concomitantly blocked PDGFRα-induced Src activation. Inhibition of Src by Src inhibitors attenuated PDGF-A-stimulated phosphorylation of Akt and Dyn2 and glioma cell migration. Additionally, mutations of binding sites to PI3K, SHP-2 or Src of PDGFRα impaired PDGFRα-stimulated phosphorylation of Akt and Dyn2, and Dyn2 association with activated PDGFRα. Taken together, this study identifies Dyn2 as an effector that mediates PDGFRα-SHP-2-induced glioma tumor growth and invasion, suggesting that targeting the PDGFRα-SHP-2-Dyn2 pathway may be beneficial to patients with malignant glioblastomas. © 2012 Macmillan Publishers Limited All rights reserved.


Kurklinsky S.,Mayo Graduate School | Chen J.,Center for Basic Research in Digestive Diseases | McNiven M.A.,Center for Basic Research in Digestive Diseases
Journal of Neurochemistry | Year: 2011

Neuronal growth cone (GC) migration and targeting are essential processes for the formation of a neural network during embryonic development. Currently, the mechanisms that support directed motility of GCs are not fully defined. The large GTPase dynamin and an interacting actin-binding protein, cortactin, have been localized to GCs, although the function performed by this complex is unclear. We have found that cortactin and the ubiquitous form of dynamin (Dyn) 2 exhibit a striking co-localization at the base of the transition zone of advancing GCs of embryonic hippocampal neurons. Confocal and total internal reflection fluorescence microscopies demonstrate that this basal localization represents point contacts. Exogenous expression of wild-type Dyn2 and cortactin leads to large, exceptionally flat, and static GCs, whereas disrupting this complex has no such effect. We find that excessive GC spreading is induced by Dyn2 and cortactin over-expression and substantial recruitment of the point contact-associated, actin-binding protein α-actinin1 to the ventral GC membrane. The distributions of other point contact proteins such as vinculin or paxillin appear unchanged. Immunoprecipitation experiments show that both Dyn2 and cortactin reside in a complex with a-actinin1. These findings provide new insights into the role of Dyn2 and the actin cytoskeleton in GC adhesion and motility. © 2011 Mayo Clinic Journal of Neurochemistry © 2011 International Society for Neurochemistry.


Cao H.,Center for Basic Research in Digestive Diseases | Chen J.,Center for Basic Research in Digestive Diseases | Krueger E.W.,Center for Basic Research in Digestive Diseases | McNiven M.A.,Center for Basic Research in Digestive Diseases
Molecular and Cellular Biology | Year: 2010

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. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


McNiven M.A.,Center for Basic Research in Digestive Diseases
Trends in Cell Biology | Year: 2013

Substantial progress has been made in recent years toward understanding the molecular mechanisms by which tumor cells, and the supporting stroma, degrade confining matrix during migration. Significant attention has been focused on understanding the biology of several dynamic and distinct, but remarkably related, cell structures that include lamellipodia, focal adhesions (FAs), filopodia, podosomes, and invadopodia. How these invasive organelles assemble and function is a topic of intense study. Most exciting has been the recent progress made by combining advanced microscope technologies with a wide variety of different 3D matrices, tissue explants, or even living model organisms. From these approaches, it has become increasingly evident that the conventional definitions of these invasive structures may be less clear than was previously thought. © 2012 Elsevier Ltd.


Juran B.D.,Center for Basic Research in Digestive Diseases | Lazaridis K.N.,Center for Basic Research in Digestive Diseases
Seminars in Liver Disease | Year: 2014

The etiology of the autoimmune liver disease primary biliary cirrhosis (PBC) remains largely unresolved, owing in large part to the complexity of interaction between environmental and genetic contributors underlying disease development. Observations of disease clustering, differences in geographical prevalence, and seasonality of diagnosis rates suggest the environmental component to PBC is strong, and epidemiological studies have consistently found cigarette smoking and history of urinary tract infection to be associated with PBC. Current evidence implicates molecular mimicry as a primary mechanism driving loss of tolerance and subsequent autoimmunity in PBC, yet other environmentally influenced disease processes are likely to be involved in pathogenesis. In this review, the authors provide an overview of current findings and touch on potential mechanisms behind the environmental component of PBC. Copyright © 2014 by Thieme Medical Publishers, Inc.


Schroeder B.,Mayo Medical School | Schroeder B.,Center for Basic Research in Digestive Diseases | Weller S.G.,Mayo Medical School | Weller S.G.,Center for Basic Research in Digestive Diseases | And 5 more authors.
EMBO Journal | Year: 2010

The epidermal growth factor receptor (EGFR) is over-expressed in a variety of human cancers. Downstream signalling of this receptor is tightly regulated both spatially and temporally by controlling its internalization and subsequent degradation. Internalization of the EGFR requires dynamin 2 (Dyn2), a large GTPase that deforms lipid bilayers, leading to vesicle scission. The adaptor protein CIN85 (cbl-interacting protein of 85 kDa), which has been proposed to indirectly link the EGFR to the endocytic machinery at the plasma membrane, is also thought to be involved in receptor internalization. Here, we report a novel and direct interaction between Dyn2 and CIN85 that is induced by EGFR stimulation and, most surprisingly, occurs late in the endocytic process. Importantly, disruption of the CIN85-Dyn2 interaction results in accumulation of internalized EGFR in late endosomes that become aberrantly elongated into distended tubules. Consistent with the accumulation of this receptor is a sustention of downstream signalling cascades. These findings provide novel insights into a previously unknown protein complex that can regulate EGFR traffic at very late stages of the endocytic pathway. © 2010 European Molecular Biology Organization.


Wang Y.,Center for Basic Research in Digestive Diseases | McNiven M.A.,Center for Basic Research in Digestive Diseases
Journal of Cell Biology | Year: 2012

Tumor cell migration and the concomitant degradation of extracellular matrix (ECM) are two essential steps in the metastatic process. It is well established that focal adhesions (FAs) play an important role in regulating migration; however, whether these structures contribute to matrix degradation is not clear. In this study, we report that multiple cancer cell lines display degradation of ECM at FA sites that requires the targeted action of MT1-MMP. Importantly, we have found that this MT1-MMP targeting is dependent on an association with a FAK- p130Cas complex situated at FAs and is regulated by Src-mediated phosphorylation of Tyr 573 at the cytoplasmic tail of MT1. Disrupting the FAK-p130Cas-MT1 complex significantly impairs FA-mediated degradation and tumor cell invasion yet does not appear to affect invadopodia formation or function. These findings demonstrate a novel function for FAs and also provide molecular insights into MT1-MMP targeting and function. © 2012 Wang and McNiven.


Cao H.,Center for Basic Research in Digestive Diseases | Krueger E.W.,Center for Basic Research in Digestive Diseases | Mcniven M.A.,Center for Basic Research in Digestive Diseases | Mcniven M.A.,Mayo Medical School
Hepatology | Year: 2011

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. © 2011 American Association for the Study of Liver Diseases.

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