Structure And Function Of Dynamin, A 100kd GTPase Involved In Endocytosis
参与胞吞作用的 100kd GTP 酶 Dynamin 的结构和功能
基本信息
- 批准号:10697785
- 负责人:
- 金额:$ 81.05万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:Adrenal GlandsAllelesArchitectureAreaBiological AssayCanadaCell membraneCellsCentronuclear myopathyCharcot-Marie-Tooth DiseaseChemicalsChromaffin CellsClathrinCollaborationsCountryCoupledCryoelectron MicroscopyCrystallizationDefectDevicesDictyostelium discoideum dynamin ADiseaseDrosophila genusDynaminDynamin 2Dynamin IElectron MicroscopeElectronsEndocytosisEnzymesEventF-ActinFamily memberFreezingGTP BindingGuanosine TriphosphateGuanosine Triphosphate PhosphohydrolasesHereditary Spastic ParaplegiaHomologous GeneHumanHydrolysisLengthLinkLipid BilayersLipid BindingLipidsLiposomesMammalian CellMapsMediatingMembraneModelingMolecularMolecular ConformationMutationNational Heart, Lung, and Blood InstituteNational Human Genome Research InstituteNational Institute of Neurological Disorders and StrokeNeckNew YorkNew York CityNutrientPH DomainPeripheral Nervous System DiseasesPlayPolymersPositioning AttributeProtein FamilyReactionRecyclingReportingResolutionRoleSamplingServicesSignal TransductionSiteSpeedStructureSurfaceSynaptic MembranesSynaptic VesiclesTemperatureTestingTimeTomogramTubeUnited States National Institutes of HealthUniversitiesVesicleWorkbasecoated pitconstrictioncrosslinkdensitydetectordisease-causing mutationfallsganghigh resolution imagingin vivoinorganic phosphateinsightmembermillisecondmutantnoveloverexpressionshibire gene productsimulationstructural biologythree dimensional structuretimelinetrafficking
项目摘要
The dynamin family of proteins consists of unique GTPases involved in membrane fission and fusion events throughout the cell. The founding member, dynamin, is crucial for endocytosis, synaptic membrane recycling, membrane trafficking within the cell and more recently, has been associated with filamentous actin. Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue of the shibire gene product in Drosophila. A temperature sensitive shibire allele causes a defect in clathrin-mediated endocytosis. Since then, overexpressing human dynamin mutants in mammalian cells was found to block clathrin-mediated endocytosis.
Over the years, our cryo-electron microscopy (cryoEM) structural work has played a leading role in dissecting the function of dynamin in membrane fission. We have shown that purified dynamin readily assembles into rings and spirals and it forms similar structures on liposomes, generating dynamin-lipid tubes that constrict upon GTP hydrolysis. A potential mechanism for dynamin constriction was revealed when we solved the three-dimensional structure of dynamin in the non-constricted and constricted states by cryo-electron microscopy (cryo-EM). These results suggest dynamin wraps around the necks of budding vesicles as a helical polymer and upon GTP hydrolysis undergoes a significant constriction that ultimately leads to membrane fission.
In 2018, we solved a high-resolution cryo-EM structure of the membrane-associated helical polymer of human dynamin-1 in the GTP-bound state (3.75 Angstroms). Images for the high-resolution structure were collected at the New York Structural Biology Center (NYSBC) in New York City using a FEI Krios electron microscope with a K2 direct electron detector. The dynamin helical structure allowed us to build an atomic model of the assembled dynamin polymer bound to lipid. Comparing soluble crystal structures to our new high-resolution cryo-EM structure revealed conformational changes that occur upon assembly and lipid binding. The structure defines the 1-start helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays in collaboration with Dr. Justin Taraska, NHLBI. The inner lumen of the dynamin-lipid tube is 7 nm compared to 20 nm observed in the apo state.
In 2020, we examined the speed of dynamin constriction using a novel time-resolved machine at the New York Structural Biology Center. For this collaboration with Drs. Carragher and Potter, we sprayed dynamin tubes and GTP simultaneously onto grids and froze the sample in the milli-second range and found dynamin undergoes constriction and falls off the lipid within 150 ms (Dandey et al, 2020). Compared to previous results that showed dynamin constricted within seconds using the traditional freezing devices, we now have a tighter timeline for dynamin-mediated constriction and fission during endocytosis.
In 2021, we solved two structures of a GTPase-defective dynamin mutant (K44A), full-length and delta-PRD, in the presence of GTP, to 3.6 Angstrom resolution, which allowed us to build an atomic model. The K44A structure resembles a previous low-resolution map of WT dynamin in a post-hydrolysis state (10 Angstroms). The high-resolution cryoEM density indicates dynamin is in the GDP-bound state, further constricts the underlying lipid bilayer to achieve an inner lumen of 3.4 nm and assembles as a 2-start helix. Constriction of the membrane to 3.4 nm lumen is reaching the theoretical limit required for spontaneous membrane fission, supporting the model that dynamin alone can cause membrane fission. The K44A structure also reveals how a 2-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck.
In 2021, we contributed to two successful collaborations. For the first collaboration with Dr. Justin Taraska (NHLBI) we generated tomograms of unroofed cells grown on EM grids to explore the architecture of endocytic structures on the plasma membrane. As a result, we were able to show that clathrin sites maintain a constant surface area and flat lattices are loosely packed allowing for spontaneous curvature (Dev Cell, 2021). The tomograms from unroofed cells also allowed us to visualize dynamin helical structures associated with the clathrin-coated pits in vivo. We are currently comparing the in vivo dynamin helical assemblies to our K44A-dynamin high-resolution cryoEM structure. In the second collaboration with Dr. Ling-Gang Wu (NINDS), we demonstrated that dynamin assembles around large circumferences, >200nm, mimicking large necks of a novel non-coated vesicle-budding mechanism in adrenal chromaffin cells (Nat Comm, 2022).
In previous years, we collaborated with Drs. Sandra Schmid (UT Southwestern) and Vadim Frolov (U Basque Country) to explore the effect of dynamins powerstroke defined by the large swing of the BSE in dynamin. To dissect the fission reaction into stages, we utilized intra-molecular chemical cross-linking to stabilize dynamin in a conformation mimicking its transition-state. We found that dynamin trapped in the transition state is unable to mediate full fission but forms stable hemifission intermediates without phosphate release. Dynamin assembly and augmented membrane insertion of its pleckstrin homology domain drives the hemifission state. Our findings, which are consistent with molecular simulations of the fission reaction, reveal a second, unappreciated energy barrier for full fission. Thus additional conformational dynamics are required after hemifission that enable dynamin to utilize the energy of GTP hydrolysis to complete the fission reaction.
Previously, we also collaborated with Drs. Sambuughin (Uniformed Services University), Goldfarb (NINDS, NIH), Renwick (Queens University, Kingston Canada), Platonov (Ammosov North-Eastern Federal University, Russian Federation) and Toro (NHGRI, NIH) to characterized a dynamin mutant that leads to a rare case of Hereditary Spastic Paraplegia (HSP). This was the first report linking a mutation in dynamin-2 to HSP. In addition, the mutation is in a region of dynamin distinct from all other dynamin-2 disease causing mutations.
动力蛋白家族由独特的gtpase组成,参与整个细胞的膜裂变和融合事件。动力蛋白是细胞内吞作用、突触膜循环、细胞内膜运输的关键成员,最近又与丝状肌动蛋白联系在一起。Dynamin在果蝇中被发现是shibire基因产物的哺乳动物同源物时,首次涉及到内吞作用。温度敏感的shibire等位基因导致网格蛋白介导的内吞作用缺陷。从那时起,在哺乳动物细胞中过度表达人动力蛋白突变体被发现可以阻断网格蛋白介导的内吞作用。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Jenny E Hinshaw其他文献
Jenny E Hinshaw的其他文献
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{{ truncateString('Jenny E Hinshaw', 18)}}的其他基金
DYNAMIN STRUCTURES: ENDOCYTOSIS AND VESCILE BUDDING
动力结构:胞吞作用和囊泡出芽
- 批准号:
6120574 - 财政年份:1999
- 资助金额:
$ 81.05万 - 项目类别:
RECYCLING OF COAT PROTEINS FROM CLATHRIN COATED VESICLES
从网格蛋白包被的囊泡中回收包被蛋白
- 批准号:
2171368 - 财政年份:1994
- 资助金额:
$ 81.05万 - 项目类别:
STRUCTURE AND FUNCTION OF DYNAMIN, A 100KD GTPASE INVOLVED IN ENDOCYTOSIS
参与内吞作用的 100KD GTP 酶 Dynamin 的结构和功能
- 批准号:
6105945 - 财政年份:
- 资助金额:
$ 81.05万 - 项目类别:
Structure And Function Of Dynamin, A 100kd GTPase Involved In Endocytosis
参与胞吞作用的 100kd GTP 酶 Dynamin 的结构和功能
- 批准号:
7967677 - 财政年份:
- 资助金额:
$ 81.05万 - 项目类别:
Structural analysis of dynamins involved in mitochondrial morphology
参与线粒体形态的动力的结构分析
- 批准号:
8553580 - 财政年份:
- 资助金额:
$ 81.05万 - 项目类别:
Structural analysis of Dnm1, a dynamin involved in mitochondrial fission
Dnm1(一种参与线粒体裂变的动力)的结构分析
- 批准号:
7593749 - 财政年份:
- 资助金额:
$ 81.05万 - 项目类别:
Structure And Function Of Dynamin, A 100kd GTPase Involved In Endocytosis
参与胞吞作用的 100kd GTP 酶 Dynamin 的结构和功能
- 批准号:
7734266 - 财政年份:
- 资助金额:
$ 81.05万 - 项目类别:
Structural analysis of Dnm1, a dynamin involved in mitochondrial fission
Dnm1(一种参与线粒体裂变的动力)的结构分析
- 批准号:
7967689 - 财政年份:
- 资助金额:
$ 81.05万 - 项目类别:
Structure And Function Of Dynamin, A 100kd GTPase Involved In Endocytosis
参与胞吞作用的 100kd GTP 酶 Dynamin 的结构和功能
- 批准号:
8349873 - 财政年份:
- 资助金额:
$ 81.05万 - 项目类别:
Structure And Function Of Dynamin, A 100kd GTPase Involved In Endocytosis
参与胞吞作用的 100kd GTP 酶 Dynamin 的结构和功能
- 批准号:
9553263 - 财政年份:
- 资助金额:
$ 81.05万 - 项目类别:
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