Shaun G. Weller

Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis

ABSTRACT The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.

Actin and Arf1-dependent recruitment of a cortactin–dynamin complex to the Golgi regulates post-Golgi transport

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.

Inhibition of epidermal growth factor receptor kinase induces protease-dependent apoptosis in human colon cancer cells

BACKGROUND & AIMS The epidermal growth factor receptor (EGFR) is under investigation as a therapeutic target for cancers. Colon cancer cell lines are variably dependent on autocrine stimulation of EGFR. We therefore examined the effects of a selective EGFR tyrosine kinase inhibitor, PD 153035, on proliferation and survival of five colon cancer cell lines whose autonomous proliferation is either EGFR ligand dependent or EGFR ligand independent. METHODS Effects of inhibitors were screened by MTS growth assays, [3H]thymidine incorporation, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay, fluorescence microscopy, immunoblotting, and in vitro protease assays. RESULTS PD 153035 caused dose-dependent cytostasis (200 nmol/L to 1 micromol/L) and apoptosis (>10 micromol/L) in ligand-dependent cell lines and caused variable apoptosis (>10 micromol/L) but no cytostasis in ligand-independent cell lines. Apoptosis induced by 10 micromol/L PD 153035 was not associated with induction of p53 protein expression but was accompanied by activation of caspases that cleave poly(ADP-ribose) polymerase, lamin B1, and Bcl-2. Inhibition of caspase 3-like protease activity by DEVD-fluoromethylketone significantly delayed the onset of PD 153035-induced apoptosis. CONCLUSIONS The EGFR tyrosine kinase inhibitor PD 153035 induces cytostasis and caspase-dependent apoptosis in EGFR ligand-dependent colon cancer cell lines. These observations encourage further investigation of EGFR tyrosine kinase inhibitors for treatment of colorectal neoplasms.

Src kinase regulates the integrity and function of the Golgi apparatus via activation of dynamin 2

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.

The small GTPase Rab7 as a central regulator of hepatocellular lipophagy

Autophagy is a central mechanism by which hepatocytes catabolize lipid droplets (LDs). Currently, the regulatory mechanisms that control this important process are poorly defined. The small guanosine triphosphatase (GTPase) Rab7 has been implicated in the late endocytic pathway and is known to associate with LDs, although its role in LD breakdown has not been tested. In this study, we demonstrate that Rab7 is indispensable for LD breakdown (“lipophagy”) in hepatocytes subjected to nutrient deprivation. Importantly, Rab7 is dramatically activated in cells placed under nutrient stress; this activation is required for the trafficking of both multivesicular bodies and lysosomes to the LD surface during lipophagy, resulting in the formation of a lipophagic “synapse.” Depletion of Rab7 leads to gross morphological changes of multivesicular bodies, lysosomes, and autophagosomes, consequently leading to attenuation of hepatocellular lipophagy. Conclusion: These findings provide additional support for the role of autophagy in hepatocellular LD catabolism while implicating the small GTPase Rab7 as a key regulatory component of this essential process. (Hepatology 2015;61:1896–1907)

Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes.

Dynamin 2 is required for starvation-mediated breakdown of lipid droplets in hepatocytes by promoting vesiculation of autolysosomal tubules to release protolysosomes.

Caveolae mediate growth factor-induced disassembly of adherens junctions to support tumor cell dissociation.

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.

A Dyn2-CIN85 complex mediates degradative traffic of the EGFR by regulation of late endosomal budding.

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.

Distinct protein kinase C isozymes signal mitogenesis and apoptosis in human colon cancer cells.

BACKGROUND & AIMS Protein kinase C (PKC) is a family of serine-threonine kinases that transmit signals from cell surface receptors. To determine if distinct PKC isozymes transmit proliferative and/or apoptotic signals in colon cancer cells, we examined the effects of 3 PKC agonists, phorbol 12-myristate 13 acetate (PMA), ingenol 3,20-dibenzoate (IDB), and bistratene A, and a selective PKC inhibitor, GF 109203X, on proliferation, apoptosis, and activation of individual PKC isozymes in 5 colon cancer cell lines. METHODS Effects were assayed by a formazan-based colorimetric assay, [(3)H]thymidine incorporation, fluorescent nuclear staining, annexin V binding, DNA fragmentation assay, and immunoblotting of cytoplasmic and membrane fractions for PKC isozymes. RESULTS Two cell lines, SNU-C1 and SNU-C4, showed proliferative responses to PMA (0.1-1 nmol/L) and IDB (10-1000 nmol/L) and marked apoptotic responses to PMA (>5 nmol/L) and bistratene A (>1 micromol/L). GF 109203X blocked proliferative and apoptotic effects of PMA with distinct IC(50)s. Proliferative concentrations of PMA and IDB caused translocation of PKCepsilon alone, whereas apoptotic concentrations of PMA and bistratene A induced translocation of PKCdelta. CONCLUSIONS Activation of PKCepsilon and PKCdelta triggers proliferative and apoptotic signals, respectively, in SNU-C4 colon cancer cells. These 2 isozymes may play important opposing roles in normal homeostasis and neoplastic transformation of the colorectal epithelium.

Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma

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.