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PubMed1996

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.

Amino Acid Sequence↗
PubMed1995

The regulation of endocytosis: identifying dynamin's binding partners.

Dynamin is a large GTPase implicated in controlling the initial stages of endocytosis. Its mechanism of action remains uncertain, but it is expected to interact cyclically with components of the endocytic machinery. Dynamin binds to a number of different macromolecules, including microtubules, SH3-domain-containing proteins, and acidic phospholipids. We interpret these findings in terms of a cooperative interaction between dynamin and components of coated and non-coated pits.

Journal Article↗
PubMed2006

The dynamin-like protein Vps1p of the yeast Saccharomyces cerevisiae associates with peroxisomes in a Pex19p-dependent manner.

Dynamins and dynamin-like proteins play important roles in organelle division. In Saccharomyces cerevisiae, the dynamin-like protein Vps1p (vacuolar protein sorting protein 1) is involved in peroxisome fission, as cells deleted for the VPS1 gene contain reduced numbers of enlarged peroxisomes. What relationship Vps1p has with peroxisomes remains unclear. Here we show that Vps1p interacts with Pex19p, a peroxin that acts as a shuttling receptor for peroxisomal membrane proteins or as a chaperone assisting the assembly/stabilization of proteins at the peroxisome membrane. Vps1p contains two putative Pex19p recognition sequences at amino acids 509-523 and 633-647. Deletion of the first (but not the second) sequence results in reduced numbers of enlarged peroxisomes in cells, as in vps1delta cells. Deletion of either sequence has no effect on vacuolar morphology or vacuolar protein sorting, suggesting that the peroxisome and vacuole biogenic functions of Vps1p are separate and separable. Substitution of proline for valine at position 516 of Vps1p abrogates Pex19p binding and gives the peroxisome phenotype of vps1delta cells. Microscopic analysis showed that overexpression of Pex19p or redirection of Pex19p to the nucleus does not affect the normal cellular distribution of Vps1p in the cytosol and in punctate structures that are not peroxisomes, suggesting that Pex19p does not function in targeting Vps1p to peroxisomes. Subcellular fractionation showed that a fraction of Vps1p is associated with peroxisomes and that deletion or mutation of the first Pex19p recognition sequence abrogates this association. Our results are consistent with Pex19p acting as a chaperone to stabilize the association of Vps1p with peroxisomes and not as a receptor involved in targeting Vps1p to peroxisomes.

Amino Acid Sequence↗
PubMed2001

Arabidopsis dynamin-like 2 that binds specifically to phosphatidylinositol 4-phosphate assembles into a high-molecular weight complex in vivo and in vitro.

Arabadopsis dynamin-like (ADL) 2, a member of the high-molecular weight (M(r)) dynamin family found in Arabidopsis, has been shown to be targeted to the plastid. In the chloroplast, most of the ADL2 was present in the fraction containing the envelope membranes when analyzed by suborganellar fractionation. Sucrose gradient and gel filtration experiments showed that when associated with membranes, ADL2 existed as a high-M(r) complex, whereas the soluble form existed as a monomer. The recombinant ADL2 expressed in Escherichia coli was present as a high-M(r) form and showed higher GTPase activity at a low NaCl concentration, whereas ADL2 existed as a low-M(r) form with a low level of GTPase activity at a high NaCl concentration. Electron microscopy studies revealed that the purified recombinant ADL2 formed spiral-coiled structures or rings. In the presence of guanosine-5'-O-(3-thio)triphosphate, these structures were transformed into a long rod structure. In contrast, in the presence of GDP, these structures disassembled into oligomers that were shown to be tetramer with 4-fold symmetry. Finally, a lipid-binding assay revealed that recombinant ADL2 purified from E. coli bound specifically to phosphatidylinositol 4-phosphate. Together, these results demonstrated that the biochemical properties of ADL2 were very similar to those of dynamin and other related proteins. Based on this similarity, we propose that ADL2 may be involved in vesicle formation at the chloroplast envelope membrane.

Arabidopsis↗
PubMed2000

Mu-opioid agonist inhibition of kappa-opioid receptor-stimulated extracellular signal-regulated kinase phosphorylation is dynamin-dependent in C6 glioma cells.

In previous studies we found that mu-opioids, acting via mu-opioid receptors, inhibit endothelin-stimulated C6 glioma cell growth. In the preceding article we show that the kappa-selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with mu-opioid agonists for 1 h results in the inhibition of kappa-opioid mitogenic signaling. The mu-selective agonist endomorphin-1 attenuates kappa-opioid-stimulated DNA synthesis, phosphoinositide turnover, and extracellular signal-regulated kinase phosphorylation. To investigate the role of receptor endocytosis in signaling, we have examined the effects of dynamin-1 and its GTPase-defective, dominant suppressor mutant (K44A) on opioid modulation of extracellular signal-regulated kinase phosphorylation in C6 cells. Overexpression of dynamin K44A in C6 cells does not affect kappa-opioid phosphorylation of extracellular signal-regulated kinase. However, it does block the inhibitory action on kappa-opioid signaling mediated by the kappa-opioid receptor. Our results are consistent with a growing body of evidence of the opposing actions of mu- and kappa-opioids and provide new insight into the role of opioid receptor trafficking in signaling.

Animals↗
PubMed2002

Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin-sensitive step and interaction with the AP-2 adaptor complex.

The pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP-VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I-K44A impaired GFP-VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I-K44A generated vesicles. A fusion protein containing the VAChT C-terminal tail precipitated the AP-2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C-terminal tail of VAChT interacts with the micro subunit of AP-2 in a di-leucine (L485A/L486A) dependent fashion. These observations suggest that the di-leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.

Adaptor Proteins, Vesicular Transport↗
PubMed1998

The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast.

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.

Actins↗
PubMed2000

Evidence that dynamin-2 functions as a signal-transducing GTPase.

The role of dynamin GTPases in the regulation of receptor-mediated endocytosis is well established. Here, we present new evidence that the ubiquitously expressed isoform dynamin-2 (dyn2) can also function in a signal transduction pathway(s). A </=5-fold increase of dyn2 relative to endogenous levels activates the transcription factor p53 and induces apoptosis, as demonstrated by reduced cell proliferation, DNA fragmentation, and caspase-3 activation. Dyn2-triggered apoptosis occurs only in dividing cells and is p53 dependent. A mutant defective in GTP binding does not trigger apoptosis, indicating that increased levels of dyn2.GTP, rather than protein levels per se, are required to transduce signals that activate p53. A truncated dyn2 lacking the COOH-terminal proline/arginine-rich domain (PRD), which interacts with many SH3 domain-containing partners implicated in both endocytosis and signal transduction, triggers apoptosis even more potently than the wild-type. This observation provides additional support for the importance of the NH(2)-terminal GTPase domain for the apoptotic phenotype. All described effects are dyn2-specific because >200-fold overexpression of dyn1, the 70% identical neuronal isoform, has no effect. Our data suggest that dyn2 can act as a signal transducing GTPase affecting transcriptional regulation.

Adenoviridae↗
PubMed2003

Analysis of post-translational modification and characterization of the domain structure of dynamin A from Dictyostelium discoideum.

The post-translational modifications of the 96 kDa protein dynamin A from Dictyostelium discoideum were analyzed using Q-TOF mass spectrometry. The accurate molecular mass of the intact protein revealed a covalent modification causing an additional mass of 42 Da. The modification could be identified as N-terminal acetylation by tandem mass spectrometry. Extracted ion chromatograms for the a(1) and b(1) ion of the tryptic T1 peptide were used to detect the acetylated peptide within 54 nanoelectrospray ionization tandem mass spectra. Owing to the accurate molecular mass of the intact protein, additional covalent modifications could be excluded. In addition to the covalent modification, the domain structure of dynamin A was determined by applying a combination of limited proteolysis, sodium dodecylsulfate polyacrylamide gel electrophoresis, automated tandem mass spectrometry and protein database searching.

Acetylation↗
PubMed1998

Human dynamin-like protein interacts with the glycogen synthase kinase 3beta.

Members of the dynamin superfamily are implicated in vesicle trafficking. Using human glycogen synthase kinase 3 beta (Gsk-3 beta) as bait in the yeast two-hybrid system, we identified a novel human dynamin-like protein IV (HdynIV). When the full-length cDNA of HdynIV was sequenced, it showed that HdynIV's carboxyl terminal lacks a proline-rich domain that can bind to Gsk-3 beta. By Northern blot analysis and isoform-specific PCR, we found that HdynIV is expressed ubiquitously in all human tissues examined. Two transcripts of 2.4 and 4.4 kb are shown to be more abundant in heart, brain, and skeletal muscle. Interestingly, the 2.4-kb transcript is expressed more distinctly in the fetal liver than in the adult liver, suggesting that this protein might play a role during development. In the present report, we have demonstrated that HdynIV interacts with the Gsk-3 beta through its carboxyl-terminal region, implying than HdynIV may also be involved in cell signaling.

Adult↗
PubMed1997

Requirement for dynamin during Notch signaling in Drosophila neurogenesis.

Singling out of a unique neural precursor from a group of equivalent cells, during Drosophila neurogenesis, involves Notch-mediated lateral signaling. During this process, activation of the Notch signaling pathway leads to repression of neural development. Disruption of this signaling pathway results in the development of an excess of neural cells. The loss of activity of dynamin, which is encoded by the gene shibire and is required for endocytosis, results in a similar phenotype. Here we have investigated the requirement of shibire function for Notch signaling during the segregation of sensory bristles on the notum of the fly. Overexpression of different constitutively active forms of Notch in shibire mutant flies indicates that shibire function is not necessary for transduction of the signal downstream of Notch, even when the receptor is integrated in the plasma membrane. However, when wild-type Notch is activated by its ligand Delta, dynamin is required in both signaling and receiving cells for normal singling out of precursors. This suggests an active role of the signaling cell for ligand-mediated receptor endocytosis in the case of transmembrane ligands. We discuss the possible implications of these results for normal functioning of Notch-mediated lateral signaling.

Animals↗
PubMed1997

The main-chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: analysis of 15N relaxation with monomer/dimer equilibration.

The backbone dynamics of the pleckstrin homology (PH) domain from dynamin were studied by 15N NMR relaxation (R1 and R2) and steady state heteronuclear 15N [1H] nuclear Overhauser effect measurements at 500 and 600 MHz, at protein concentrations of 1.7 mM and 300 microM, and by molecular dynamics (MD) simulations. The analysis was performed using the model-free approach. The method was extended in order to account for observed partial (equilibrium) dimerization of the protein at NMR concentrations. A model is developed that takes into account both rapid monomer-dimer exchange and anisotropy of the over-all rotation of the dimer. The data show complex dynamics of the dynamin PH domain. Internal motions in elements of the secondary structure are restricted, as inferred from the high value of the order parameter (S2 approximately 0.9) and from the local correlation time < 100 ps. Of the four extended loop regions that are disordered in the NMR-derived solution structure of the protein, loops beta 1/beta 2 and beta 5/beta 6 are involved in a large-amplitude (S2 down to 0.2 to 0.3) subnanosecond to nanosecond time-scale motion. Reorientation of the loops beta 3/beta 4 and beta 6/beta 7, in contrast, is restricted, characterized by the values of order parameter S2 approximately 0.9 more typical of the protein core. These loops, however, are involved in much slower processes of motion resulting in a conformational exchange on a microsecond to submillisecond time scale. The motions of the terminal regions (residues 1 to 10, 122 to 125) are practically unrestricted (S2 down to 0.05, characteristic times in nanosecond time scale), suggesting that these parts of the sequence do not participate in the protein fold. The analysis shows a larger sensitivity of the 15N relaxation data to protein microdynamic parameters (S2, tau loc) when protein molecular mass (tau c) increases. The use of negative values of the steady state 15N[1H] NOEs as an indicator of the residues not belonging to the folded structure is suggested. The amplitudes of local motion observed in the MD simulation are in a good-agreement with the NMR data for the amide NH groups located in the protein core.

Blood Proteins↗
PubMedDate not supplied

Normal mitochondrial structure and genome maintenance in yeast requires the dynamin-like product of the MGM1 gene.

The isolation and characterization of MGM1, an yeast gene with homology to members of the dynamin gene family, is described. The MGM1 gene is located on the right arm of chromosome XV between STE4 and PTP2. Sequence analysis revealed a single open reading frame of 902 residues capable of encoding a protein with an approximate molecular mass of 101 kDa. Loss of MGM1 resulted in slow growth on rich medium, failure to grow on non-fermentable carbon sources, and loss of mitochondrial DNA. The mitochondria also appeared abnormal when visualized with an antibody to a mitochondrial-matrix marker. MGM1 encodes a dynamin-like protein involved in the propagation of functional mitochondria in yeast.

Base Sequence↗
PubMed2003

Modulation of Fas receptor proteins and dynamin during opiate addiction and induction of opiate withdrawal in rat brain.

The Fas receptor is involved in the regulation of apoptosis but also can function as a non-apoptotic signal transducer. This study was mainly designed to quantitate Fas proteins in rat brain during heroin addiction and opiate withdrawal. In rat, mouse and human brains, and in SH-SY5Y cells, similar forms of Fas were immunodetected with different antibodies (i.e., 35 kDa native Fas and 48- and 51-kDa glycosylated Fas). Acute (2 h) treatments with the micro-opioid receptor agonists heroin (10 mg/kg) and morphine (30 mg/kg) increased the immunodensity of native Fas (124% and 36%) but not that of glycosylated Fas in the cerebral cortex. Chronic (5 days) heroin (5-30 mg/kg) and morphine (10-100 mg/kg) were also associated with increased native Fas (76% and 45%) and with different expressions of glycosylated Fas. In heroin-dependent rats, opiate withdrawal (48 h) resulted in a sustained increase in native Fas (107%) and in up-regulation of 51 kDa glycosylated Fas (51%). Acute treatments with selective delta-receptor (SNC-80, 10 mg/kg) or kappa-receptor (U 50488-H, 10 mg/kg) agonists did not alter the content of native or glycosylated Fas. Chronic pentazocine (10-80 mg/kg, 5 days), a mixed opiate drug and sigma(1) receptor agonist, decreased native (48%) and glycosylated (38-82%) Fas proteins. Similarly, the selective sigma(1) agonist (+)-SKF 10047 also decreased native Fas (37%) and the effect was blocked by the sigma(1) antagonist BD 1063. Brain dynamin was up-regulated by acute and/or chronic heroin (30-39%), morphine (47-85%), pentazocine (51%) and heroin withdrawal (74%). The main results indicate that chronic heroin/morphine treatment and heroin withdrawal are associated with up-regulation of 35 kDa native Fas (and with different expressions of glycosylated Fas), and also with concomitant increases of dynamin in rat brain.

Animals↗
PubMed1995

The function of dynamin in endocytosis.

Temperature-sensitive shibire mutants of Drosophila melanogaster become rapidly paralyzed upon a shift to the restrictive temperature, which is due to a block in synaptic vesicle endocytosis. The shibire gene encodes the GTPase dynamin. Recent studies have shown that dynamin forms rings at the neck of invaginated clathrin-coated pits, and have suggested that a conformational change in the ring, which correlates with GTP hydrolysis, plays an essential role in vesicle fission.

Animals↗
PubMed2004

Drosophila Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans.

The Drosophila transforming growth factor beta (TGF-beta) homolog Decapentaplegic (Dpp) acts as a morphogen that forms a long-range concentration gradient to direct the anteroposterior patterning of the wing. Both planar transcytosis initiated by Dynamin-mediated endocytosis and extracellular diffusion have been proposed for Dpp movement across cells. In this work, we found that Dpp is mainly extracellular, and its extracellular gradient coincides with its activity gradient. We demonstrate that a blockage of endocytosis by the dynamin mutant shibire does not block Dpp movement but rather inhibits Dpp signal transduction, suggesting that endocytosis is not essential for Dpp movement but is involved in Dpp signaling. Furthermore, we show that Dpp fails to move across cells mutant for dally and dally-like (dly), two Drosophila glypican members of heparin sulfate proteoglycan (HSPG). Our results support a model in which Dpp moves along the cell surface by restricted extracellular diffusion involving the glypicans Dally and Dly.

Animals↗
PubMed2004

SNARE filtering by dynamin.

Fission and fusion are the two elementary steps of membrane traffic. The mechanoenzyme dynamin acts at the fission step, but, in this issue of Cell, suggest an additional role of dynamin in preparing membranes for fusion.

Adenosine Triphosphate↗
PubMed2005

Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins.

Cell membranes undergo continuous curvature changes as a result of membrane trafficking and cell motility. Deformations are achieved both by forces extrinsic to the membrane as well as by structural modifications in the bilayer or at the bilayer surface that favor the acquisition of curvature. We report here that a family of proteins previously implicated in the regulation of the actin cytoskeleton also have powerful lipid bilayer-deforming properties via an N-terminal module (F-BAR) similar to the BAR domain. Several such proteins, like a subset of BAR domain proteins, bind to dynamin, a GTPase implicated in endocytosis and actin dynamics, via SH3 domains. The ability of BAR and F-BAR domain proteins to induce tubular invaginations of the plasma membrane is enhanced by disruption of the actin cytoskeleton and is antagonized by dynamin. These results suggest a close interplay between the mechanisms that control actin dynamics and those that mediate plasma membrane invagination and fission.

Actins↗