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PubMed1998

Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p.

Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynamin-related GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis-Golgi GTPase Rab1A, but not to the staining for the trans-Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1(K38E)) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.

Animals↗
PubMed2005

Elucidation of clathrin-mediated endocytosis in tetrahymena reveals an evolutionarily convergent recruitment of dynamin.

Ciliates, although single-celled organisms, contain numerous subcellular structures and pathways usually associated with metazoans. How this cell biological complexity relates to the evolution of molecular elements is unclear, because features in these cells have been defined mainly at the morphological level. Among these ciliate features are structures resembling clathrin-coated, endocytic pits associated with plasma membrane invaginations called parasomal sacs. The combination of genome-wide sequencing in Tetrahymena thermophila with tools for gene expression and replacement has allowed us to examine this pathway in detail. Here we demonstrate that parasomal sacs are sites of clathrin-dependent endocytosis and that AP-2 localizes to these sites. Unexpectedly, endocytosis in Tetrahymena also involves a protein in the dynamin family, Drp1p (Dynamin-related protein 1). While phylogenetic analysis of AP subunits indicates a primitive origin for clathrin-mediated endocytosis, similar analysis of dynamin-related proteins suggests, strikingly, that the recruitment of dynamin-family proteins to the endocytic pathway occurred independently during the course of the ciliate and metazoan radiations. Consistent with this, our functional analysis suggests that the precise roles of dynamins in endocytosis, as well as the mechanisms of targeting, differ in metazoans and ciliates.

Animals↗
PubMed1996

Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.

Presynaptic terminals contain several specialized compartments, which have been described by electron microscopy. We show in an identified Drosophila neuromuscular synapse that several of these compartments-synaptic vesicle clusters, presynaptic plasma membrane, presynaptic cytosol, and axonal cytoskeleton-labeled by specific reagents may be resolved from one another by laser scanning confocal microscopy. Using a panel of compartment-specific markers and Drosophila shibire(ts1) mutants to trap an intermediate stage in synaptic vesicle recycling, we have examined the localization and redistribution of dynamin within single synaptic varicosities at the larval neuromuscular junction. Our results suggest that dynamin is not a freely diffusible molecule in resting nerve terminals; rather, it appears localized to synaptic sites by association with yet uncharacterized presynaptic components. In shi(ts1) nerve terminals depleted of synaptic vesicles, dynamin is quantitatively redistributed to the plasma membrane. It is not, however, distributed uniformly over presynaptic plasmalemma; instead, fluorescence images show "hot spots" of dynamin on the plasma membrane of vesicle-depleted nerve terminals. We suggest that these dynamin-rich domains may mark the active zones for synaptic vesicle endocytosis first described at the frog neuromuscular junction.

Animals↗
PubMed1997

Identification and subcellular localization of a novel mammalian dynamin-related protein homologous to yeast Vps1p and Dnm1p.

The dynamin family of GTP-binding proteins are implicated in vesicular transport. These include mammalian dynamins I, II, III, and yeast Vps1p and Dnm1p. Dynamin is involved in the formation of clathrin-coated vesicles at the plasma membrane. On the other hand, Vps1p and Dnm1p appear to be involved in transport from the late Golgi compartment to vacuoles and in an endocytic process, respectively. In this study, we identified a novel human protein, named Dnm1p/Vps1p-like protein (DVLP). It resembled more closely Dnm1p and Vps1p than dynamins not only in the primary structure but also in the domain organization. DVLP mRNA was expressed ubiquitously, suggesting that this protein plays a fundamental role in cellular function. Immunofluorescence analysis of cells expressing epitope-tagged DVLP revealed that it showed a diffused perinuclear staining pattern that was not superimposed on that of the marker protein for the Golgi apparatus, trans-Golgi network, lysosomes, endosomes, or endoplasmic reticulum. These data suggest that DVLP is not involved in the formation of known coated vesicles.

Amino Acid Sequence↗
PubMed2003

Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA.

To investigate the role of clathrin in coated vesicle formation, a cell line with inducible expression of clathrin heavy chain (CHC) antisense RNA was produced. After 18 h of CHC antisense RNA expression, the internalization of transferrin was inhibited by 90%. Although the amount of CHC was reduced by only 10%, the frequency of clathrin-coated pits at the cell surface increased by a factor of 3-5, and clathrin-coated structures also accumulated on a pleiomorphic, multivesicular, endosomal compartment. Remarkably, the coated pits were connected to the cell surface by long, tubular necks wrapped by dynamin rings, and the level of dynamin in the CHC antisense RNA-expressing cells was up-regulated 10-fold. In contrast, the amount of several other proteins associated with clathrin coat formation was unaffected. Thus, this study demonstrates that CHC antisense RNA causes accumulation of clathrin-coated pits with dynamin rings around the neck in intact cells not transfected with dynamin mutants, suggesting the existence of a previously uncharacterized functional interplay between clathrin and dynamin.

Animals↗
PubMed2003

Dynamin 3 is a component of the postsynapse, where it interacts with mGluR5 and Homer.

The dynamins comprise a large family of mechanoenzymes known to participate in membrane modeling events. All three conventional dynamin genes (Dyn1, Dyn2, Dyn3) are expressed in mammalian brain and produce more than 27 different dynamin proteins as a result of alternative splicing. Past studies have suggested that Dyn1 participates in specialized neuronal functions such as rapid synaptic vesicle recycling, while Dyn2 may mediate the conventional clathrin-mediated uptake of surface receptors. Currently, the distribution, expression, and function of Dyn3 in neurons, or in any other cell type, are completely undefined. Here, we demonstrate that Dyn1 and Dyn3 localize differentially in the synapse. Dyn1 concentrates within the presynaptic compartment, while Dyn3 localizes to dendritic spine tips. Within the postsynaptic density (PSD), we found Dyn3, but not Dyn1, to be part of a biochemically isolated complex comprised of Homer and metabotropic glutamate receptors. Finally, although dominant-negative Dyn3 did not seem to inhibit receptor endocytosis, overexpression of a specific Dyn3 spliced variant in mature neurons caused a marked remodeling of dendritic spines. These data suggest that Dyn3 is a postsynaptic dynamin and, like its binding partner Homer, plays a significant role in dendritic spine morphogenesis and remodeling.

Amino Acid Sequence↗
PubMed2001

Agonist-promoted trafficking of human bradykinin receptors: arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells.

In this study, we analysed the agonist-promoted trafficking of human B(2) (B(2)R) and B(1) (B(1)R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B(2)R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B(2)R using radioligand binding and by imaging of B(2)R-GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B(1)R was sequestered to a considerably lesser extent upon binding of des-Arg(10)-kallidin. B(2)R sequestration was rapid (half-life approximately 5 min) and reached a steady-state level that was significantly lower than that of BK sequestration. B(2)R sequestration was minimally inhibited by K44A dynamin (22.4+/-3.7%), and was insensitive to arrestin-(319-418), which are dominant-negative mutants of dynamin I and beta-arrestin respectively. Furthermore, the B(2)R-mediated sequestration of BK was completely insensitive to both mutants, as was the association of BK with a caveolae-enriched fraction of the cells. On the other hand, agonist-promoted sequestration of the beta(2)-adrenergic receptor was dramatically inhibited by K44A dynamin (81.2+/-16.3%) and by arrestin-(319-418) (36.9+/-4.4%). Our results show that B(2)R is sequestered to a significantly greater extent than is B(1)R upon agonist treatment in HEK293 cells. Furthermore, B(2)R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and beta-arrestin-independent manner and, at least in part, involves caveolae.

Arrestins↗
PubMed2001

Protein phosphorylation is required for endocytosis in nerve terminals: potential role for the dephosphins dynamin I and synaptojanin, but not AP180 or amphiphysin.

Dynamin I and at least five other nerve terminal proteins, amphiphysins I and II, synaptojanin, epsin and eps15 (collectively called dephosphins), are coordinately dephosphorylated by calcineurin during endocytosis of synaptic vesicles. Here we have identified a new dephosphin, the essential endocytic protein AP180. Blocking dephosphorylation of the dephosphins is known to inhibit endocytosis, but the role of phosphorylation has not been determined. We show that the protein kinase C (PKC) antagonists Ro 31-8220 and Go 7874 block the rephosphorylation of dynamin I and synaptojanin that occurs during recovery from an initial depolarizing stimulus (S1). The rephosphorylation of AP180 and amphiphysins 1 and 2, however, were unaffected by Ro 31-8220. Although these dephosphins share a single phosphatase, different protein kinases phosphorylated them after nerve terminal stimulation. The inhibitors were used to selectively examine the role of dynamin I and/or synaptojanin phosphorylation in endocytosis. Ro 31-8220 and Go 7874 did not block the initial S1 cycle of endocytosis, but strongly inhibited endocytosis following a second stimulus (S2). Therefore, phosphorylation of a subset of dephosphins, which includes dynamin I and synaptojanin, is required for the next round of stimulated synaptic vesicle retrieval.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

U50,488H-induced internalization of the human kappa opioid receptor involves a beta-arrestin- and dynamin-dependent mechanism. Kappa receptor internalization is not required for mitogen-activated protein kinase activation.

Agonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat kappa opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human kappa receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat kappa opioid receptor. U50, 488H-induced human kappa receptor internalization was time- and concentration-dependent, with 30-40% of the receptors internalized following a 30-min exposure to 1 microM U50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50, 488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50, 488H-induced internalization. In contrast, incubation with sucrose (0.4-0.8 M) significantly reduced U50,488H-induced internalization of the kappa receptor. While co-expression of the wild type GRK2, beta-arrestin, or dynamin I had no effect on kappa receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, beta-arrestin (319-418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of beta-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human kappa receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat kappa receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human kappa opioid receptor in Chinese hamster ovary cells occurs via a GRK-, beta-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the kappa receptor is not required for activation of MAP kinase.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1999

The calcineurin-dynamin 1 complex as a calcium sensor for synaptic vesicle endocytosis.

Exocytosis of synaptic vesicles is calcium-dependent, with synaptotagmin serving as the calcium sensor. Endocytosis of synaptic vesicles has also been postulated as a calcium-dependent process; however, an endocytic calcium sensor has not been found. We now report a physical association between the calcium-dependent phosphatase calcineurin and dynamin 1, a component of the synaptic endocytic machinery. The calcineurin-dynamin 1 interaction is calcium-dependent, with an EC(50) for calcium in the range of 0.1-0. 4 microM. Disruption of the calcineurin-dynamin 1 interaction inhibits clathrin-mediated endocytosis. Thus, the calcium-dependent formation of the calcineurin-dynamin 1 complex, delivered to the other endocytic coat proteins, provides a calcium-sensing mechanism that facilitates endocytosis.

Animals↗
PubMed2002

Interactions of phocein with nucleoside-diphosphate kinase, Eps15, and Dynamin I.

Phocein, an intracellular protein interacting with striatin, bears a few homologies with the sigma-subunits of clathrin adaptor proteins (Baillat, G., Moqrich, A., Castets, F., Baude, A., Bailly, Y., Benmerah, A., and Monneron, A. (2001) Mol. Biol. Cell 12, 663-673). Using phocein as a bait in a yeast two-hybrid screen, we identified two novel interacting proteins, nucleoside-diphosphate kinase (NDPK) and Eps15. Immunoprecipitation and pull-down experiments involving native and/or recombinant phocein and, respectively, NDPK and Eps15, biochemically validated their interactions. NDPK and Eps15 were recently shown to be functional neighbors of dynamin. Dynamin I is shown here to directly interact with NDPK through its C-terminal proline-rich domain, whereas recombinant phocein associates with native dynamin I. Immunocytochemical studies of rat embryonic hippocampal neurons demonstrated partial co-localization of phocein and dynamin I. Phocein thus appears to be a component of the complexes involved in some steps of the vesicular traffic machinery.

Animals↗
PubMed1998

A human dynamin-related protein controls the distribution of mitochondria.

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.

Animals↗
PubMed2001

A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis.

Dynamin, a high-molecular-weight GTPase, plays a critical role in vesicle formation at the plasma membrane during endocytosis in animal cells. Here we report the identification of a new dynamin homolog in Arabidopsis named Arabidopsis dynamin-like 6 (ADL6). ADL6 is quite similar to dynamin I in its structural organization: a conserved GTPase domain at the N terminus, a pleckstrin homology domain at the center, and a Pro-rich motif at the C terminus. In the cell, a majority of ADL6 is associated with membranes. Immunohistochemistry and in vivo targeting experiments revealed that ADL6 is localized to the Golgi apparatus. Expression of the dominant negative mutant ADL6[K51E] in Arabidopsis protoplasts inhibited trafficking of cargo proteins destined for the lytic vacuole and caused them to accumulate at the trans-Golgi network. In contrast, expression of ADL6[K51E] did not affect trafficking of a cargo protein, H(+)-ATPase:green fluorescent protein, destined for the plasma membrane. These results suggest that ADL6 is involved in vesicle formation for vacuolar trafficking at the trans-Golgi network but not for trafficking to the plasma membrane in plant cells.

Amino Acid Sequence↗
PubMed1999

Dynamin is required for recombinant adeno-associated virus type 2 infection.

Recombinant adeno-associated virus (rAAV) vectors for gene therapy of inherited disorders have demonstrated considerable potential for molecular medicine. Recent identification of the viral receptor and coreceptors for AAV type 2 (AAV-2) has begun to explain why certain organs may demonstrate higher efficiencies of gene transfer with this vector. However, the mechanisms by which AAV-2 enters cells remain unknown. In the present report, we have examined whether the endocytic pathways of rAAV-2 are dependent on dynamin, a GTPase protein involved in clathrin-mediated internalization of receptors and their ligands from the plasma membrane. Using a recombinant adenovirus expressing a dominant-inhibitory form of dynamin I (K44A), we have demonstrated that rAAV-2 infection is partially dependent on dynamin function. Overexpression of mutant dynamin I significantly inhibited AAV-2 internalization and gene delivery, but not viral binding. Furthermore, colocalization of rAAV and transferrin in the same endosomal compartment provides additional evidence that clathrin-coated pits are the predominant pathway for endocytosis of AAV-2 in HeLa cells.

Cell Line, Transformed↗
PubMed2000

Characterization of the mouse dynamin I gene promoter and identification of sequences that direct expression in neuronal cells.

Dynamin I is expressed at high levels in brain and its expression is regulated during the developmental stages of brain. To elucidate the molecular mechanism by which the expression is tissue-specifically regulated, we cloned the 5'-flanking region of the mouse dynamin I gene and determined the nucleotide sequence of 1036 bases upstream from the translation start site. Transient transfection studies with a chloramphenicol acetyltransferase reporter gene in neuroblastoma NS20Y and Lewis lung cells demonstrated that the 5'-flanking region has a cell-type-specific promoter activity. Deletion analyses demonstrated that the minimal promoter activity was detected in the proximal region 195 bp upstream of the translation initiation codon (-90 to +105). The minimal promoter was embedded in a GC-rich region (75% GC content), in which an Sp1-binding motif and a nuclear factor (NF)-kappa B-like element (NE-1) were found, but it lacked TATA and CAAT boxes. Mutational analysis and electrophoretic mobility-shift assay analysis revealed that Sp1 binds to the Sp1 site and that this element is critical for the promoter activity of the dynamin I gene. We found that the NE-1 sequence is required for the expression of the dynamin I gene but NEBP (NE-1-binding protein), which binds to the NE-1 sequence, is not NF-kappa B. We also found that one base in the NE-1 sequence (the underlined G residue in GGGATTCGCGGA) is critical for binding specificity to discriminate between NEBP and NF-kappa B. By UV cross-linking analysis, we found that NEBP is an approx. 104 kDa nuclear protein.

Animals↗
PubMed1993

Sea urchin egg 100-kDa dynamin-related protein: identification of and localization to intracellular vesicles.

We have initiated a study to identify possible tubulin dimer binding proteins from unfertilized sea urchin eggs utilizing tubulin dimer coupled to Sepharose 4B. A 100-kDa polypeptide has been isolated and its interaction with tubulin/microtubules examined. Solution assembly studies indicated the 100-kDa polypeptide potentiated microtubule assembly. The 100-kDa protein also bundled and cosedimented with polymerized microtubules. Rabbit polyclonal anti-egg 100-kDa antibodies cross-reacted with a 100-kDa polypeptide in the eggs of several different sea urchin species and with mammalian brain dynamin, a 100-kDa microtubule binding protein. These biochemical characterizations and immunological results suggest that the sea urchin 100-kDa microtubule binding protein appears to be related to the dynamin protein family. To gain insight into the functional role of sea urchin egg 100-kDa dynamin-related protein, we immunolocalized egg 100-kDa protein in cryosections and isolated cortices of unfertilized eggs. Egg 100-kDa protein was localized to the monolayer of cortical granules that underlie the plasma membrane and to the inner cytoplasm. Immunolocalizations of egg 100-kDa protein in cortices isolated from fertilized eggs detailed the redistribution of the egg 100-kDa onto cortically positioned vesicles. Egg 100-kDa dynamin-related protein was expressed throughout early development with approximately equivalent amounts present in the unfertilized egg and embryo through early gastrulation, followed by an abrupt decrease in the level of expression by the pluteus larval stage.

Animals↗
PubMed1998

Expression of mutant dynamin protects cells against diphtheria toxin but not against ricin.

Diphtheria toxin is believed to enter sensitive mammalian cells via receptor-mediated endocytosis from clathrin-coated pits, while ricin can enter via both clathrin-dependent and clathrin-independent endocytosis. The present study has confirmed this by determining the toxin sensitivity of COS-7y cells which were transiently overexpressing a trans dominant negative mutant of dynamin, a GTPase required for the budding of clathrin-coated vesicles from the plasma membrane. Cells overexpressing wild-type dynamin showed normal receptor-mediated endocytosis of transferrin and remained sensitive to both diphtheria toxin and ricin. Cells overexpressing a mutant dynamin defective in GTP binding and hydrolysis were unable to endocytose transferrin and were protected against diphtheria toxin, but they remained completely sensitive to ricin intoxication. Treating non-transfected cells or cells overexpressing mutant dynamin with nystatin caused a redistribution of the caveolae membrane marker protein VIP21-caveolin from the cell surface to intracellular locations, but did not affect their sensitivity to ricin. The redistribution of caveolin seen after nystatin treatment may reflect the disappearance of caveolae. If this is the case, caveolae are not responsible for the endocytosis of ricin. An alternative clathrin-independent route may operate for ricin, since cellular uptake, intracellular transport, and translocation into the cytosol remain unaffected when clathrin-dependent endocytosis is effectively blocked.

Animals↗
PubMed1991

Predominant and developmentally regulated expression of dynamin in neurons.

We have cloned a cDNA for dynamin, a 100 kd microtubule-associated motor protein whose 5' region contains a GTP-binding motif homologous to that of the Mx proteins, from a rat brain library and analyzed its expression. Dynamin mRNA is 3.6 kb and is preferentially expressed in the brain after postnatal day 7, parallel to the developmental increase of the protein. In situ hybridization revealed high levels of dynamin transcripts in neural cells in the cerebellar cortex, hippocampus (particularly in the CA3 area), and cerebral cortex. The transcripts appeared in cerebellar granular cells only after they had ceased dividing and had migrated to the inner granular layer. We show that dynamin is expressed predominantly in neural cells after elongation of their processes, suggesting a role especially in mature neurons.

Adrenal Gland Neoplasms↗