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PubMed2000

The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study.

Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.

Animals↗
PubMed1999

Impairment of dynamin's GAP domain stimulates receptor-mediated endocytosis.

Dynamin is a GTP-hydrolysing protein that is an essential participant in clathrin-mediated endocytosis by cells. It self-assembles into 'collars' in vitro which also formin vivo at the necks of invaginated coated pits. This self-assembly stimulates dynamin's GTPase activity and it has been proposed that dynamin hydrolyses GTP in order to generate the force needed to sever vesicles from the plasma membrane. A mechanism is now described in which self-assembly of dynamin is coordinated by a domain of dynamin with a GTPase-activating function. Unexpectedly, when dynamin mutants defective in self-assembly-stimulated GTPase activity are overexpressed, receptor-mediated endocytosis is accelerated. The results indicate that dynamin, like other members of the GTPase superfamily, functions as a molecular regulator in receptor-mediated endocytosis, rather than as a force-generating GTPase.

Amino Acid Sequence↗
PubMed1999

Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring.

The GTPase dynamin plays an essential part in endocytosis by catalysing the fission of nascent clathrin-coated vesicles from the plasma membrane. Using preformed phosphatidylinositol-4,5-bisphosphate-containing lipid nanotubes as a membrane template for dynamin self-assembly, we investigate the conformational changes that arise during GTP hydrolysis by dynamin. Electron microscopy reveals that, in the GTP-bound state, dynamin rings appear to be tightly packed together. After GTP hydrolysis, the spacing between rings increases nearly twofold. When bound to the nanotubes, dynamin's GTPase activity is cooperative and is increased by three orders of magnitude compared with the activity of unbound dynamin. An increase in the Kcat (but not the K(m) of GTP hydrolysis accounts for the pronounced cooperativity. These data indicate that a novel, lengthwise ('spring-like') conformational change in a dynamin helix may participate in vesicle fission.

Animals↗
PubMed2004

The stimulatory action of amphiphysin on dynamin function is dependent on lipid bilayer curvature.

Amphiphysin is a major dynamin-binding partner at the synapse; however, its function in fission is unclear. Incubation of large unilamellar liposomes with mice brain cytosol led to massive formation of small vesicles, whereas cytosol of amphiphysin 1 knockout mice was much less efficient in this reaction. Vesicle formation from large liposomes by purified dynamin was also strongly enhanced by amphiphysin. In the presence of liposomes, amphiphysin strongly affected dynamin GTPase activity and the recruitment of dynamin to the liposomes, but this activity was highly dependent on liposome size. Deletion from amphiphysin of its central proline-rich stretch dramatically potentiated its effect on dynamin, possibly by relieving an inhibitory intramolecular interaction. These results suggest a model in which maturation of endocytic pits correlates with the oligomerization of dynamin with either amphiphysin or other proteins with similar domain structure. Formation of these complexes is coupled to the activation of dynamin GTPase activity, thus explaining how deep invagination of the pit leads to fission.

Animals↗
PubMed2006

Nitric oxide regulates endocytosis by S-nitrosylation of dynamin.

The GTPase dynamin regulates endocytic vesicle budding from the plasma membrane, but the molecular mechanisms involved remain incompletely understood. We report that dynamin, which interacts with NO synthase, is S-nitrosylated at a single cysteine residue (C607) after stimulation of the beta(2) adrenergic receptor. S-nitrosylation increases dynamin self-assembly and GTPase activity and facilitates its redistribution to the membrane. A mutant protein bearing a C607A substitution does not self-assemble properly or increase its enzymatic activity in response to NO. In NO-generating cells, expression of dynamin C607A, like the GTPase-deficient dominant-negative K44A dynamin, inhibits both beta(2) adrenergic receptor internalization and bacterial invasion. Furthermore, exogenous or endogenously produced NO enhances internalization of both beta(2) adrenergic and epidermal growth factor receptors. Thus, NO regulates endocytic vesicle budding by S-nitrosylation of dynamin. Collectively, our data suggest a general NO-dependent mechanism by which the trafficking of receptors may be regulated and raise the idea that pathogenic microbes and viruses may induce S-nitrosylation of dynamin to facilitate cellular entry.

Cell Line↗
PubMed1998

Differential interactions of the growth factor receptor-bound protein 2 N-SH3 domain with son of sevenless and dynamin. Potential role in the Ras-dependent signaling pathway.

In this paper, we show that the 36-45 surface-exposed sequence WYKAELNGKD of growth factor receptor-bound protein 2 (Grb2) N-SH3 domain inhibits the interaction between Grb2 and a 97-kDa protein identified as dynamin. Moreover, the peptide GPPPQVPSRPNR from dynamin also blocks the binding of dynamin to the proline-rich recognition platform of Grb2. Mutations in the 36-45 motif show that Glu-40 is critical for dynamin recognition. These observations were confirmed by immunoprecipitation experiments, carried out using ER 22 cells. It was also observed that the proline-rich peptide from dynamin was unable to dissociate the Grb2.Sos complex, whereas the proline-rich peptide from Son of sevenless (Sos) inhibited Grb2. dynamin interaction. A time-dependent stimulation of epidermal growth factor receptor overexpressing clone 22 (ER 22) cells by epidermal growth factor resulted in an immediate increase of the Grb2.Sos complex and a concomitant decrease in Grb2.dynamin. This suggests that the recruitment of Grb2.Sos to the membrane, triggered by epidermal growth factor stimulation, activates the Ras-dependent signaling and simultaneously enhances free dynamin levels, leading to both receptor internalization and endocytotic processes.

Adaptor Proteins, Signal Transducing↗
PubMed2003

The single dynamin-like protein of Trypanosoma brucei regulates mitochondrial division and is not required for endocytosis.

Members of the evolutionarily conserved dynamin-related GTPase family mediate numerous cellular membrane remodeling events. Dynamin family functions include the scission of clathrin-coated pits from the plasma membrane, mitochondrial fission, and chloroplast division. Here we report that the divergent eukaryote Trypanosoma brucei possesses a single dynamin family gene, which we have designated TbDLP. Furthermore, a single dynamin family gene is also found in the Leishmania major and Trypanosoma vivax genomes, indicating that this is a conserved feature among the kinetoplastida. TbDLP is most homologous to the DMN/DRP family of dynamin-like proteins. Indirect immunofluorescence microscopy reveals that TbDLP is distributed in punctate structures within the cell that partially co-localize with the mitochondrion when labeled with MitoTracker. To define TbDLP function, we have used RNA interference to silence the TbDLP gene. Reduction of TbDLP protein levels causes a profound alteration in mitochondrial morphology without affecting the structure of other membrane-bound compartments, including the endocytic and exocytic apparatus. The mitochondrial profiles present in wild type trypanosomes fuse and collapse in the mutant cells, and by electron microscopy the mitochondria are found to contain an accumulation of constriction sites. These findings demonstrate TbDLP functions in division of the mitochondrial membrane. Most significantly, as TbDLP is the sole member of the dynamin family in this organism, scission of clathrin-coated pits involved in protein trafficking through the highly active endocytic system in trypanosomes must function in the absence of dynamin. The evolutionary implications of these findings are discussed.

Amino Acid Sequence↗
PubMed2006

Differential regulation of interleukin 5-stimulated signaling pathways by dynamin.

Through the yeast two-hybrid screen we have identified dynamin-2 as a molecule that interacts with the alpha subunit of the interleukin (IL) 5 receptor. Dynamin-2 is a GTPase that is critical for endocytosis. We have shown that dynamin-2 interacts with the IL-5 receptor-associated tyrosine kinases, Lyn and JAK2, in eosinophils. Tyrosine phosphorylation of dynamin is markedly enhanced upon IL-5 stimulation. The inhibition of tyrosine kinases results in complete abolition of ligand-induced receptor endocytosis. Inhibition of dynamin by a dominant-negative mutant or by small interfering RNA results in enhancement of IL-5-stimulated ERK1/2 signaling and cell proliferation. In contrast, the absence of a functional dynamin does not affect STAT5 or AKT phosphorylation or cell survival. Thus, we have identified specific functions for dynamin in the IL-5 signaling pathway and demonstrated its role in receptor endocytosis and termination of the ERK1/2 signaling pathway.

Cell Proliferation↗
PubMed1993

Effects of mutant rat dynamin on endocytosis.

Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non-coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2-terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over-expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

Dynamin-mediated internalization of caveolae.

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.

Amino Acid Sequence↗
PubMed2001

Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages.

Abundant evidence has shown that the GTPase dynamin is required for receptor-mediated endocytosis, but its exact role in endocytic clathrin-coated vesicle formation remains to be established. Whereas dynamin GTPase domain mutants that are defective in GTP binding and hydrolysis are potent dominant-negative inhibitors of receptor-mediated endocytosis, overexpression of dynamin GTPase effector domain (GED) mutants that are selectively defective in assembly-stimulated GTPase-activating protein activity can stimulate the formation of constricted coated pits and receptor-mediated endocytosis. These apparently conflicting results suggest that a complex relationship exists between dynamin's GTPase cycle of binding and hydrolysis and its role in endocytic coated vesicle formation. We sought to explore this complex relationship by generating dynamin GTPase mutants predicted to be defective at distinct stages of its GTPase cycle and examining the structural intermediates that accumulate in cells overexpressing these mutants. We report that the effects of nucleotide-binding domain mutants on dynamin's GTPase cycle in vitro are not as predicted by comparison to other GTPase superfamily members. Specifically, GTP and GDP association was destabilized for each of the GTPase domain mutants we analyzed. Nonetheless, we find that overexpression of dynamin mutants with subtle differences in their GTPase properties can lead to the accumulation of distinct intermediates in endocytic coated vesicle formation.

Amino Acid Substitution↗
PubMed2003

Dynamin regulates focal exocytosis in phagocytosing macrophages.

Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion ("focal" exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5'-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase*amphiphysin-SH3 domain, but not a mutated form that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis. Immunochemical analysis of endogenous dynamin distribution in macrophages revealed a substantial particulate pool, some of which localized to a presumptive endosomal compartment. Expression of enhanced green fluorescent protein*dynamin-2 showed a motile dynamin pool, a fraction of which migrated toward and within the phagosomal cup. These results suggest that dynamin is involved in the production and/or movement of vesicles from an intracellular organelle to the cell surface to support membrane expansion around the engulfed particle.

Actins↗
PubMed2005

Dynamin forms a Src kinase-sensitive complex with Cbl and regulates podosomes and osteoclast activity.

Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption.

Actins↗
PubMed2001

Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.

Binding Sites↗
PubMed2005

Regulatory mechanisms of dynamin-dependent endocytosis.

Extensive studies on endocytosis in the last decade have resulted in identification of several key molecules that function in clathrin- and dynamin-dependent endocytosis. Most endocytic molecules contain multiple binding motifs that mediate protein-protein or protein-lipid interactions, which must be modulated spatially and temporally during endocytosis. Regulation of these interactions is the molecular basis of regulatory mechanisms involved in endocytosis. This review first describes current models of the mechanism of dynamin-dependent fission, then introduces several mechanisms that modulate dynamin GTPase activity and dynamin-dependent vesicle formation. Such mechanisms include regulation by inositol phospholipids, especially phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], and their metabolism. It concludes by describing the regulation of dynamin 1 by its binding partner, amphiphysin 1, and regulation by cyclin-dependent kinase 5 (Cdk5)-dependent phosphorylation of dynamin 1 and amphiphysin 1. These mechanisms help endocytic molecules to function properly, and cooperatively regulate dynamin-dependent endocytosis.

Animals↗
PubMed2006

Increased association of dynamin II with myosin II in ras transformed NIH3T3 cells.

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin II has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin II and myosin II. The dynamin II expression level was higher when co-expressed with myosin II in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin II interacted with myosin II. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin II and myosin II gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

Animals↗
PubMed1998

Role of dynamin in the formation of transport vesicles from the trans-Golgi network.

Dynamin guanosine triphosphatases support the scission of clathrin-coated vesicles from the plasmalemma during endocytosis. By fluorescence microscopy of cultured rat hepatocytes, a green fluorescent protein-dynamin II fusion protein localized with clathrin-coated vesicles at the Golgi complex. A cell-free assay was utilized to demonstrate the role of dynamin in vesicle formation at the trans-Golgi. Addition of peptide-specific anti-dynamin antibodies to the assay mixture inhibited both constitutive exocytic and clathrin-coated vesicle formation. Immunodepletion of dynamin proteins also inhibited vesicle formation, and budding efficiency was restored upon readdition of purified dynamin. These data suggest that dynamin participates in the formation of distinct transport vesicles from the trans-Golgi network.

Adenosine Triphosphate↗
PubMed2005

Ionizing radiation-induced adenovirus infection is mediated by Dynamin 2.

Specific viral targeting into intrahepatic tumors remains critical for adenovirus gene therapy in liver cancer. We previously showed that ionizing radiation increases adenovirus uptake and transgene expression in cells and colon cancer xenografts. Here, we tested whether radiation induces viral uptake through virus-cell membrane interaction. We found that radiation (8 Gy) induced adenoviral gene transfer in rat hepatocytes (WB) and human colon carcinoma cells (LoVo). This induction (24.4- and 6.5-fold, respectively) and viral uptake were significantly diminished by preincubation with antibody for Dynamin 2 but not for Coxsackie adenovirus receptor or for integrin alpha(v). Radiation-induced Dynamin 2 expression was detected by immunohistochemical staining and by increased mRNA levels for Dynamin 2 in WB (1.5-fold) and LoVo (2.2-fold) cells. Specific small interference RNA (siRNA) transfection significantly inhibited Dynamin 2 expression in various tumor cell lines (LoVo, D54, and MCF-7) and abolished the radiation induction of Dynamin 2. Likewise, radiation-induced viral gene transfer in these cells (6.5-, 5.5-, and 9.0-fold, respectively) was significantly reduced in siRNA-transfected cells (2.7-, 3.7-, and 5.0-fold, respectively). Moreover, viral uptake in LoVo tumor xenografts was significantly increased in s.c. tumors (10.9-fold) when adenovirus was given i.v. at 24 hours after tumor irradiation, coincident with an elevated Dynamin 2 expression in irradiated tumors. These data suggest that ionizing radiation induces adenovirus gene transfer in cells and tumor xenografts by regulating viral uptake, potentially through interaction with cellular Dynamin 2 and thus should provide insight into improving adenovirus targeting in tumors.

Adenoviridae↗