23 results on '"Marijn G. J. Ford"'
Search Results
2. Light Scattering Techniques to Assess Self-Assembly and Hydrodynamics of Membrane Trafficking Proteins
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Marijn G J, Ford and Rajesh, Ramachandran
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Molecular Weight ,Membranes ,Hydrodynamics ,Proteins ,Endosomes - Abstract
Light scattering methods permit the determination of molar mass and hydrodynamic radius for a protein from first principles. They are, therefore, particularly useful for the biophysical characterization of any protein. Molar mass and hydrodynamic radius determinations may be used to demonstrate that the protein of interest multimerizes. In the endomembrane system, reversible and regulated assembly and multimerization of proteins is critical for building coats required for vesicle budding, for the function of membrane remodeling machines, for fission and fusion and for assembling and disassembling trafficking intermediates. Light scattering methods have therefore significantly contributed to the understanding of the underlying trafficking processes. Herein, we describe methods to express and purify the recombinant fungal SNX-BAR Mvp1, a membrane remodeling protein required for retrograde trafficking at the endosome. Using Mvp1 as an example, we provide protocols for determining its molar mass and hydrodynamic radius by multiangle static light scattering and dynamic light scattering, respectively. These methods can be applied directly to the study of other membrane trafficking proteins, yielding a wealth of biophysical and biochemical information.
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- 2022
3. The structural biology of the dynamin‐related proteins: New insights into a diverse, multitalented family
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Joshua S. Chappie and Marijn G. J. Ford
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Dynamins ,macromolecular substances ,GTPase ,Computational biology ,Biology ,Biochemistry ,Article ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Genetics ,Animals ,Humans ,Molecular Biology ,030304 developmental biology ,Dynamin ,0303 health sciences ,Binding Sites ,Cryoelectron Microscopy ,Cell Biology ,Cell biology ,Molecular Docking Simulation ,Structural biology ,Guanosine Triphosphate ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Dynamin-related proteins are multidomain, mechanochemical GTPases that self-assemble and orchestrate a wide array of cellular processes. Over the past decade, structural insights from X-ray crystallography and cryo-electron microscopy have reshaped our mechanistic understanding of these proteins. Here we provide a historical perspective on these advances that highlights the structural attributes of different dynamin family members and explores how these characteristics affect GTP hydrolysis, conformational coupling, and oligomerization. We also discuss a number of lingering challenges remaining in the field that suggest future directions of study.
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- 2019
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4. Purification of the Dynamin-Related Protein Vps1 Using Mammalian and Bacterial Expression Systems
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Natalia V, Varlakhanova and Marijn G J, Ford
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GTP-Binding Proteins ,Recombinant Fusion Proteins ,Genetic Vectors ,Escherichia coli ,Vesicular Transport Proteins ,Animals ,Gene Expression ,Humans ,Chaetomium ,Cloning, Molecular ,Transfection ,Cell Line - Abstract
The dynamin-related proteins (DRPs) are self-assembling membrane remodeling machines that are indispensable for fundamental cellular trafficking and homeostatic processes. We describe in this chapter protocols developed in our laboratory for purification of full-length and minimal constructs of Chaetomium thermophilum Vps1, the model fungal DRP, using mammalian and Escherichia coli expression systems.
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- 2020
5. The cryo-EM structure of the SNX-BAR Mvp1 tetramer
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Peijun Zhang, Dapeng Sun, Rajesh Ramachandran, Natalia V. Varlakhanova, Marijn G. J. Ford, and Bryan A. Tornabene
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Models, Molecular ,0301 basic medicine ,genetic structures ,Endosome ,Science ,Dimer ,Sorting Nexins ,Biophysics ,General Physics and Astronomy ,Nerve Tissue Proteins ,Endosomes ,Saccharomyces cerevisiae ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein Domains ,Tetramer ,Humans ,BAR domain ,Binding site ,lcsh:Science ,Binding Sites ,Mitral Valve Prolapse ,Multidisciplinary ,Chemistry ,Cell Membrane ,Cryoelectron Microscopy ,General Chemistry ,Transport protein ,Protein Transport ,030104 developmental biology ,Amphiphysin ,lcsh:Q ,Structural biology ,030217 neurology & neurosurgery - Abstract
Sorting nexins (SNX) are a family of PX domain-containing proteins with pivotal roles in trafficking and signaling. SNX-BARs, which also have a curvature-generating Bin/Amphiphysin/Rvs (BAR) domain, have membrane-remodeling functions, particularly at the endosome. The minimal PX-BAR module is a dimer mediated by BAR-BAR interactions. Many SNX-BAR proteins, however, additionally have low-complexity N-terminal regions of unknown function. Here, we present the cryo-EM structure of the full-length SNX-BAR Mvp1, which is an autoinhibited tetramer. The tetramer is a dimer of dimers, wherein the membrane-interacting BAR surfaces are sequestered and the PX lipid-binding sites are occluded. The N-terminal low-complexity region of Mvp1 is essential for tetramerization. Mvp1 lacking its N-terminus is dimeric and exhibits enhanced membrane association. Membrane binding and remodeling by Mvp1 therefore requires unmasking of the PX and BAR domain lipid-interacting surfaces. This work reveals a tetrameric configuration of a SNX-BAR protein that provides critical insight into SNX-BAR function and regulation., SNX-BAR proteins are a family of PX and BAR domain-containing proteins with pivotal roles in trafficking processes. Here authors present the cryo-EM structure of the full-length fungal SNX-BAR Mvp1, which is an autoinhibited tetramer and provides critical insight into SNX-BAR function and regulation.
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- 2020
6. Feedback regulation of TORC1 by its downstream effectors Npr1 and Par32
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Marijn G. J. Ford, Bryan A. Tornabene, and Natalia V. Varlakhanova
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0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Amino Acid Transport Systems ,Amino Acid Motifs ,Regulator ,Saccharomyces cerevisiae ,Mechanistic Target of Rapamycin Complex 1 ,Biology ,Conserved sequence ,03 medical and health sciences ,Molecular Biology ,Conserved Sequence ,Cell Nucleus ,Feedback, Physiological ,Sirolimus ,Kinase ,Permease ,Effector ,Cell Membrane ,fungi ,Articles ,Cell Biology ,Subcellular localization ,Signaling ,Cell biology ,Amino acid permease ,Protein Transport ,030104 developmental biology ,Mutation ,Phosphorylation ,Protein Kinases ,Protein Processing, Post-Translational ,Subcellular Fractions - Abstract
TORC1 (target of rapamycin complex) integrates complex nutrient signals to generate and fine-tune a growth and metabolic response. Npr1 (nitrogen permease reactivator) is a downstream effector kinase of TORC1 that regulates the stability, activity, and trafficking of various nutrient permeases including the ammonium permeases Mep1, Mep2, and Mep3 and the general amino acid permease Gap1. Npr1 exerts its regulatory effects on Mep1 and Mep3 via Par32 (phosphorylated after rapamycin). Activation of Npr1 leads to phosphorylation of Par32, resulting in changes in its subcellular localization and function. Here we demonstrate that Par32 is a positive regulator of TORC1 activity. Loss of Par32 renders cells unable to recover from exposure to rapamycin and reverses the resistance to rapamycin of Δ npr1 cells. The sensitivity to rapamycin of cells lacking Par32 is dependent on Mep1 and Mep3 and the presence of ammonium, linking ammonium metabolism to TORC1 activity. Par32 function requires its conserved repeated glycine-rich motifs to be intact but, surprisingly, does not require its localization to the plasma membrane. In all, this work elucidates a novel mechanism by which Npr1 and Par32 exert regulatory feedback on TORC1.
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- 2018
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7. Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly.
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Eva M Schmid, Marijn G J Ford, Anne Burtey, Gerrit J K Praefcke, Sew-Yeu Peak-Chew, Ian G Mills, Alexandre Benmerah, and Harvey T McMahon
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Biology (General) ,QH301-705.5 - Abstract
Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.
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- 2006
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8. Structural and functional characterization of the dominant negative P‐loop lysine mutation in the dynamin superfamily protein Vps1
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Natalia V. Varlakhanova, Marijn G. J. Ford, Joshua S. Chappie, Bryan A. Tornabene, and Christopher J. Hosford
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Alanine ,Dynamins ,Models, Molecular ,0303 health sciences ,Chemistry ,Lysine ,030302 biochemistry & molecular biology ,Mutant ,Wild type ,Vesicular Transport Proteins ,Context (language use) ,GTPase ,Articles ,Chaetomium ,Biochemistry ,Cell biology ,Fungal Proteins ,03 medical and health sciences ,DNM1 ,stomatognathic system ,Mutation (genetic algorithm) ,Mutation ,Molecular Biology ,030304 developmental biology ,Dynamin - Abstract
Dynamin-superfamily proteins (DSPs) are large self-assembling mechanochemical GTPases that harness GTP hydrolysis to drive membrane remodeling events needed for many cellular processes. Mutation to alanine of a fully conserved lysine within the P-loop of the DSP GTPase domain results in abrogation of GTPase activity. This mutant has been widely used in the context of several DSPs as a dominant-negative to impair DSP-dependent processes. However, the precise deficit of the P-loop K to A mutation remains an open question. Here, we use biophysical, biochemical and structural approaches to characterize this mutant in the context of the endosomal DSP Vps1. We show that the Vps1 P-loop K to A mutant binds nucleotide with an affinity similar to wild type but exhibits defects in the organization of the GTPase active site that explain the lack of hydrolysis. In cells, Vps1 and Dnm1 bearing the P-loop K to A mutation are defective in disassembly. These mutants become trapped in assemblies at the typical site of action of the DSP. This work provides mechanistic insight into the widely-used DSP P-loop K to A mutation and the basis of its dominant-negative effects in the cell.
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- 2020
9. Purification of the Dynamin-Related Protein Vps1 Using Mammalian and Bacterial Expression Systems
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Natalia V. Varlakhanova and Marijn G. J. Ford
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0106 biological sciences ,0303 health sciences ,Bl21 de3 ,Chemistry ,medicine.disease_cause ,01 natural sciences ,Cell biology ,03 medical and health sciences ,Chaetomium thermophilum ,Membrane remodeling ,medicine ,Escherichia coli ,Homeostasis ,030304 developmental biology ,010606 plant biology & botany ,Dynamin - Abstract
The dynamin-related proteins (DRPs) are self-assembling membrane remodeling machines that are indispensable for fundamental cellular trafficking and homeostatic processes. We describe in this chapter protocols developed in our laboratory for purification of full-length and minimal constructs of Chaetomium thermophilum Vps1, the model fungal DRP, using mammalian and Escherichia coli expression systems.
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- 2020
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10. Author response for 'The Structural Biology of the Dynamin‐Related Proteins: New Insights into a Diverse, Multi‐Talented Family'
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Joshua S. Chappie and Marijn G. J. Ford
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Structural biology ,Computational biology ,Biology ,Dynamin - Published
- 2019
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11. Structures of the fungal dynamin-related protein Vps1 reveal a unique, open helical architecture
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Frances Joan D. Alvarez, Tyler M. Brady, Natalia V. Varlakhanova, Christopher J. Hosford, Marijn G. J. Ford, Joshua S. Chappie, Peijun Zhang, and Bryan A. Tornabene
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0301 basic medicine ,Endosome ,Vesicular Transport Proteins ,Saccharomyces cerevisiae ,GTPase ,Biology ,Crystallography, X-Ray ,Article ,Protein Structure, Secondary ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,GTP-Binding Proteins ,Hydrolase ,Compartment (development) ,Research Articles ,Dynamin ,Cryoelectron Microscopy ,Cell Biology ,030104 developmental biology ,Helix ,Biophysics ,030217 neurology & neurosurgery ,Function (biology) - Abstract
How specific dynamin-related proteins (DRPs) are tailored to their cellular targets is an open question. Varlakhanova et al. present structures of the fungal DRP Vps1, which functions at the endosomal compartment. The crystal and cryoEM structures reveal a unique DRP architecture that highlights structural flexibilities of DRP self-assembly., Dynamin-related proteins (DRPs) are large multidomain GTPases required for diverse membrane-remodeling events. DRPs self-assemble into helical structures, but how these structures are tailored to their cellular targets remains unclear. We demonstrate that the fungal DRP Vps1 primarily localizes to and functions at the endosomal compartment. We present crystal structures of a Vps1 GTPase–bundle signaling element (BSE) fusion in different nucleotide states to capture GTP hydrolysis intermediates and concomitant conformational changes. Using cryoEM, we determined the structure of full-length GMPPCP-bound Vps1. The Vps1 helix is more open and flexible than that of dynamin. This is due to further opening of the BSEs away from the GTPase domains. A novel interface between adjacent GTPase domains forms in Vps1 instead of the contacts between the BSE and adjacent stalks and GTPase domains as seen in dynamin. Disruption of this interface abolishes Vps1 function in vivo. Hence, Vps1 exhibits a unique helical architecture, highlighting structural flexibilities of DRP self-assembly.
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- 2018
12. Ivy1 is a negative regulator of Gtr-dependent TORC1 activation
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Marijn G. J. Ford, Bryan A. Tornabene, and Natalia V. Varlakhanova
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0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Glutamine ,EGO complex ,TORC1 signaling ,Saccharomyces cerevisiae ,GTPase ,TORC1 complex ,Biology ,03 medical and health sciences ,Microautophagy ,Monomeric GTP-Binding Proteins ,chemistry.chemical_classification ,Cell growth ,Intracellular Membranes ,Cell Biology ,Amino acid ,Cell biology ,030104 developmental biology ,chemistry ,Vacuoles ,Carrier Proteins ,Dimerization ,Protein Binding ,Transcription Factors ,Research Article - Abstract
The highly conserved TORC1 complex controls cell growth in response to nutrients, especially amino acids. The EGO complex activates TORC1 in response to glutamine and leucine. Here, we demonstrate that the I-BAR domain-containing protein Ivy1 colocalizes with Gtr1 and Gtr2, a heterodimer of small GTPases that are part of the EGO complex. Ivy1 is a negative regulator of Gtr-induced TORC1 activation, and is contained within puncta associated with the vacuolar membrane in cells grown in nutrient-rich medium or after brief nitrogen starvation. Addition of glutamine to nitrogen-starved cells leads to dissipation of Ivy1 puncta and redistribution of Ivy1 throughout the vacuolar membrane. Continued stimulation with glutamine results in concentration of Ivy1 within vacuolar membrane invaginations and its spatial separation from the EGO complex components Gtr1 and Gtr2. Disruption of vacuolar membrane invagination is associated with persistent mislocalization of Ivy1 across the vacuolar membrane and inhibition of TORC1 activity. Together, our findings illustrate a novel negative-feedback pathway that is exerted by Ivy1 on Gtr-dependent TORC1 signaling and provide insight into a potential molecular mechanism underlying TORC1 activation by vacuolar membrane remodeling.
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- 2018
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13. Pib2 and EGO Complex are both required for activation of TORC1
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Michael J. Mihalevic, Marijn G. J. Ford, Kara A. Bernstein, and Natalia V. Varlakhanova
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0301 basic medicine ,Saccharomyces cerevisiae Proteins ,EGO complex ,Saccharomyces cerevisiae ,TORC1 complex ,Phosphatidylinositol 3-Kinases ,03 medical and health sciences ,chemistry.chemical_compound ,Autophagy ,Phosphatidylinositol ,PI3K/AKT/mTOR pathway ,biology ,Cell growth ,Intracellular Membranes ,Cell Biology ,biology.organism_classification ,Cell biology ,Enzyme Activation ,Glutamine ,Protein Transport ,030104 developmental biology ,chemistry ,Biochemistry ,Vacuoles ,Leucine ,Apoptosis Regulatory Proteins ,Signal Transduction ,Transcription Factors ,Research Article - Abstract
The TORC1 complex is a key regulator of cell growth and metabolism in Saccharomyces cerevisiae . The vacuole-associated EGO complex couples activation of TORC1 to the availability of amino acids, specifically glutamine and leucine. The EGO complex is also essential for reactivation of TORC1 following rapamycin-induced growth arrest and for its distribution on the vacuolar membrane. Pib2, a FYVE-containing phosphatidylinositol 3-phosphate (PI3P)-binding protein, is a newly discovered and poorly characterized activator of TORC1. Here, we show that Pib2 is required for reactivation of TORC1 following rapamycin-induced growth arrest. Pib2 is required for EGO complex-mediated activation of TORC1 by glutamine and leucine as well as for redistribution of Tor1 on the vacuolar membrane. Therefore, Pib2 and the EGO complex cooperate to activate TORC1 and connect phosphoinositide 3-kinase (PI3K) signaling and TORC1 activity.
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- 2017
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14. Membrane fission by dynamin: what we know and what we need to know
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Jenny E. Hinshaw, Thomas D. Pollard, Vadim A. Frolov, Oliver Daumke, Adam Frost, Pietro De Camilli, Harry H. Low, Elizabeth H. Chen, Tom Kirchhausen, Martin Lenz, Christopher G. Burd, Sandra L. Schmid, Harvey T. McMahon, Philip Robinson, Aurélien Roux, Bruno Antonny, Michael M. Kozlov, Katja Faelber, Marijn G. J. Ford, Christien J. Merrifield, Institut de pharmacologie moléculaire et cellulaire (IPMC), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Department of Cell Biology [New Haven], Yale University School of Medicine-Howard Hughes Medical Institute (HHMI), Laboratoire de Physique Théorique et Modèles Statistiques (LPTMS), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11), Institute for Integrative Biology of the Cell, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Physico-Chimie-Curie (PCC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC), Université Nice Sophia Antipolis (1965 - 2019) (UNS), Yale School of Medicine [New Haven, Connecticut] (YSM)-Howard Hughes Medical Institute (HHMI), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Wellcome Trust
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0301 basic medicine ,Dynamins ,endocrine system ,GTP' ,membrane fission ,GTPase ,Review ,macromolecular substances ,Biology ,Endocytosis ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Membrane fission ,Organelle ,dynamin ,Molecular motor ,endocytosis ,Animals ,Humans ,Membrane & Intracellular Transport ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,Dynamin ,[PHYS]Physics [physics] ,08 Information And Computing Sciences ,General Immunology and Microbiology ,General Neuroscience ,Cell Membrane ,Helical polymer ,11 Medical And Health Sciences ,06 Biological Sciences ,Cell biology ,molecular motor ,030104 developmental biology ,ddc:540 ,Guanosine Triphosphate ,biological phenomena, cell phenomena, and immunity ,Developmental Biology - Abstract
The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.
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- 2016
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15. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes
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Barbara M.F. Pearse, Philip R. Evans, Marijn G. J. Ford, Matthew K. Higgins, Yvonne Vallis, Harvey T. McMahon, Adele Gibson, Colin R. Hopkins, and David J. Owen
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Multidisciplinary ,Epsin ,biology ,biology.protein ,Ap180 ,Clathrin adaptor proteins ,Monomeric Clathrin Assembly Proteins ,ENTH domain ,Endocytosis ,Clathrin coat ,Clathrin ,Cell biology - Abstract
Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH 2 -terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns(4,5)P 2 ] via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH 2 -terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P 2 -binding site. Because AP180 could bind to PtdIns(4,5)P 2 and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.
- Published
- 2016
16. The crystal structure of dynamin
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Simon Jenni, Marijn G. J. Ford, and Jodi Nunnari
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Models, Molecular ,Protein Conformation ,Molecular Sequence Data ,Endocytic cycle ,GTPase ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,Article ,Protein structure ,Animals ,Humans ,Amino Acid Sequence ,Dynamin I ,Dynamin ,Multidisciplinary ,Nucleotides ,Membrane structure ,Protein Structure, Tertiary ,Rats ,Pleckstrin homology domain ,Amino Acid Substitution ,Biochemistry ,Biophysics ,Guanosine Triphosphate ,Protein Multimerization ,Crystallization ,Biogenesis ,Protein Binding - Abstract
Dynamin-related proteins (DRPs) are multi-domain GTPases that function via oligomerization and GTP-dependent conformational changes to play central roles in regulating membrane structure across phylogenetic kingdoms. How DRPs harness self-assembly and GTP-dependent conformational changes to remodel membranes is not understood. Here we present the crystal structure of an assembly-deficient mammalian endocytic DRP, dynamin 1, lacking the proline-rich domain, in its nucleotide-free state. The dynamin 1 monomer is an extended structure with the GTPase domain and bundle signalling element positioned on top of a long helical stalk with the pleckstrin homology domain flexibly attached on its opposing end. Dynamin 1 dimer and higher order dimer multimers form via interfaces located in the stalk. Analysis of these interfaces provides insight into DRP family member specificity and regulation and provides a framework for understanding the biogenesis of higher order DRP structures and the mechanism of DRP-mediated membrane scission events.
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- 2011
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17. Curvature of clathrin-coated pits driven by epsin
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Gerrit J. K. Praefcke, Philip R. Evans, Ian G. Mills, Brian J. Peter, Yvonne Vallis, Harvey T. McMahon, and Marijn G. J. Ford
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Models, Molecular ,Phosphatidylinositol 4,5-Diphosphate ,Epsin ,Molecular Sequence Data ,Vesicular Transport Proteins ,Inositol 1,4,5-Trisphosphate ,Crystallography, X-Ray ,Clathrin ,Clathrin coat ,Biopolymers ,Animals ,Humans ,Amino Acid Sequence ,ENTH domain ,Multidisciplinary ,biology ,Vesicle ,Neuropeptides ,Brain ,Membrane Proteins ,Coated Pits, Cell-Membrane ,Membrane budding ,Endocytosis ,Protein Structure, Tertiary ,Rats ,Cell biology ,Adaptor Proteins, Vesicular Transport ,Microscopy, Electron ,Drosophila melanogaster ,Membrane curvature ,Liposomes ,Mutation ,biology.protein ,Ap180 ,Carrier Proteins ,Protein Binding - Abstract
Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P(2) in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic alpha-helix in epsin is coupled to PtdIns(4,5)P(2) binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.
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- 2002
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18. Solitary and repetitive binding motifs for the AP2 complex alpha-appendage in amphiphysin and other accessory proteins
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Peter H. Li, M. Madan Babu, Ian G. Mills, Marijn G. J. Ford, Lene E. Olesen, Harvey T. McMahon, Gerrit J. K. Praefcke, Yvonne Vallis, and Eva M. Schmid
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Dynamins ,Amino Acid Motifs ,Nerve Tissue Proteins ,Biology ,Biochemistry ,Adaptor Protein Complex alpha Subunits ,Membrane Lipids ,Chlorocebus aethiops ,Animals ,Humans ,Adaptor Protein Complex beta Subunits ,Molecular Biology ,Signal transducing adaptor protein ,Coated Pits, Cell-Membrane ,Cell Biology ,Transmembrane protein ,Endocytosis ,Transport protein ,Protein Structure, Tertiary ,Rats ,Membrane curvature ,Multiprotein Complexes ,Amphiphysin ,COS Cells ,Biophysics ,Sequence motif - Abstract
Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and beta-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the alpha-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the alpha-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the alpha-appendage.
- Published
- 2007
19. The Conserved Isoleucine-Valine-Phenylalanine Motif Couples Activation State and Endocytic Functions of β-Arrestins
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Joshua Z. Rappoport, Alexandre Benmerah, Sanford M. Simon, Stefano Marullo, Marijn G. J. Ford, Anne Burtey, Mark G.H. Scott, Eva M. Schmid, Harvey T. McMahon, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), University of Bergen (UiB), Université d'Oslo, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Potsdam Institute for Climate Impact Research (PIK), Laboratory of Cellular Biophysics, Rockefeller University [New York], Institut Cochin (UMR_S567 / UMR 8104), and Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
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[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,Arrestins ,[SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging ,Phenylalanine ,Clathrin adaptor complex ,Amino Acid Motifs ,Molecular Sequence Data ,Endocytic cycle ,[SDV.SA.AGRO]Life Sciences [q-bio]/Agricultural sciences/Agronomy ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Biochemistry ,Clathrin ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,[SDV.EE.ECO]Life Sciences [q-bio]/Ecology, environment/Ecosystems ,Structural Biology ,Chlorocebus aethiops ,Genetics ,Arrestin ,Animals ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Amino Acid Sequence ,Isoleucine ,Binding site ,[SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials ,Molecular Biology ,Conserved Sequence ,beta-Arrestins ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,G protein-coupled receptor ,[SDV.EE.SANT]Life Sciences [q-bio]/Ecology, environment/Health ,0303 health sciences ,[SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health ,Sequence Homology, Amino Acid ,biology ,030302 biochemistry & molecular biology ,Signal transducing adaptor protein ,Valine ,Cell Biology ,Cell biology ,COS Cells ,biology.protein ,Clathrin adaptor proteins ,[SDV.AEN]Life Sciences [q-bio]/Food and Nutrition ,HeLa Cells ,[SDV.EE.IEO]Life Sciences [q-bio]/Ecology, environment/Symbiosis - Abstract
Beta-arrestins (betaarrs) play a central role in the regulation of G-protein-coupled receptors (GPCRs). Their binding to phosphorylated activated GPCRs induces a conformational transition to an active state resulting in the release of their flexible C-terminal tail. Binding sites for clathrin and the adaptor protein (AP)-2 clathrin adaptor complex are then unmasked, which drive the recruitment of betaarrs-GPCR complexes into clathrin-coated pits (CCPs). A conserved isoleucine-valine-phenylalanine (IVF) motif of the C-terminal tail controls betaarr activation through intramolecular interactions. Here, we provide structural, biochemical and functional evidence in living cells that the IVF motif also controls binding to AP-2. While the F residue is directly involved in AP-2 binding, substitutions of I and V residues, markedly enhanced affinity for AP-2 resulting in active betaarr mutants, which are constitutively targeted to CCPs in the absence of any GPCR activation. Conformational change and endocytic functions of betaarrs thus appear to be coordinated via the complex molecular interactions established by the IVF motif.
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- 2007
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- View/download PDF
20. Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature
- Author
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Philip R. Evans, P. Jonathan G. Butler, Oliver Daumke, Balachandra G. Hegde, Ralf Langen, Helen M. Kent, Harvey T. McMahon, Rohit Mittal, Marijn G. J. Ford, and William Mike Henne
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Models, Molecular ,Dimer ,Molecular Sequence Data ,Antiparallel (biochemistry) ,Curvature ,Fatty Acid-Binding Proteins ,Protein Structure, Secondary ,chemistry.chemical_compound ,Protein structure ,Structural Biology ,BAR domain ,Humans ,Amino Acid Sequence ,Molecular Biology ,Chemistry ,Cell Membrane ,Electron Spin Resonance Spectroscopy ,Membrane Proteins ,Proteins ,Crystallography ,Membrane ,Membrane protein ,Membrane curvature ,Liposomes ,Dimerization ,Hydrophobic and Hydrophilic Interactions - Abstract
SummaryA spectrum of membrane curvatures exists within cells, and proteins have evolved different modules to detect, create, and maintain these curvatures. Here we present the crystal structure of one such module found within human FCHo2. This F-BAR (extended FCH) module consists of two F-BAR domains, forming an intrinsically curved all-helical antiparallel dimer with a Kd of 2.5 μM. The module binds liposomes via a concave face, deforming them into tubules with variable diameters of up to 130 nm. Pulse EPR studies showed the membrane-bound dimer is the same as the crystal dimer, although the N-terminal helix changed conformation on membrane binding. Mutation of a phenylalanine on this helix partially attenuated narrow tubule formation, and resulted in a gain of curvature sensitivity. This structure shows a distant relationship to curvature-sensing BAR modules, and suggests how similar coiled-coil architectures in the BAR superfamily have evolved to expand the repertoire of membrane-sculpting possibilities.
- Published
- 2007
21. Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly
- Author
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Sew-Yeu Peak-Chew, Eva M. Schmid, Gerrit J. K. Praefcke, Harvey T. McMahon, Alexandre Benmerah, Anne Burtey, Ian G. Mills, Marijn G. J. Ford, Potsdam Institute for Climate Impact Research (PIK), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), University of Bergen (UiB), Université d'Oslo, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Cochin (UMR_S567 / UMR 8104), and Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Models, Molecular ,Protein Folding ,[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,Arrestins ,Eukaryotes ,[SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging ,Amino Acid Motifs ,Vesicular Transport Proteins ,[SDV.SA.AGRO]Life Sciences [q-bio]/Agricultural sciences/Agronomy ,Coated Pit ,Plasma protein binding ,Ligands ,Molecular Biology/Structural Biology ,Protein Structure, Secondary ,Mice ,0302 clinical medicine ,Protein Interaction Mapping ,Biology (General) ,beta-Arrestins ,ComputingMilieux_MISCELLANEOUS ,0303 health sciences ,biology ,Adaptor Protein Complex beta Subunits ,Systems Biology ,General Neuroscience ,Clathrin-Coated Vesicles ,Coated Pits, Cell-Membrane ,Cell biology ,Synopsis ,Clathrin adaptor proteins ,General Agricultural and Biological Sciences ,Protein Binding ,Research Article ,QH301-705.5 ,Molecular Sequence Data ,Protein domain ,Adaptor Protein Complex 2 ,Biophysics ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Models, Biological ,Clathrin ,General Biochemistry, Genetics and Molecular Biology ,Protein–protein interaction ,03 medical and health sciences ,[SDV.EE.ECO]Life Sciences [q-bio]/Ecology, environment/Ecosystems ,Arrestin ,Animals ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Amino Acid Sequence ,[SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials ,030304 developmental biology ,[SDV.EE.SANT]Life Sciences [q-bio]/Ecology, environment/Health ,Binding Sites ,[SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health ,General Immunology and Microbiology ,Cell Biology ,Protein Structure, Tertiary ,Adaptor Proteins, Vesicular Transport ,biology.protein ,[SDV.AEN]Life Sciences [q-bio]/Food and Nutrition ,030217 neurology & neurosurgery ,HeLa Cells ,Neuroscience ,[SDV.EE.IEO]Life Sciences [q-bio]/Ecology, environment/Symbiosis - Abstract
Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors., Formation of clathrin-coated vesicles, important in endocytosis, relies on accessory proteins assembled by adaptor protein complex 2 (AP2). Here, mass spectrometry and crystallization identifies proteins recruited by AP2's β-appendage for this purpose.
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- 2006
- Full Text
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22. Evolving nature of the AP2 alpha-appendage hub during clathrin-coated vesicle endocytosis
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M. Madan Babu, Eva M. Schmid, Ian G. Mills, Lene E. Olesen, Yvonne Vallis, Marijn G. J. Ford, Gerrit J. K. Praefcke, Jennifer L. Gallop, Harvey T. McMahon, and Sew-Yeu Peak-Chew
- Subjects
Models, Molecular ,Proteomics ,Amino Acid Motifs ,Adaptor Protein Complex 2 ,Coated vesicle ,Nerve Tissue Proteins ,Plasma protein binding ,Synaptojanin ,Crystallography, X-Ray ,Ligands ,Clathrin ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Mass Spectrometry ,Article ,Chlorocebus aethiops ,Animals ,Enzyme Inhibitors ,Molecular Biology ,Binding Sites ,General Immunology and Microbiology ,biology ,General Neuroscience ,Vesicle ,Signal transducing adaptor protein ,Water ,Clathrin-Coated Vesicles ,Endocytosis ,Phosphoric Monoester Hydrolases ,Cell biology ,Protein Structure, Tertiary ,Rats ,COS Cells ,biology.protein ,Mutagenesis, Site-Directed ,Ap180 ,Vesicle scission ,Protein Binding - Abstract
Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called 'top' and 'side' sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.
- Published
- 2004
23. An integrated structural analysis of dynamin assembly
- Author
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Marijn G. J. Ford, Jodi Nunnari, and Simon Jenni
- Subjects
Chemistry ,Biophysics ,Instrumentation ,Dynamin - Abstract
Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.
- Published
- 2012
- Full Text
- View/download PDF
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