McNiven, Mark A.

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.

Suprastimulation of pancreatic acini is a well-known model for pancreatitis, and it is characterized by actin reorganization and cell blebbing. Currently, however, the mechanisms underlying regulation of these aberrant cytoskeletal and membrane dynamics and how they contribute to cell injury are unclear. We observed that suprastimulation results in a rapid activation of Src and relocalization of the actin-binding protein cortactin from the apical to the basolateral domain at the necks of membrane blebs. Furthermore, Src-mediated cortactin tyrosine phosphorylation was markedly increased after suprastimulation. Pretreatment of acini with Src inhibitors or expression of a cortactin tyrosine phospho-inhibitory mutant reduced actin redistribution and bleb formation induced by suprastimulation in vitro. Importantly, inhibition of Src activity in rat models of suprastimulation-induced pancreatitis substantially reduced disease severity, as indicated by a reduction in serum amylase and pancreatic edema and a striking improvement in tissue histology. These findings indicate a novel, disease-relevant role for Src-mediated cortactin phosphorylation in aberrant reorganization of the actin cytoskeleton, a mechanism that is likely to have implications in other types of cell injury. In addition, they suggest a potential use for Src inhibitors as an approach to reduce cell injury.

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.

An elaborate vesicle transport system supports the active exchange of membranes and protein cargo between the plasma membrane and the trans-Golgi network. Many observations suggest that highly conserved mechanisms are used in vesicle formation and scission. Such similarity is found both at the level of the receptor-ligand sequestration process that uses clathrin and associated polymeric and monomeric adaptor proteins, and in the machinery used to deform and vesiculate lipid membranes.

Abstract Internalization and subsequent trafficking of receptor tyrosine kinases (RTKs) play an important role in the modulation of growth factor–stimulated signaling events that affect different cellular processes, from cell growth and mitosis to motility and invasion. The intracellular transport of these receptors has traditionally been viewed as being initiated via clathrin-coated pits. However, nonclathrin pathways have been implicated as well, although these remain poorly understood. Most recently, the formation of dynamic, transient endocytic membrane structures termed circular dorsal ruffles or “dorsal waves” have been reported to selectively sequester and internalize a large percentage of a specific RTK from the surface of growth factor–stimulated cells. This process is dependent on dynamin and cortactin, two endocytic proteins that are also associated with the actin cytoskeleton, whereas it is independent of traditional coat proteins, such as clathrin and caveolin. Additionally, dorsal wave formation requires the participation and remodeling of a dynamic actin cytoskeleton. Most importantly, the formation of these structures may be less frequent in tumor cells and thereby have significant effects on receptor signaling and cell growth. (Cancer Res 2006; 66(23): 11094-6)

Dynamin is a 100-kDa microtubule-activated GTPase originally isolated from mammalian brain that has been proposed to be crucial in the early steps of endocytosis. Previous studies on the primary structure, biochemical properties, and functional role of dynamin indicated that it was neuron-specific. However, using an antibody against a synthetic peptide representing an enzymatic region of rat brain dynamin (D100), we identified a 100-kDa protein doublet in rat liver, suggesting that dynamin exists as different isoforms that are distinct from the brain counterpart. We then initiated a search for distinctive dynamin isoforms with antibodies and cDNA probes. A 500-bp PCR-generated cDNA probe corresponding to the enzymatic region of the rat brain dynamin-encoding gene was used to isolate six overlapping clones from a rat liver cDNA library that together span the complete coding sequence of another dynamin gene, "Dyn2." Sequence analyses reveal that dynamin 2 (Dyn2) is 75% identical to brain dynamin at the DNA level and is 79% identical at the protein level. By Northern blot analysis and isoform-specific PCR, Dyn2 was found ubiquitously in adult rat tissues as two transcripts of 3.5 kb and 4 kb; the highest levels were found in testis. These results indicate that dynamin proteins are encoded by at least two genes expressed differentially in mammalian tissues and that the expression of Dyn2, and not of brain dynamin, accounts for the ubiquitous distribution of dynamin in rat tissues.

The large GTPase dynamin, long known for its role in endocytosis, has most recently been implicated as a facilitator of cell migration and invasion. Recent observations link dynamin to the cycle of membrane expansion and retraction essential for cell motility. Its role in actin polymerization, membrane deformation and vesiculation, and focal adhesion dynamics are all important for this process, and the new findings provide exciting directions for studies of this ubiquitous and diverse protein family.

Abstract Cells form transient, circular dorsal ruffles or “waves” in response to stimulation of receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor. These dynamic structures progress inward on the dorsal surface and disappear, occurring concomitantly with a marked reorganization of F-actin. The cellular function of these structures is largely unknown. Here we show that EGF-induced waves selectively sequester and internalize ∼50% of ligand-bound EGFR from the cell surface. This process requires receptor phosphorylation, active phosphatidylinositol 3-kinase, and dynamin 2, although clathrin-coated pits or caveolae are not required. Epithelial and fibroblast cells stimulated with EGF sequestered EGFR rapidly into waves that subsequently generated numerous receptor-positive tubular-vesicular structures. Electron microscopy confirmed that waves formed along the dorsal membrane surface and extended numerous tubules into the cytoplasm. These findings characterize a structure that selectively sequesters large numbers of activated EGFR for their subsequent internalization, independent of traditional endocytic mechanisms such as clathrin pits or caveolae. (Cancer Res 2006; 66(7): 3603-10)

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.

ABSTRACT Vascular endothelial growth factor receptor‐2 (VEGFR‐2, also known as KDR) is a receptor tyrosine kinase (RTK) regulating mitogenic, chemotactic, permeability, and survival signals in vascular endothelial cells (EC) in response to its ligand, vascular permeability factor/VEGF (VPF/VEGF), arguably the most important angiogenic cytokine. However, the compartmentalization of KDR in EC and the mechanisms regulating this process have not been well defined. Here, we demonstrate that KDR is present on the plasma membrane, on endosomes, and in the perinuclear region of EC and colocalizes with early endosomal antigen (EEA1), caveolin‐1, and dynamin‐2, a signal transducing GTPase involved in receptor endocytosis. Furthermore, we also observed that dynamin‐2 coimmunoprecipitates with KDR and is required for EC signaling/survival. Interestingly, EC overexpressing a mutant form of dynamin deficient in GTP binding (K44A) caused a selective inhibition in KDR protein level and endosomal vesicle formation and induced cell cycle arrest by inducing p21. Taken together, our findings suggest that dynamin‐2 regulates KDR expression and function and hence plays an important role in VPF/VEGF mediated angiogenesis.