Large GTPase Dynamin Research Articles

S2032 Pancreatic Tumor Cells Exhibit An Aberrantly Organized Golgi Apparatus That Is Regulated By SRC Kinase and the Large GTPase Dynamin 2

BCL6 and nuclear translocation of HB-EGF-CTF in gastric cancers. Method: We examined BCL6 and cyclinD2 expression level under the 12-0-tetradecanoylphorbor-13-acetate (TPA) stimulation. KB-R7785 was used to suppress HB-EGF-CTF nuclear translocation. We also investigated the interaction and translocation of BCL6 with HB-EGF-CTF by using immunofluorescence microscopy and immunoprecipitation. Furthermore, we immunohistochemically studied 100 surgical specimens of advanced gastric cancers to analyze the expression of HB-EGF, BCL6, and cyclin D2. Results: TPA treatment resulted in BCL6 degradation and cyclin D2 up-regulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF. The HB-EGF-CTF nuclear translocation lead to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and the ubiquitin/proteasome pathway mediated the BCL6 degradation. siRNA targeting BCL6 revealed that the upregulation of cyclin D2 depends on BCL6 degradation. BCL6 interacts with nuclear translocated HB-EGF-CTF and the nuclear export and degradation of BCL6 (caused by this interaction and the ubiquitin/proteasome pathway) induces cyclin D2 up-regulation. In human gastric cancer tissues, BCL6 expression is associated with differentiated gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also induced cyclin D2 expression In Vivo (human cancer tissues). Conclusion: Inhibition of HB-EGF-CTF nuclear translocation and the resulting maintenance of BCL6 function are important for the regulation of gastric cancer progression.

S2042 Focal Adhesion Dynamics in Invasive Pancreatic Tumor Cells Are Regulated By SRC Activation of the Large GTPase Dynamin 2

S2042 Focal Adhesion Dynamics in Invasive Pancreatic Tumor Cells Are Regulated By SRC Activation of the Large GTPase Dynamin 2

The Role of Dynamin in the Clearance of Apoptotic Cells

In animals, cells undergoing apoptosis are rapidly engulfed and degraded by phagocytes. The removal of apoptotic cells provides a safe means for eliminating unwanted and dangerous cells from the body. Furthermore, it prevents tissue injury, and inflammatory and auto‐immune responses induced by the lingering dead cells. In a genetic screen for mutations that affect the removal of apoptotic cells in the nematode C. elegans. We isolated fourteen loss‐of‐function alleles of dyn‐1, which encodes the C. elegans ortholog of large GTPase dynamin. Our cell biological characterizations indicate that DYN‐1 is essential for both the engulfment and degradation of apoptotic cells. DYN‐1 promotes the incorporation of endosomes to the extending pseudopods during engulfment, and the recruitment and incorporation of both endosomes and lysosomes to phagosomes containing apoptotic cells. Phosphatidylinositol 3‐phosphate (PI(3)P) and the small GTPase RAB‐7 mediate the specific function of DYN‐1 in phagosome maturation. Our in vitro and in vivo analyses demonstrate that the GTP hydrolysis and self‐assembly activities of DYN‐1 control distinct aspects of DYN‐1's function in phagosome maturation. Furthermore, the phagocytic receptor CED‐1 recruits DYN‐1 to the site of engulfment. These findings indicate that DYN‐1 is a key component of a pathway that regulates vesicle transport during the removal of apoptotic cells.

Dynamin 2 Regulates Granule Exocytosis during NK Cell-Mediated Cytotoxicity

NK cells are innate immune cells that can eliminate their targets through granule release. In this study, we describe a specialized role for the large GTPase Dynamin 2 (Dyn2) in the regulation of these secretory events leading to cell-mediated cytotoxicity. By modulating the expression of Dyn2 using small interfering RNA or by inhibiting its activity using a pharmacological agent, we determined that Dyn2 does not regulate conjugate formation, proximal signaling, or granule polarization. In contrast, during cell-mediated killing, Dyn2 localizes with lytic granules and polarizes to the NK cell-target interface where it regulates the final fusion of lytic granules with the plasma membrane. These findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity.

Local Actin-Dependent Endocytosis Is Zygotically Controlled to Initiate Drosophila Cellularization

In early Drosophila embryos, several mitotic cycles proceed with aborted cytokinesis before a modified cytokinesis, called cellularization, finally divides the syncytium into individual cells. Here, we find that scission of endocytic vesicles from the plasma membrane (PM) provides a control point to regulate the furrowing events that accompany this development. At early mitotic cycles, local furrow-associated endocytosis is controlled by cell cycle progression, whereas at cellularization, which occurs in a prolonged interphase, it is controlled by expression of the zygotic gene nullo. nullo mutations impair cortical F-actin accumulation and scission of endocytic vesicles, such that membrane tubules remain tethered to the PM and deplete structural components from the furrows, precipitating furrow regression. Thus, Nullo regulates scission to restrain endocytosis of proteins essential for furrow stabilization at the onset of cellularization. We propose that developmentally regulated endocytosis can coordinate actin/PM remodeling to directly drive furrow dynamics during morphogenesis.

The closure of Pak1-dependent macropinosomes requires the phosphorylation of CtBP1/BARS

Membrane fission is an essential process in membrane trafficking and other cellular functions. While many fissioning and trafficking steps are mediated by the large GTPase dynamin, some fission events are dynamin independent and involve C-terminal-binding protein-1/brefeldinA-ADP ribosylated substrate (CtBP1/BARS). To gain an insight into the molecular mechanisms of CtBP1/BARS in fission, we have studied the role of this protein in macropinocytosis, a dynamin-independent endocytic pathway that can be synchronously activated by growth factors. Here, we show that upon activation of the epidermal growth factor receptor, CtBP1/BARS is (a) translocated to the macropinocytic cup and its surrounding membrane, (b) required for the fission of the macropinocytic cup and (c) phosphorylated on a specific serine that is a substrate for p21-activated kinase, with this phosphorylation being essential for the fission of the macropinocytic cup. Importantly, we also show that CtBP1/BARS is required for macropinocytic internalization and infection of echovirus 1. These results provide an insight into the molecular mechanisms of CtBP1/BARS activation in membrane fissioning, and extend the relevance of CtBP1/BARS-induced fission to human viral infection.

Phagocytic Receptor CED-1 Initiates a Signaling Pathway for Degrading Engulfed Apoptotic Cells

Apoptotic cells in animals are engulfed by phagocytic cells and subsequently degraded inside phagosomes. To study the mechanisms controlling the degradation of apoptotic cells, we developed time-lapse imaging protocols in developing Caenorhabditis elegans embryos and established the temporal order of multiple events during engulfment and phagosome maturation. These include sequential enrichment on phagocytic membranes of phagocytic receptor cell death abnormal 1 (CED-1), large GTPase dynamin (DYN-1), phosphatidylinositol 3-phosphate (PI(3)P), and the small GTPase RAB-7, as well as the incorporation of endosomes and lysosomes to phagosomes. Two parallel genetic pathways are known to control the engulfment of apoptotic cells in C. elegans. We found that null mutations in each pathway not only delay or block engulfment, but also delay the degradation of engulfed apoptotic cells. One of the pathways, composed of CED-1, the adaptor protein CED-6, and DYN-1, controls the rate of enrichment of PI(3)P and RAB-7 on phagosomal surfaces and the formation of phagolysosomes. We further identified an essential role of RAB-7 in promoting the recruitment and fusion of lysosomes to phagosomes. We propose that RAB-7 functions as a downstream effector of the CED-1 pathway to mediate phagolysosome formation. Our work suggests that phagocytic receptors, which were thought to act specifically in initiating engulfment, also control phagosome maturation through the sequential activation of multiple effectors such as dynamin, PI(3)P, and Rab GTPases.

Dynamin 2 is involved in Angiotensin II‐induced NADPH oxidase activity mediated through cortactin in vascular smooth muscle cells

Although the actin cytoskeleton has been implicated in the control of NADPH oxidase activation induced by angiotensin II (Ang II), very little is known about the detailed molecular mechanisms. The dynamin family of large GTPases has been suggested in the formation of nascent vesicles in endocytic pathways. However, there is no report about the role of the large GTPase dynamin 2 (Dyn2) on the activity of NADPH oxidase induced by Ang II in vascular smooth muscle cells (VSMC). In this study we found that Dyn2 was colocalized with cortactin, the actin‐binding protein, by immunofluorencence. By performing immunoprecipitation, Ang II stimulation enhanced the association between Dyn2 and cortactin. Importantly, both cortactin siRNA and Dyn2 siRNA significantly inhibited Ang II‐induced ROS production, whereas cytochalasin D and latrunculin A suppressed the ROS formation by Ang II. Furthermore, Dyn2 siRNA significantly inhibited Ang II‐stimulated redox‐sensitive Akt phosphorylation without affecting redox‐insensitive ERK1/2 phosphorylation. These findings provide the first demonstration that Dyn2 is involved in Ang II‐stimulation NADPH oxidase activation mediated through cortactin, which may play an important role in temple‐spatial role in ROS‐dependent Ang II signaling in vascular smooth muscle cells.

Activity‐dependent control of bulk endocytosis by protein dephosphorylation in central nerve terminals

Bulk endocytosis is the process by which nerve terminals retrieve large amounts of synaptic vesicle membrane during periods of strong stimulation intensity. The process is rapidly activated and is most probably calcium dependent in a similar manner to synaptic vesicle exocytosis. This article briefly summarizes the current knowledge of bulk endocytosis with respect to its activation, kinetics and molecular mechanism. It also presents recent data from our laboratory showing that the dephosphorylation of a group of endocytosis proteins called the dephosphins by the Ca(2+)-dependent protein phosphatase calcineurin is key to the activity-dependent stimulation of the process. Possible downstream effectors of calcineurin are discussed such as the large GTPase dynamin I and its phosphorylation-dependent interaction partner syndapin I.

Perturbation of the vesicle cycle in reticulospinal synapses of lamprey after presynaptic microinjections of GTPγS

The synaptic vesicle cycle sustains neurotransmission and keeps exo- and endocytosis in synapses in dynamic equilibrium. GTP-binding proteins function as key regulators of this cycle. The large GTPase dynamin is implicated in the fission of clathrin-coated vesicles from presynaptic membrane during endocytosis. The present study addresses the effect of the nonhydrolysable GTP analog GTPγS on assembly of the dynamin fission complex in situ. Intraaxonal microinjections of GTPγS induced the following distinct ultrastructural changes in the synapses: the number of synaptic vesicles in a cluster decreased while the number of the docked vesicles at the active zone increased; at the same time, the clathrin-coated intermediates also increased in number, indicating the inhibition of synaptic vesicle recycling. Unusual clathrin-coated intermediates were found. At low concentrations of GTPγS, they were presented by long tubules wreathed with a dynamin helix (spiral) and topped with a clathrin-coated vesicle. At high concentrations of GTPγS the tubular structures were much shorter and branched, with each branch topped with a clathrin-coated vesicle. The spiral pitch and the tubule diameter were significantly reduced as the concentration of GTPγS built up (23.1 ± 0.4 and 26.6 ± 0.4 nm, respectively, at low and 19.0 ± 0.5 and 23.3 ± 0.4 nm at high concentration of GTPγS, p < 0.001). We suggest that these ultrastructural changes reflect different steps in dynamin-mediated fission of clathrin-coated vesicles and propose a model for this process. The model implies that at first, GTP hydrolysis leads to a fast elongation of the helix due to a straightening of its dynamin dimmers. This entails an increase both in a pitch and a diameter of the dynamin helix. The shift in diameter disrupts local hydrophobic interactions between the inner and the outer lipid layers of the membrane at the sites of dynamin binding. Concurrent stretching of the helix and the clathrin-coated vesicle’s neck disintegrates the neck membrane and results finally in a release of the clathrincoated vesicle.

The dynamin middle domain is critical for tetramerization and higher-order self-assembly

The large multidomain GTPase dynamin self-assembles around the necks of deeply invaginated coated pits at the plasma membrane and catalyzes vesicle scission by mechanisms that are not yet completely understood. Although a structural role for the 'middle' domain in dynamin function has been suggested, it has not been experimentally established. Furthermore, it is not clear whether this putative function pertains to dynamin structure in the unassembled state or to its higher-order self-assembly or both. Here, we demonstrate that two mutations in this domain, R361S and R399A, disrupt the tetrameric structure of dynamin in the unassembled state and impair its ability to stably bind to and nucleate higher-order self-assembly on membranes. Consequently, these mutations also impair dynamin's assembly-dependent stimulated GTPase activity.

Dynamin is required for F-actin assembly and pedestal formation by enteropathogenic Escherichia coli (EPEC)

After attaching to human intestinal epithelial cells, enteropathogenic Escherichia coli (EPEC) induces the formation of an actin-rich pedestal-like structure. The signalling pathway leading to pedestal formation is initiated by the bacterial protein Tir, which is inserted into the host cell plasma membrane. The domain exposed on the cell surface binds to another bacterial protein, intimin, while one of the cytoplasmic domains binds the adaptor protein Nck. This leads to recruitment of other cytoskeletal proteins including neural Wiskott-Aldrich syndrome protein (N-WASP) and Arp2/3, resulting in focused actin polymerization at the site of bacterial attachment. In this study we investigated the role of the large GTPase dynamin 2 (Dyn2) in pedestal formation. We found that in HeLa cells, both endogenous and overexpressed Dyn2 were recruited to sites of EPEC attachment. Recruitment of endogenous Dyn2 required the presence of Tir, Nck and N-WASP but was independent of cortactin and Arp2/3. Knock-down of Dyn2 expression by RNA interference reduced actin polymerization and pedestal formation. Overexpression of dominant-negative mutants of Dyn2 also reduced pedestal formation and prevented recruitment of N-WASP, Arp3 and cortactin, but not Nck. Together, our results indicate that Dyn2 is an integral component of the signalling cascade leading to actin polymerization in EPEC pedestals.

Dynamin as a mover and pincher during cell migration and invasion

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.

NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS

Intracellular trafficking of endothelial nitric oxide synthase (eNOS) between different compartments is incompletely understood. Recently, we described a novel eNOS-interacting protein, NOSTRIN, which upon overexpression drives eNOS away from the plasma membrane towards intracellular compartments. Sequence similarity of NOSTRIN and pacsins/syndapins suggested a role for NOSTRIN in endocytosis. Accordingly, we show here that NOSTRIN interacts with the large GTPase dynamin and the actin nucleation promoting factor N-WASP by means of its SH3 domain, which also represents the docking site for eNOS. Via a coiled-coil region in the C-terminal portion of the protein, NOSTRIN oligomerizes, mainly forming trimers, which would allow simultaneous interaction with multiple binding partners of the SH3 domain. Consistent with this notion, expression of dynamin-2-GFP in CHO cells stably expressing eNOS (CHO-eNOS) results in recruitment of eNOS to dynamin-positive structures, only when NOSTRIN is present as well. Similarly, when N-WASP-GFP and NOSTRIN are co-expressed in CHO-eNOS cells, both proteins strongly co-localize with eNOS and are recruited to structures running along actin filaments. If, however, the actin cytoskeleton is depolymerized by cytochalasin D, NOSTRIN and eNOS are associated with extended structures in the cell periphery, possibly being unable to leave the plasma membrane. Together, these results indicate that NOSTRIN may facilitate endocytosis of eNOS by coordinating the function of dynamin and N-WASP.

Caveolin-1 Interacts Directly with Dynamin-2

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.

Secretion of hepatitis B virus surface (HBsAg) and HBeAg in HepG2.2.15 is dependent upon the large GTPase dynamin: Ahmad S Abdulkarim, Hong Cao, Mark A McNiven, Mayo Clinic, Rochester, MN

Secretion of hepatitis B virus surface (HBsAg) and HBeAg in HepG2.2.15 is dependent upon the large GTPase dynamin: Ahmad S Abdulkarim, Hong Cao, Mark A McNiven, Mayo Clinic, Rochester, MN

Participation of the large GTPase dynamin in actin comet formation, morphology, and movement in living hepatocytes: James D Orth, E W Krueger, Mark A McNiven, Mayo Clinic, Rochester, MN

Participation of the large GTPase dynamin in actin comet formation, morphology, and movement in living hepatocytes: James D Orth, E W Krueger, Mark A McNiven, Mayo Clinic, Rochester, MN

The yeast dynamin-related GTPase Vps1p functions in the organization of the actin cytoskeleton via interaction with Sla1p.

Recent studies have suggested that the function of the large GTPase dynamin in endocytosis in mammalian cells may comprise a modulation of actin cytoskeleton. The role of dynamin in actin cytoskeleton organization in the yeast Saccharomyces cerevisiae has remained undefined. In this report, we found that one of the yeast dynamin-related proteins, Vps1p, is required for normal actin cytoskeleton organization. At both permissive and non-permissive temperatures, the vps1 mutants exhibited various degrees of phenotypes commonly associated with actin cytoskeleton defects: depolarized and aggregated actin structures, hypersensitivity to the actin cytoskeleton toxin latrunculin-A, randomized bud site selection and chitin deposition, and impaired efficiency in the internalization of membrane receptors. Over-expression of the GTPase mutants of vps1 also led to actin abnormalities. Consistent with these actin-related defects, Vps1p was found to interact physically, and partially co-localize, with the actin-regulatory protein Sla1p. The normal cellular localization of Sla1p required Vps1p and could be altered by over-expression of a region of Vps1p that was involved in the interaction with Sla1p. The same region also promoted mis-sorting of the vacuolar protein carboxypeptidase Y upon over-expression. These findings suggest that the functions of the dynamin-related protein Vps1p in actin cytoskeleton dynamics and vacuolar protein sorting are probably related to each other.

The syndapin protein family: linking membrane trafficking with the cytoskeleton.

Syndapins--also called PACSINs--are highly conserved Src-homology 3 (SH3)-domain-containing proteins that seem to exist in all multicellular eukaryotes. They interact with the large GTPase dynamin and several other proteins implicated in vesicle trafficking. Syndapin-dynamin complexes appear to play an important role in vesicle fission at different donor membranes, including the plasma membrane (endocytosis) and Golgi membranes. In addition, syndapins are implicated in later steps of vesicle cycling in neuronal and non-neuronal cells. Syndapins also interact with N-WASP, a potent activator of the Arp2/3 complex that forms a critical part of the actin polymerization machinery. Syndapin oligomers can thereby couple bursts of actin polymerization with the vesicle fission step involving dynamins. This allows newly formed vesicles to move away from the donor membrane driven by actin polymerization. Syndapins also engage in additional interactions with molecules involved in several signal transduction pathways, producing crosstalk at the interface between membrane trafficking and the cytoskeleton. Given the distinct expression patterns of the different syndapins and their splice forms, these proteins could have isoform-specific functions.

Agonist-induced trafficking of the low-affinity formyl peptide receptor FPRL1.

The formyl peptide-like receptor FPRL1 is a member of the chemoattractant subfamily of G protein- coupled receptors involved in regulating leukocyte migration in inflammation. To elucidate mechanisms underlying the internalization of ligand-bound FPRL1 and possible receptor recycling, we characterized the endocytic itinerary of FPRL1. We show that agonist-triggered internalization from the plasma membrane into intracellular compartments is prevented by perturbation of clathrin-mediated endocytosis, such as expression of the dominant-negative clathrin Hub mutant, siRNA-mediated depletion of cellular clathrin and expression of a dominant-negative mutant of the large GTPase dynamin. Internalized FPRL1 co-localized with endocytosed transferrin and the small GTPases Rab4 and Rab11 in vesicular structures most resembling recycling endosomes. Recycling of FPRL1 was significantly reduced by pretreatment with PI3-kinase inhibitors. Thus, ligand-bound FPRL1 undergoes primarily clathrin-mediated and dynamin-dependent endocytosis and the receptor recycles via a rapid PI3-kinase-sensitive route as well as pathways involving perinuclear recycling endosomes.