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Annual Review of Cell and Developmental... 2000Dynamin, a 100-kDa GTPase, is an essential component of vesicle formation in receptor-mediated endocytosis, synaptic vesicle recycling, caveolae internalization, and... (Review)
Review
Dynamin, a 100-kDa GTPase, is an essential component of vesicle formation in receptor-mediated endocytosis, synaptic vesicle recycling, caveolae internalization, and possibly vesicle trafficking in and out of the Golgi. In addition to the GTPase domain, dynamin also contains a pleckstrin homology domain (PH) implicated in membrane binding, a GTPase effector domain (GED) shown to be essential for self-assembly and stimulated GTPase activity, and a C-terminal proline-rich domain (PRD), which contains several SH3-binding sites. Dynamin partners bind to the PRD and may either stimulate dynamin's GTPase activity or target dynamin to the plasma membrane. Purified dynamin readily self-assembles into rings or spirals. This striking structural property supports the hypothesis that dynamin wraps around the necks of budding vesicles where it plays a key role in membrane fission. The focus of this review is on the relationship between the GTPase and self-assembly properties of dynamin and its cellular function.
Topics: Animals; Dynamins; GTP Phosphohydrolases; Humans; Membrane Fusion; Phosphorylation; Protein Structure, Tertiary
PubMed: 11031245
DOI: 10.1146/annurev.cellbio.16.1.483 -
The Journal of Biological Chemistry Sep 1996GTP hydrolysis by dynamin is required to drive coated vesicle budding at the plasma membrane. A diverse set of molecules including microtubules, grb2, and acidic...
GTP hydrolysis by dynamin is required to drive coated vesicle budding at the plasma membrane. A diverse set of molecules including microtubules, grb2, and acidic phospholipids stimulate dynamin GTPase activity in vitro, although the physiological relevance of these effectors remains to be determined. Dynamin has been shown to assemble around microtubules, the most potent stimulatory molecule, into structures indistinguishable by electron microscopy from collars captured in vivo at the necks of endocytic coated pits. Under low ionic strength conditions purified dynamin self-assembles into rings and helical stacks of rings. Here we show that dynamin self-assembly stimulates its GTPase activity as much as 10-fold. Thus, we identify dynamin, itself, as the first effector of dynamin GTPase activity known to be physiologically relevant. Assembled dynamin's stimulated GTPase activity is not dependent on the direct interaction of high affinity GTP binding sites since a mutant defective in GTP binding and hydrolysis can coassemble with and stimulate GTP hydrolysis by wild-type dynamin. Finally, we find that GTP destabilizes assembled dynamin structures, suggesting that the activated rates of GTP hydrolysis reflect a continuing cycle of assembly, GTP hydrolysis, and disassembly.
Topics: Cell Line; Dynamins; Electrophoresis, Polyacrylamide Gel; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hydrogen-Ion Concentration; Hydrolysis; Microtubules; Protein Conformation; Substrate Specificity
PubMed: 8798389
DOI: 10.1074/jbc.271.37.22310 -
Biomedicine & Pharmacotherapy =... Sep 2023Mitochondrial dysfunction, especially in terms of mitochondrial dynamics, has been reported to be closely associated with neuronal outcomes and neurological impairment... (Review)
Review
Mitochondrial dysfunction, especially in terms of mitochondrial dynamics, has been reported to be closely associated with neuronal outcomes and neurological impairment in cerebral ischemia/hypoxia injury. Dynamin-related protein 1 (Drp1) is a cytoplasmic GTPase that mediates mitochondrial fission and participates in neuronal cell death, calcium signaling, and oxidative stress. The neuroprotective role of Drp1 inhibition has been confirmed in several central nervous system disease models, demonstrating that targeting Drp1 may shed light on novel approaches for the treatment of cerebral ischemia/hypoxia injury. In this review, we aimed to highlight the roles of Drp1 in programmed cell death, oxidative stress, mitophagy, and mitochondrial function to provide a better understanding of mitochondrial disturbances in cerebral ischemia/hypoxia injury, and we also summarize the advances in novel chemical compounds targeting Drp1 to provide new insights into potential therapies for cerebral ischemia/hypoxia injury.
Topics: Humans; Brain Ischemia; Dynamins; Hypoxia; Mitochondria; Neurons; Animals
PubMed: 37516018
DOI: 10.1016/j.biopha.2023.115247 -
Traffic (Copenhagen, Denmark) Aug 2014Dynamin is a 96-kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin...
Dynamin is a 96-kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3-domain-containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP- and actin-dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin-actin interactions and dynamin's GTPase activity in the regulation of dynamin oligomerization in cells.
Topics: Actins; Amino Acid Sequence; Animals; COS Cells; Chlorocebus aethiops; Dynamins; Guanosine Triphosphate; Mice; Molecular Sequence Data; Protein Binding; Protein Multimerization; Protein Structure, Tertiary
PubMed: 24891099
DOI: 10.1111/tra.12178 -
Methods in Molecular Biology (Clifton,... 2022This protocol describes the chemical synthesis of the dynamin inhibitors Dynole 34-2 and Acrylo-Dyn 2-30, and their chemical scaffold matched partner inactive compounds....
This protocol describes the chemical synthesis of the dynamin inhibitors Dynole 34-2 and Acrylo-Dyn 2-30, and their chemical scaffold matched partner inactive compounds. The chosen active and inactive paired compounds represent potent dynamin inhibitors and very closely related dynamin-inactive compounds, with the synthesis of three of the four compounds readily possible via a common intermediate. Combined with the assay data provided, this allows the interrogation of dynamin in vitro and potentially in vivo.
Topics: Cyanoacrylates; Dynamins; Endocytosis; Indoles
PubMed: 35099803
DOI: 10.1007/978-1-0716-1916-2_17 -
European Journal of Medicinal Chemistry Feb 2023Wiskostatin (1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol) (1) is a carbazole-based compound reported as a specific and relatively potent inhibitor of...
Wiskostatin (1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol) (1) is a carbazole-based compound reported as a specific and relatively potent inhibitor of the N-WASP actin remodelling complex (S-isomer EC = 4.35 μM; R-isomer EC = 3.44 μM). An NMR solution structure showed that wiskostatin interacts with a cleft in the regulatory GTPase binding domain of N-WASP. However, numerous studies have reported wiskostatin's actions on membrane transport and cytokinesis that are independent of the N-WASP-Arp2/3 complex pathway, but offer limited alternative explanation. The large GTPase, dynamin has established functional roles in these pathways. This study reveals that wiskostatin and its analogues, as well as other carbazole-based compounds, are inhibitors of helical dynamin GTPase activity and endocytosis. We characterise the effects of wiskostatin on in vitro dynamin GTPase activity, in-cell endocytosis, and determine the importance of wiskostatin functional groups on these activities through design and synthesis of libraries of wiskostatin analogues. We also examine whether other carbazole-based scaffolds frequently used in research or the clinic also modulate dynamin and endocytosis. Understanding off-targets for compounds used as research tools is important to be able to confidently interpret their action on biological systems, particularly when the target and off-targets affect overlapping mechanisms (e.g. cytokinesis and endocytosis). Herein we demonstrate that wiskostatin is a dynamin inhibitor (IC 20.7 ± 1.2 μM) and a potent inhibitor of clathrin mediated endocytosis (IC = 6.9 ± 0.3 μM). Synthesis of wiskostatin analogues gave rise to 1-(9H-carbazol-9-yl)-3-((4-methylbenzyl)amino)propan-2-ol (35) and 1-(9H-carbazol-9-yl)-3-((4-chlorobenzyl)amino)propan-2-ol (43) as potent dynamin inhibitors (IC = 1.0 ± 0.2 μM), and (S)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8a) and (R)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8b) that are amongst the most potent inhibitors of clathrin mediated endocytosis yet reported (IC = 2.3 ± 3.3 and 2.1 ± 1.7 μM, respectively).
Topics: Dynamin I; Dynamins; Carbazoles; GTP Phosphohydrolases; Actins; Clathrin; Endocytosis
PubMed: 36577213
DOI: 10.1016/j.ejmech.2022.115001 -
Biological Chemistry Nov 2018Dynamins are essential as membrane remodelers in various cellular processes, like receptor-mediated endocytosis, synaptic vesicle recycling and spermatogenesis.... (Review)
Review
Dynamins are essential as membrane remodelers in various cellular processes, like receptor-mediated endocytosis, synaptic vesicle recycling and spermatogenesis. Moreover, dynamin is involved in the internalization of numerous viruses and in the motility of several cancer cell lines. As tools for dissecting the underlying mechanisms of these important biological processes and as potential future therapeutics, small molecules have been developed in the last two decades that modulate the functions of dynamin. In this review we give an overview of the compound classes that are currently in use and describe how they affect dynamin function.
Topics: Animals; Dynamins; Humans; Models, Molecular; Molecular Structure; Small Molecule Libraries
PubMed: 30067507
DOI: 10.1515/hsz-2018-0257 -
Molecular Membrane Biology 1996Dynamin is a neuronal phosphoprotein and a GTPase enzyme which mediates late stages of endocytosis in both neural and non-neural cells. Current knowledge about dynamin... (Review)
Review
Dynamin is a neuronal phosphoprotein and a GTPase enzyme which mediates late stages of endocytosis in both neural and non-neural cells. Current knowledge about dynamin is reviewed with particular emphasis on its structure and regulation with respect to phosphorylation, protein-protein interactions and phospholipid binding. The major themes are the biochemical regulation of dynamin, its effects on dynamin's GTPase activity and how this might relate to assembling the 'fission ring' that brings about vesicle retrieval. Dynamin I is an isoform of the enzyme primarily located in the central and peripheral nervous systems, where it is enriched in areas of abundant synaptic contacts. Dynamin I undergoes protein-protein interactions via its proline-rich domain at the C-terminus and these can elevate its N-terminal GTPase activity. Dynamin I interacts with multiple proteins in the nerve terminal, including SH3 domain-containing proteins such as amphiphysin and potentially with other proteins such as betagamma subunits. These regulate its role in endocytosis by targeting dynamin I to specific subcellular locations of retrieval. Dynamin I is phosphorylated in vivo by PKC and dephosphorylated on depolarization and calcium influx into nerve terminals in parallel with the coupled events of exocytosis and endocytosis. In late stages of synaptic vesicle retrieval dynamin I undergoes stimulated assembly into a collar, or fission ring, that surrounds the neck of recycling synaptic vesicles. Activation of GTP hydrolysis probably then generates the free synaptic vesicle, which can be refilled with neurotransmitters. This targeting and assembly may involve sequential steps including recruitment of AP-2 to synaptotagmin on the synaptic vesicle, and recruitment of amphiphysin, dynamin I, and synaptojanin. In addition to synaptic vesicle retrieval, dynamin has been associated with intracellular events mediated by growth factor receptors, insulin receptors and the beta-adrenergic receptor. This is likely to reflect targeting of these receptors for endocytosis soon after their activation. However, does it also suggest a broader role for dynamin in other aspects of intracellular signalling pathways?
Topics: Animals; Dynamin I; Dynamins; Endocytosis; GTP Phosphohydrolases; Humans; Signal Transduction
PubMed: 9116759
DOI: 10.3109/09687689609160598 -
Cell Dec 2008The GTPase dynamin is critically involved in membrane fission during endocytosis. How does dynamin use the energy of GTP hydrolysis for membrane remodeling? By...
The GTPase dynamin is critically involved in membrane fission during endocytosis. How does dynamin use the energy of GTP hydrolysis for membrane remodeling? By monitoring the ionic permeability through lipid nanotubes (NT), we found that dynamin was capable of squeezing NT to extremely small radii, depending on the NT lipid composition. However, long dynamin scaffolds did not produce fission: instead, fission followed GTPase-dependent cycles of assembly and disassembly of short dynamin scaffolds and involved a stochastic process dependent on the curvature stress imposed by dynamin. Fission happened spontaneously upon NT release from the scaffold, without leakage. Our calculations revealed that local narrowing of NT could induce cooperative lipid tilting, leading to self-merger of the inner monolayer of NT (hemifission), consistent with the absence of leakage. We propose that dynamin transmits GTP's energy to periodic assembling of a limited curvature scaffold that brings lipids to an unstable intermediate.
Topics: Animals; Cell Membrane; Dynamins; Endocytosis; Guanosine Triphosphate; Intracellular Membranes; Lipid Bilayers; Lipid Metabolism; Models, Biological; Nanotubes; Nucleotides
PubMed: 19084269
DOI: 10.1016/j.cell.2008.11.028 -
Annual Review of Biophysics 2013In eukaryotic cells, membrane compartments are split into two by membrane fission. This ensures discontinuity of membrane containers and thus proper... (Review)
Review
In eukaryotic cells, membrane compartments are split into two by membrane fission. This ensures discontinuity of membrane containers and thus proper compartmentalization. The first proteic machinery implicated in catalyzing membrane fission was dynamin. Dynamin forms helical collars at the neck of endocytic buds. This structural feature suggested that the helix of dynamin could constrict in order to promote fission of the enclosed membrane. However, verifying this hypothesis revealed itself to be a challenge, which inspired many in vitro and in vivo studies. The primary goal of this review is to discuss recent structural and physical data from biophysical studies that have refined our understanding of the dynamin mechanism. In addition to the constriction hypothesis, other models have been proposed to explain how dynamin induces membrane fission. We present experimental data supporting these various models and assess which model is the most probable.
Topics: Animals; Biomechanical Phenomena; Cell Membrane; Dynamins; Eukaryotic Cells; Intracellular Membranes; Models, Biological; Polymerization; Protein Structure, Secondary
PubMed: 23541160
DOI: 10.1146/annurev-biophys-050511-102247