2024
Photosensitive Nanoprobes for Rapid High Purity Isolation and Size‐Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations
Weerakkody J, Tseng T, Topper M, Thoduvayil S, Radhakrishnan A, Pincet F, Kyriakides T, Gunasekara R, Ramakrishnan S. Photosensitive Nanoprobes for Rapid High Purity Isolation and Size‐Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations. Advanced Functional Materials 2024, 34 PMID: 39372670, PMCID: PMC11452007, DOI: 10.1002/adfm.202400390.Peer-Reviewed Original ResearchExtracellular vesicle subpopulationsVesicle subpopulationsIsolation of vesiclesPurity extracellular vesiclesRelease of vesiclesAnalysis of nucleic acidsNear-native formLarge-scale isolationLipid nanoprobesDownstream analysisPurity isolationEfficient isolationVesiclesSynthetic vesiclesNucleic acidsExtracellular vesiclesIsolation methodIsolatesBiomarker discoveryExposure to lightSubpopulationsEnrichmentProteinComplex biological mediaCleavageKinetic study of membrane protein interactions: from three to two dimensions
Adrien V, Reffay M, Taulier N, Verchère A, Monlezun L, Picard M, Ducruix A, Broutin I, Pincet F, Urbach W. Kinetic study of membrane protein interactions: from three to two dimensions. Scientific Reports 2024, 14: 882. PMID: 38195620, PMCID: PMC10776792, DOI: 10.1038/s41598-023-50827-5.Peer-Reviewed Original ResearchConceptsMembrane proteinsMembrane protein interactionsProtein-protein interactionsProtein complexesProtein interactionsMembrane environmentOpposite membranesBacterial efflux pumpsProtein behaviorProtein systemsMolecular interactionsEfflux pumpsProteinExploration distanceMembraneFluorescence recovery experimentsInteractionBinding rateBinding constantsComplexes
2023
Synaptophysin chaperones the assembly of 12 SNAREpins under each ready-release vesicle
Bera M, Radhakrishnan A, Coleman J, Sundaram R, Ramakrishnan S, Pincet F, Rothman J. Synaptophysin chaperones the assembly of 12 SNAREpins under each ready-release vesicle. Proceedings Of The National Academy Of Sciences Of The United States Of America 2023, 120: e2311484120. PMID: 37903271, PMCID: PMC10636311, DOI: 10.1073/pnas.2311484120.Peer-Reviewed Original ResearchConceptsSpecific molecular functionsSynaptic vesicle protein synaptophysinTarget membrane bilayerSensor synaptotagminSNARE proteinsMolecular functionsMembrane proteinsSNAREpinsReceptor vesiclesSingle-molecule measurementsGene knockoutMembrane bilayerLipid bilayersProtein synaptophysinVesiclesDetergent extractsHexamer structureSYPMechanism of actionProteinAssemblyChaperonesSynaptotagminExocytosisBilayersDirect determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer
Panda A, Giska F, Duncan A, Welch A, Brown C, McAllister R, Hariharan P, Goder J, Coleman J, Ramakrishnan S, Pincet F, Guan L, Krishnakumar S, Rothman J, Gupta K. Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer. Nature Methods 2023, 20: 891-897. PMID: 37106230, PMCID: PMC10932606, DOI: 10.1038/s41592-023-01864-5.Peer-Reviewed Original ResearchConceptsIntegral membrane proteinsMembrane proteinsOligomeric organizationOligomeric stateNative mass spectrometry analysisFunctional oligomeric stateKey membrane componentMass spectrometry analysisNMS analysisTarget membraneLipid bindingMembrane componentsProteolipid vesiclesMembrane compositionLipid compositionSpectrometry analysisLipid membranesNeurotransmitter releaseProteinMembraneLipidsMembrane propertiesDirect determinationBilayersTransporters
2022
The Get1/2 insertase forms a channel to mediate the insertion of tail-anchored proteins into the ER
Heo P, Culver J, Miao J, Pincet F, Mariappan M. The Get1/2 insertase forms a channel to mediate the insertion of tail-anchored proteins into the ER. Cell Reports 2022, 42: 111921. PMID: 36640319, PMCID: PMC9932932, DOI: 10.1016/j.celrep.2022.111921.Peer-Reviewed Original ResearchConceptsTransmembrane domainTA proteinsSingle C-terminal transmembrane domainC-terminal transmembrane domainTail-anchored (TA) proteinsTail-anchored proteinsEndoplasmic reticulum membraneGet3Reticulum membraneChannel functionInsertaseBulk fluorescenceAqueous channelsProteinChannel activityMutation analysisMembraneMicrofluidic assayTranslocaseYeastComplexesInsertionTranslocationHydrophilic segmentsBindingActuating tension-loaded DNA clamps drives membrane tubulation
Liu L, Xiong Q, Xie C, Pincet F, Lin C. Actuating tension-loaded DNA clamps drives membrane tubulation. Science Advances 2022, 8: eadd1830. PMID: 36223466, PMCID: PMC9555772, DOI: 10.1126/sciadv.add1830.Peer-Reviewed Original ResearchConceptsDNA clampMembrane tubulationMembrane dynamicsMembrane-remodeling eventsVesicle tubulationConformational changesSpatiotemporal controlDNA signalsCell membraneDNA nanostructuresTubulationMembrane deformationClosed stateOpen stateSelf-assembled DNA nanostructuresOrganismsProteinMembrane tubeArtificial systemsTube widthMembraneDynamicsThe beginning and the end of SNARE‐induced membrane fusion
Mion D, Bunel L, Heo P, Pincet F. The beginning and the end of SNARE‐induced membrane fusion. FEBS Open Bio 2022, 12: 1958-1979. PMID: 35622519, PMCID: PMC9623537, DOI: 10.1002/2211-5463.13447.Peer-Reviewed Original Research
2020
In vitro physiological membrane‐on‐a‐chip and its application in cell and neuronal biology
Heo P, Rothman J, Pincet F. In vitro physiological membrane‐on‐a‐chip and its application in cell and neuronal biology. The FASEB Journal 2020, 34: 1-1. DOI: 10.1096/fasebj.2020.34.s1.08637.Peer-Reviewed Original ResearchNeuronal biologyTail-anchored proteinsPost-translational insertionMost biological processesAreas of biologyVesicle traffickingProtein insertionER membranePhysiological lipid compositionBiological processesMolecular mechanismsBiologyLipid compositionPhysiological membranesModel membranesMembraneInvaluable insightsCellsTraffickingProteinInsertionPatch-clamp amplifierΑ-synucleinopathiesCompositionBilayersFreezing and piercing of in vitro asymmetric plasma membrane by α-synuclein
Heo P, Pincet F. Freezing and piercing of in vitro asymmetric plasma membrane by α-synuclein. Communications Biology 2020, 3: 148. PMID: 32235856, PMCID: PMC7109109, DOI: 10.1038/s42003-020-0883-7.Peer-Reviewed Original ResearchMeSH KeywordsAlpha-SynucleinCell MembraneElectric CapacitanceFluorescence Recovery After PhotobleachingHydrophobic and Hydrophilic InteractionsLab-On-A-Chip DevicesMembrane FluidityMembrane LipidsMembrane PotentialsMembranes, ArtificialMicrofluidic Analytical TechniquesNeuronsProtein AggregatesProtein Aggregation, PathologicalProtein BindingProtein ConformationStructure-Activity RelationshipSynucleinopathiesConceptsPlasma membraneMembrane-bound proteinsAccumulation of aggregatesΑ-synucleinCytosolic leafletMembrane topologyMembrane hydrophobic coreCytosolic proteinsProteinExtracellular onesHydrophobic corePathological roleDiscrete sizesMembraneLeafletsMembrane capacitanceNeurological diseasesLipidsAccumulationMicrofluidic setup
2019
Highly Reproducible Physiological Asymmetric Membrane with Freely Diffusing Embedded Proteins in a 3D‐Printed Microfluidic Setup
Heo P, Ramakrishnan S, Coleman J, Rothman JE, Fleury J, Pincet F. Highly Reproducible Physiological Asymmetric Membrane with Freely Diffusing Embedded Proteins in a 3D‐Printed Microfluidic Setup. Small 2019, 15: e1900725. PMID: 30977975, DOI: 10.1002/smll.201900725.Peer-Reviewed Original ResearchConceptsMost biological processesLipid leafletAreas of biologyEmbedded proteinsBiological processesRelevant lipidsProteinAsymmetric bilayersPhysiological conditionsModel membranesPlanar bilayersBilayer formation processInvaluable insightsBilayersConfocal microscopeMembraneLipidsTransmembraneBiologyLeafletsMicrofluidic setupRecapitulation
2018
High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis
Ramakrishnan S, Gohlke A, Li F, Coleman J, Xu W, Rothman JE, Pincet F. High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis. Langmuir 2018, 34: 5849-5859. PMID: 29694054, DOI: 10.1021/acs.langmuir.8b00116.Peer-Reviewed Original ResearchConceptsMembrane fusionFusion eventsSoluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteinsSNARE-dependent membrane fusionIndividual vesicle fusion eventsFactor attachment protein receptor proteinsN-ethylmaleimide-sensitive factor attachment protein receptor proteinsT-SNARE proteinsSingle-vesicle fusionProtein receptor proteinsVesicle fusion eventsMobility of proteinsVesicle dockingContent releaseVesicle fusionHigh-throughput monitoringPlanar membranesReceptor proteinLipid mixingProteinLipid bilayersVesiclesCorrect reconstitutionMembraneAqueous compartmentVesicle Tubulation with Self‐Assembling DNA Nanosprings
Grome M, Zhang Z, Pincet F, Lin C. Vesicle Tubulation with Self‐Assembling DNA Nanosprings. Angewandte Chemie 2018, 130: 5428-5432. DOI: 10.1002/ange.201800141.Peer-Reviewed Original ResearchMembrane-deforming proteinsDNA origami designMembrane tubulationMembrane tubulesMembrane curvatureVesicle tubulationMembrane surface coverageDNA structureLipid bilayersTubulationNanospringsTube morphologyIntricate interplayArtificial nanomachinesVesicle deformationSpherical vesiclesNanotechnologyMajor goalProteinDNAVesiclesNanomachinesBioengineeringDetergentsMorphology
2016
A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion
Xu W, Nathwani B, Lin C, Wang J, Karatekin E, Pincet F, Shih W, Rothman JE. A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion. Journal Of The American Chemical Society 2016, 138: 4439-4447. PMID: 26938705, PMCID: PMC4950518, DOI: 10.1021/jacs.5b13107.Peer-Reviewed Original ResearchConceptsMembrane fusionSoluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexCore molecular machineryMembrane fusion eventsProtein receptor complexMembrane fusion processMolecular machineryDNA origami platformTarget membraneAuxiliary proteinsIntracellular communicationDocking stepSingle-event levelReceptor complexLipid mixingSmall unilamellar vesiclesLipid bilayersSnareFundamental processesVesiclesUnilamellar vesiclesTraffickingMachineryProteinFusion
2015
Accelerating SNARE‐Mediated Membrane Fusion by DNA–Lipid Tethers
Xu W, Wang J, Rothman J, Pincet F. Accelerating SNARE‐Mediated Membrane Fusion by DNA–Lipid Tethers. Angewandte Chemie 2015, 127: 14596-14600. DOI: 10.1002/ange.201506844.Peer-Reviewed Original ResearchDNA-lipidMembrane-distal endMembrane-proximal endArtificial tetherSNARE functionCore machinerySNARE proteinsProtein functionTarget membraneMembrane fusionBiological processesNative proteinBase pairsLipid mixingMaximum fusion rateProgrammable toolsBase-pair hybridizationProteinSnareMembraneFusionMachineryTetherNucleotidesVesiclesFormation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock
Motta I, Gohlke A, Adrien V, Li F, Gardavot H, Rothman JE, Pincet F. Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock. Langmuir 2015, 31: 7091-7099. PMID: 26038815, PMCID: PMC4950989, DOI: 10.1021/acs.langmuir.5b01173.Peer-Reviewed Original ResearchConceptsGiant unilamellar vesiclesLipid-anchored proteinsOsmotic shockTrans-membrane proteinsSingle giant unilamellar vesiclesProtein substratesPeripheral proteinsSpecific lipidsDifferent proteinsPhotobleaching experimentsFluorescence recoveryCell membraneProteinLarge vesiclesPhysiological conditionsModel systemUnilamellar vesiclesPhospholipid bilayersVesiclesSimple generic methodPrevious dataMembraneHigh concentrationsLipidsBilayers
2014
Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization
Chalbi M, Barraud-Lange V, Ravaux B, Howan K, Rodriguez N, Soule P, Ndzoudi A, Boucheix C, Rubinstein E, Wolf J, Ziyyat A, Perez E, Pincet F, Gourier C. Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization. Development 2014, 141: 3732-3739. PMID: 25209248, DOI: 10.1242/dev.111534.Peer-Reviewed Original ResearchConceptsGamete fusionMammalian fertilizationMolecular mechanismsSperm protein IZUMO1Intercellular contact areaFusion machineryMembrane proteinsMembrane organizationIZUMO1Intercellular adhesionAdhesion partnersRecruitment kineticsKey playersCD9Adhesion phaseEggsAdhesion areaFertilizationFusionHuman eggsGametesMachineryAdhesionSpeciesProteinArf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting
Wilfling F, Thiam AR, Olarte MJ, Wang J, Beck R, Gould TJ, Allgeyer ES, Pincet F, Bewersdorf J, Farese RV, Walther TC. Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting. ELife 2014, 3: e01607. PMID: 24497546, PMCID: PMC3913038, DOI: 10.7554/elife.01607.Peer-Reviewed Original ResearchMeSH KeywordsADP-Ribosylation Factor 1AnimalsBiological TransportCell LineCoat Protein Complex ICOP-Coated VesiclesDrosophila melanogasterDrosophila ProteinsEndoplasmic ReticulumHumansLipaseLipid DropletsLipolysisMiceNanoparticlesParticle SizePhospholipidsRNA InterferenceSurface TensionTime FactorsTransfectionTriglyceridesConceptsCellular lipid dropletsLipid dropletsProtein machineryProtein targetingUbiquitous organellesVesicle traffickingLD surfaceSpecific proteinsKey enzymeLD morphologyMembrane precursorsMachineryMetabolic energyProteinNeutral lipidsTG storageEnzymeUnclear mechanismsAmount of phospholipidsRecent evidenceOrganellesCOPITraffickingTriacylglycerolsBuds
2013
COPI buds 60-nm lipid droplets from reconstituted water–phospholipid–triacylglyceride interfaces, suggesting a tension clamp function
Thiam AR, Antonny B, Wang J, Delacotte J, Wilfling F, Walther TC, Beck R, Rothman JE, Pincet F. COPI buds 60-nm lipid droplets from reconstituted water–phospholipid–triacylglyceride interfaces, suggesting a tension clamp function. Proceedings Of The National Academy Of Sciences Of The United States Of America 2013, 110: 13244-13249. PMID: 23901109, PMCID: PMC3746913, DOI: 10.1073/pnas.1307685110.Peer-Reviewed Original ResearchMeSH KeywordsADP-Ribosylation Factor 1AnimalsCoat Protein Complex IEscherichia coliHumansHydrophobic and Hydrophilic InteractionsLipid BilayersMicroscopy, ElectronPhosphatidylcholinesPhosphatidylethanolaminesPhospholipidsProtein TransportSf9 CellsSpectrometry, FluorescenceSpodopteraSurface TensionTransport VesiclesTriglyceridesWaterConceptsCoat protein complex ILipid dropletsCoat protein complexTransport vesicle formationProtein complex ILipid droplet targetingBud vesiclesCOPI subunitsProtein complexesCoat componentsNeighboring proteinsIntracellular traffickingVesicle formationComplex IClamp functionPossible direct interactionDirect interactionPhospholipid packingUnsuitable targetsProteinBilayer membranesCoatomerMembraneOrganellesProtomersPreparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores
Shi L, Howan K, Shen QT, Wang YJ, Rothman JE, Pincet F. Preparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores. Nature Protocols 2013, 8: 935-948. PMID: 23598444, DOI: 10.1038/nprot.2013.048.Peer-Reviewed Original ResearchConceptsFusion eventsScaffold proteinFusion poreMembrane scaffold proteinFluorescent lipidFluorescence-based approachFusion machineryAccessible lipidsCognate proteinNanodiscsPore expansionLipid bilayersProteinCargo releaseCargoLipidsPlate readerVAMP2MachineryFluorescenceSnareEncapsulated cargoDirect studyReleaseAssays
2011
CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization
Jégou A, Ziyyat A, Barraud-Lange V, Perez E, Wolf J, Pincet F, Gourier C. CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization. Proceedings Of The National Academy Of Sciences Of The United States Of America 2011, 108: 10946-10951. PMID: 21690351, PMCID: PMC3131345, DOI: 10.1073/pnas.1017400108.Peer-Reviewed Original ResearchConceptsAdhesion sitesFusion-competent sitesWild-type eggsCD9 tetraspaninEgg membrane proteinsSperm-egg fusionMembrane proteinsGamete membranesMammalian fertilizationCompetent sitesGamete interactionAdhesion eventsTight proximityEgg membraneCD9TetraspaninsEggsFertilizationMembraneStrong-interaction modesAdhesion probabilitySitesFusionInteraction modesProtein