2024
Quantitative single-molecule analysis of assembly and Ca2+-dependent disassembly of synaptotagmin oligomers on lipid bilayers
Li F, Coleman J, Redondo-Morata L, Kalyana Sundaram R, Stroeva E, Rothman J, Pincet F. Quantitative single-molecule analysis of assembly and Ca2+-dependent disassembly of synaptotagmin oligomers on lipid bilayers. Communications Biology 2024, 7: 1608. PMID: 39627539, PMCID: PMC11615320, DOI: 10.1038/s42003-024-07317-9.Peer-Reviewed Original ResearchConceptsSyt-1Lipid bilayerRing-like oligomersCa2+-evoked releaseSynaptotagmin-1Single-molecule imaging methodsSynaptic vesiclesBiochemical evidencePhysiological requirementsOligomerizationAnalysis of assembliesBilayerOligomersCa2+LipidAssemblyCa2Classes of oligomersMutationsVesiclesDisassemblyEvoked release
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 actionProteinAssemblyChaperonesSynaptotagminExocytosisBilayersDiacylglycerol-dependent hexamers of the SNARE-assembling chaperone Munc13-1 cooperatively bind vesicles
Li F, Grushin K, Coleman J, Pincet F, Rothman J. Diacylglycerol-dependent hexamers of the SNARE-assembling chaperone Munc13-1 cooperatively bind vesicles. Proceedings Of The National Academy Of Sciences Of The United States Of America 2023, 120: e2306086120. PMID: 37883433, PMCID: PMC10623011, DOI: 10.1073/pnas.2306086120.Peer-Reviewed Original ResearchRoles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery
Sundaram R, Chatterjee A, Bera M, Grushin K, Panda A, Li F, Coleman J, Lee S, Ramakrishnan S, Ernst A, Gupta K, Rothman J, Krishnakumar S. Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery. Proceedings Of The National Academy Of Sciences Of The United States Of America 2023, 120: e2309516120. PMID: 37590407, PMCID: PMC10450444, DOI: 10.1073/pnas.2309516120.Peer-Reviewed Original ResearchConceptsCore protein machineryRelease-ready vesiclesSynaptic vesicle primingVesicle primingProtein machinerySingle-molecule imagingSNAREpin assemblyFunctional intermediatesFunctional reconstitutionMunc13DiacylglycerolCoordinated actionMunc18VesiclesMachineryComplete reconstitutionNew roleSelective effectDetailed characterizationChaperonesRate of caReconstitutionVAMP2ComplexinMutationsRapid Quantification of First and Second Phase Insulin Secretion Dynamics using an In vitro Platform for Improving Insulin Therapy
Thoduvayil S, Weerakkody J, Sundaram R, Topper M, Bera M, Coleman J, Li X, Mariappan M, Ramakrishnan S. Rapid Quantification of First and Second Phase Insulin Secretion Dynamics using an In vitro Platform for Improving Insulin Therapy. Cell Calcium 2023, 113: 102766. PMID: 37295201, PMCID: PMC10450995, DOI: 10.1016/j.ceca.2023.102766.Peer-Reviewed Original ResearchConceptsCellular pathwaysSecretion dynamicsSmall molecule screeningLuciferase reporter systemInsulin secretion dynamicsHigh-throughput compoundInsulin secretionHigh-throughput quantificationReporter systemGenetic studiesLive cellsDistinct rolesInsulin therapyGlucose-stimulated insulin secretionSmall moleculesEffective insulin therapyPathwaySecretionDirect 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
Native Planar Asymmetric Suspended Membrane for Single‐Molecule Investigations: Plasma Membrane on a Chip (Small 51/2022)
Sundaram R, Bera M, Coleman J, Weerakkody J, Krishnakumar S, Ramakrishnan S. Native Planar Asymmetric Suspended Membrane for Single‐Molecule Investigations: Plasma Membrane on a Chip (Small 51/2022). Small 2022, 18 DOI: 10.1002/smll.202270277.Peer-Reviewed Original ResearchGiant plasma membrane vesiclesTotal internal reflection fluorescence microscopyMembrane protein assemblyPlasma membrane vesiclesReflection fluorescence microscopyDifferent cell typesSingle-molecule investigationsProtein functionProtein assembliesInner leafletPlasma membraneMembrane vesiclesCell typesLipid architectureFluorescence microscopyLipid membranesMolecule investigationsMembraneSilicon-based platformVesiclesAssemblyCellsBilayersLeafletsNative Planar Asymmetric Suspended Membrane for Single‐Molecule Investigations: Plasma Membrane on a Chip
Sundaram R, Bera M, Coleman J, Weerakkody J, Krishnakumar S, Ramakrishnan S. Native Planar Asymmetric Suspended Membrane for Single‐Molecule Investigations: Plasma Membrane on a Chip. Small 2022, 18: e2205567. PMID: 36328714, DOI: 10.1002/smll.202205567.Peer-Reviewed Original ResearchConceptsPlasma membraneProtein assembliesNative plasma membrane vesiclesTotal internal reflection fluorescence microscopySingle-molecule levelSingle-molecule investigationsCellular plasma membranePlasma membrane vesiclesSingle-molecule precisionReflection fluorescence microscopyKnowledge of lipidProtein complexesProtein architectureCell signalingMovement of moleculesProtein orientationLipid membranesBiological processesCellular membranesMembrane vesiclesMicroarray platformFluorescence investigationsLipid domainsFluorescence microscopyMembraneA Quantitative Native Mass Spectrometry Platform for Deconstructing Hierarchical Organization of Membrane Proteins and Lipids
Panda A, Giska F, Brown C, Coleman J, Rothman J, Gupta K. A Quantitative Native Mass Spectrometry Platform for Deconstructing Hierarchical Organization of Membrane Proteins and Lipids. The FASEB Journal 2022, 36 DOI: 10.1096/fasebj.2022.36.s1.0r472.Peer-Reviewed Original ResearchMembrane proteinsOligomeric stateSpecific lipidsBiophysical propertiesSugar transporter proteinsPhysiological membranesBacterial plasma membraneTarget membrane proteinsLipid bilayer environmentSynaptic vesicle proteinsLipid compositionMS/MS capabilitiesProtein oligomerizationCellular signalingOligomeric organizationVesicle proteinsMembrane curvaturePlasma membraneMacromolecular assembliesTransporter proteinsNative massOligomeric populationMS/MS analysisRegulatory roleDiverse setMolecular determinants of complexin clamping and activation function
Bera M, Ramakrishnan S, Coleman J, Krishnakumar SS, Rothman JE. Molecular determinants of complexin clamping and activation function. ELife 2022, 11: e71938. PMID: 35442188, PMCID: PMC9020821, DOI: 10.7554/elife.71938.Peer-Reviewed Original ResearchConceptsSynaptotagmin-1Single-vesicle fusionAccessory helixFusion clampHelical domainMolecular detailsComplexinMutational analysisVesicle releaseFusion kineticsMolecular determinantsSpecific interactionsInhibitory functionProbability of fusionRapid CaSNAREpinsAssembly processFusionClamping functionDomainHelixVesiclesFunctionMembraneInteractionA tunable lipid bilayer native MS platform for direct determination of hierarchical organization of membrane proteins and lipids at the membrane
Panda A, Giska F, Brown C, Coleman J, Rothman J, Gupta K. A tunable lipid bilayer native MS platform for direct determination of hierarchical organization of membrane proteins and lipids at the membrane. Biophysical Journal 2022, 121: 312a-313a. DOI: 10.1016/j.bpj.2021.11.1192.Peer-Reviewed Original Research
2021
Vesicle capture by membrane‐bound Munc13‐1 requires self‐assembly into discrete clusters
Li F, Sundaram R, Gatta AT, Coleman J, Ramakrishnan S, Krishnakumar SS, Pincet F, Rothman JE. Vesicle capture by membrane‐bound Munc13‐1 requires self‐assembly into discrete clusters. FEBS Letters 2021, 595: 2185-2196. PMID: 34227103, DOI: 10.1002/1873-3468.14157.Peer-Reviewed Original ResearchConceptsMunc13-1Vesicle captureSpecific plasma membrane domainsStep-wise photobleachingC-domainMunc13-1 proteinPlasma membrane domainsSynaptic vesicle dockingC-terminal CVesicle dockingMembrane domainsTIRF microscopySoluble proteinVesicle membraneActive zoneMultiple copiesSynaptic vesiclesFunctional significanceSmall unilamellar vesiclesLipid bilayersVesiclesUnilamellar vesiclesProteinDiscrete clustersCopiesNascent fusion pore opening monitored at single-SNAREpin resolution
Heo P, Coleman J, Fleury JB, Rothman JE, Pincet F. Nascent fusion pore opening monitored at single-SNAREpin resolution. Proceedings Of The National Academy Of Sciences Of The United States Of America 2021, 118: e2024922118. PMID: 33495324, PMCID: PMC7865171, DOI: 10.1073/pnas.2024922118.Peer-Reviewed Original ResearchConceptsFusion poreFusion pore openingTransient fusion poresSingle fusion eventFast neurotransmitter releaseSingle SNAREpinAsymmetric lipid bilayerProtein complexesSNAREpinsTarget membraneFusion eventsCellular traffickingVesicle fusionSmall vesiclesIon channelsLipid bilayersCargo transportKey eventsEnergy landscapePore openingNeurotransmitter releaseDiscrete sizesVesiclesSynaptic transmissionCargo release
2020
Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly
Sundaram R, Jin H, Li F, Shu T, Coleman J, Yang J, Pincet F, Zhang Y, Rothman JE, Krishnakumar SS. Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly. FEBS Letters 2020, 595: 297-309. PMID: 33222163, PMCID: PMC8068094, DOI: 10.1002/1873-3468.14006.Peer-Reviewed Original ResearchMeSH KeywordsAnimalsBinding SitesCloning, MolecularEscherichia coliGene ExpressionGenetic VectorsLipid BilayersLiposomesMiceModels, MolecularNerve Tissue ProteinsOptical TweezersPhosphatidylcholinesPhosphatidylethanolaminesPhosphatidylserinesPolyethylene GlycolsProtein BindingProtein Conformation, alpha-HelicalProtein Conformation, beta-StrandProtein Interaction Domains and MotifsRecombinant Fusion ProteinsSynaptosomal-Associated Protein 25Syntaxin 1Vesicle-Associated Membrane Protein 2ConceptsSNARE complex assemblyComplex assemblyMunc13-1 MUN domainDetailed structure-function analysisSNARE protein VAMP2Syntaxin 1/Structure-function analysisSynaptic vesicle fusionOptical tweezers studiesSNARE assemblySNARE motifMUN domainMunc18-1Syntaxin-1Munc13-1FRET spectroscopyLinker regionVesicle fusionDirect bindingPhospholipid bilayersPresynaptic membraneSNAP25AssemblyBindingRecruitsSynaptotagmin-1 membrane binding is driven by the C2B domain and assisted cooperatively by the C2A domain
Gruget C, Bello O, Coleman J, Krishnakumar SS, Perez E, Rothman JE, Pincet F, Donaldson SH. Synaptotagmin-1 membrane binding is driven by the C2B domain and assisted cooperatively by the C2A domain. Scientific Reports 2020, 10: 18011. PMID: 33093513, PMCID: PMC7581758, DOI: 10.1038/s41598-020-74923-y.Peer-Reviewed Original ResearchSynergistic roles of Synaptotagmin-1 and complexin in calcium-regulated neuronal exocytosis
Ramakrishnan S, Bera M, Coleman J, Rothman JE, Krishnakumar SS. Synergistic roles of Synaptotagmin-1 and complexin in calcium-regulated neuronal exocytosis. ELife 2020, 9: e54506. PMID: 32401194, PMCID: PMC7220375, DOI: 10.7554/elife.54506.Peer-Reviewed Original ResearchConceptsSynaptotagmin-1Vesicular fusion machinerySingle-vesicle fusionFusion of vesiclesSNARE complexFusion machineryNeuronal exocytosisOligomer bindsRegulatory proteinsVesicle fusionSNAREpinsSynchronous fusionSynaptic vesiclesNovel mechanismVesiclesComplexinKinetic delayPrimary interfaceSynergistic roleFusionExocytosisMachineryProteinBindsMechanismSynaptotagmin 1 oligomers clamp and regulate different modes of neurotransmitter release
Tagliatti E, Bello OD, Mendonça PRF, Kotzadimitriou D, Nicholson E, Coleman J, Timofeeva Y, Rothman JE, Krishnakumar SS, Volynski KE. Synaptotagmin 1 oligomers clamp and regulate different modes of neurotransmitter release. Proceedings Of The National Academy Of Sciences Of The United States Of America 2020, 117: 3819-3827. PMID: 32015138, PMCID: PMC7035618, DOI: 10.1073/pnas.1920403117.Peer-Reviewed Original ResearchMunc13 Recruits SNAP25 to Facilitate SNARE Complex Assembly
Sundaram R, Li F, Coleman J, Pincet F, Rothman J, Krishnakumar S. Munc13 Recruits SNAP25 to Facilitate SNARE Complex Assembly. Biophysical Journal 2020, 118: 400a-401a. DOI: 10.1016/j.bpj.2019.11.2274.Peer-Reviewed Original ResearchDissecting the Synergistic Roles of Synaptotagmin and Complexin in Ca2+-Regulated Exocytosis
Ramakrishnan S, Bera M, Coleman J, Pincet F, Rothman J, Krishnakumar S. Dissecting the Synergistic Roles of Synaptotagmin and Complexin in Ca2+-Regulated Exocytosis. Biophysical Journal 2020, 118: 488a. DOI: 10.1016/j.bpj.2019.11.2702.Peer-Reviewed Original ResearchMunc13 Clusters Capture Vesicles to Lipid Bilayer Membrane
Li F, Sundaram R, Coleman J, Krishnakumar S, Pincet F, Rothman J. Munc13 Clusters Capture Vesicles to Lipid Bilayer Membrane. Biophysical Journal 2020, 118: 344a. DOI: 10.1016/j.bpj.2019.11.1990.Peer-Reviewed Original Research