Eugene W. Krueger

The Mitochondrial Protein hFis1 Regulates Mitochondrial Fission in Mammalian Cells through an Interaction with the Dynamin-Like Protein DLP1

ABSTRACT The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.

The large GTPase dynamin regulates actin comet formation and movement in living cells

The large GTPase dynamin (Dyn2) has been demonstrated by us and others to interact with several different actin-binding proteins. To define how Dyn2 might participate in actin dynamics in livings cells we have expressed green fluorescent protein (GFP)-tagged Dyn2 in cultured cells and observed labeling of comet-like vesicles and macropinosomes. The comet structures progressed with a constant velocity and were reminiscent of actin comets associated with motile vesicles in cells expressing type I phosphatidylinositol phosphate 5-kinases. Based on these observations we sought to determine whether Dyn2 is an integral component of actin comets. Cells expressing type I phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a prominent colocalization of Dyn2 and actin in comet structures. Interestingly, comet formation and motility were normal in cells expressing wild-type Dyn2-GFP but altered markedly in Dyn2 mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a significant reduction in comet number, length, velocity, and efficiency of movement. In contrast, comets in cells expressing Dyn2ΔPRD-GFP appeared dark and did not incorporate the mutant Dyn2 protein, indicating that the proline-rich domain (PRD) is required for Dyn2 recruitment. Further, these comets were significantly longer and slower than those in control cells. These findings demonstrate a role for Dyn2 in actin-based vesicle motility.

Epithelial Growth Factor-induced Phosphorylation of Caveolin 1 at Tyrosine 14 Stimulates Caveolae Formation in Epithelial Cells

Caveolae are flask-shaped endocytic structures composed primarily of caveolin-1 (Cav1) and caveolin-2 (Cav2) proteins. Interestingly, a cytoplasmic accumulation of Cav1 protein does not always result in a large number of assembled caveolae organelles, suggesting a regulatory mechanism that controls caveolae assembly. In this study we report that stimulation of epithelial cells with epithelial growth factor (EGF) results in a profound increase in the number of caveolar structures at the plasma membrane. Human pancreatic tumor cells (PANC-1) and normal rat kidney cells (NRK), as a control, were treated with 30 ng/ml EGF for 0, 5, and 20 min before fixation and viewing by electron microscopy. Cells fixed without EGF treatment exhibited modest numbers of plasma membrane-associated caveolae. Cells treated with EGF for 5 or 20 min showed an 8–10-fold increase in caveolar structures, some forming long, pronounced caveolar “towers” at the cell-cell borders. It is known that Cav1 is Src-phosphorylated on tyrosine 14 in response to EGF treatment, although the significance of this modification is unknown. We postulated that phosphorylation could provide the stimulus for caveolae assembly. To this end, we transfected cells with mutant forms of Cav1 that could not be phosphorylated (Cav1Y14F) and tested if this altered protein reduced the number of EGF-induced caveolae. We observed that EGF-stimulated PANC-1 cells expressing the mutant Cav1Y14F protein exhibited a 90–95% reduction in caveolae number compared with cells expressing wild type Cav1. This study provides novel insights into how cells regulate caveolae formation and implicates EGF-based signaling cascades in the phosphorylation of Cav1 as a stimulus for caveolae assembly.

Src-Mediated Phosphorylation of Dynamin and Cortactin Regulates the “Constitutive” Endocytosis of Transferrin

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.

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.

Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

Ethanol-induced alterations of the microtubule cytoskeleton in hepatocytes

Ethanol has been predicted to alter vesicle-based protein traffic in hepatocytes, in part, via a disruption of the microtubule (MT) cytoskeleton. However, information on the effects of chronic ethanol exposure on MT function in vivo is sparse. Therefore the goal of this study was to test for ethanol-induced changes in rat liver tubulin expression, assembly, and cellular organization, using molecular, biochemical and morphological methods. The results of this study showed that tubulin mRNA and protein levels were not altered by ethanol. Tubulin, isolated from control and ethanol-fed rats, showed similar polymerization characteristics as assessed by calculation of the critical concentration for assembly and morphological structure. In contrast, the total amount of assembly-competent tubulin was reduced in livers from ethanol-fed rats compared with control rats when assessed by quantitative immunoblot analysis using a tubulin antibody. In addition, we observed that MT regrowth and organization in cultured hepatocytes treated with cold and nocodazole was markedly impaired by chronic ethanol exposure. In summary, these results indicate that tubulin levels in liver are not reduced by ethanol exposure. While there is a substantial amount of tubulin protein capable of assembling into functional MTs in ethanol-damaged livers, a marked portion of this tubulin is polymerization incompetent. This may explain why these hepatocytes exhibit a reduced number of MTs with an altered organization.

Dynamin 2 Potentiates Invasive Migration of Pancreatic Tumor Cells through Stabilization of the Rac1 GEF Vav1

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.

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.