2024
Synaptotagmin-1 and synaptotagmin-7 synergistically regulate the timing and plasticity of Ca2+-evoked vesicular release process
Bose D, Bera M, Norman C, Volynski K, Krishnakumar S. Synaptotagmin-1 and synaptotagmin-7 synergistically regulate the timing and plasticity of Ca2+-evoked vesicular release process. Biophysical Journal 2024, 123: 381a. DOI: 10.1016/j.bpj.2023.11.2327.Peer-Reviewed Original Research
2023
The release of inhibition model reproduces kinetics and plasticity of neurotransmitter release in central synapses
Norman C, Krishnakumar S, Timofeeva Y, Volynski K. The release of inhibition model reproduces kinetics and plasticity of neurotransmitter release in central synapses. Communications Biology 2023, 6: 1091. PMID: 37891212, PMCID: PMC10611806, DOI: 10.1038/s42003-023-05445-2.Peer-Reviewed Original ResearchConceptsFusion clampSV exocytosisSynaptic vesiclesNeurotransmitter releaseSNARE complexSNARE proteinsSV fusionPhysiological timescalesSynaptotagmin-1Synergistic regulationMolecular biochemistryComplete assemblyPresynaptic proteinsSynaptotagmin-7Molecular architectureCalcium bindingExocytosisDual bindingProteinCentral synapsesBindingPlasticitySynaptotagminSnareVesiclesRoles 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 caReconstitutionVAMP2ComplexinMutationsDirect 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 platformVesiclesAssemblyCellsBilayersLeafletsSynaptotagmin rings as high-sensitivity regulators of synaptic vesicle docking and fusion
Zhu J, McDargh ZA, Li F, Krishnakumar SS, Rothman JE, O’Shaughnessy B. Synaptotagmin rings as high-sensitivity regulators of synaptic vesicle docking and fusion. Proceedings Of The National Academy Of Sciences Of The United States Of America 2022, 119: e2208337119. PMID: 36103579, PMCID: PMC9499556, DOI: 10.1073/pnas.2208337119.Peer-Reviewed Original ResearchConceptsVesicle dockingPlasma membrane domainsSynaptic vesiclesCalcium sensor synaptotagminSynaptic vesicle dockingInhibitor of fusionFusion clampSensor synaptotagminMembrane domainsTrigger fusionPlasma membraneC2AB domainAnionic phospholipid bilayersNeuronal synapsesMembrane compositionPhospholipid monolayersATP levelsVesiclesExocytotic releaseDockingPhospholipid bilayersMolecular 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 functionDomainHelixVesiclesFunctionMembraneInteraction
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 clustersCopiesSymmetrical arrangement of proteins under release-ready vesicles in presynaptic terminals
Radhakrishnan A, Li X, Grushin K, Krishnakumar SS, Liu J, Rothman JE. Symmetrical arrangement of proteins under release-ready vesicles in presynaptic terminals. Proceedings Of The National Academy Of Sciences Of The United States Of America 2021, 118: e2024029118. PMID: 33468631, PMCID: PMC7865176, DOI: 10.1073/pnas.2024029118.Peer-Reviewed Original ResearchConceptsPlasma membraneSynaptic vesiclesSV fusionRelease-ready vesiclesFusion machinerySingle SNAREpinSV releaseExocytosis machineryMolecular eventsNative conditionsProtein componentsCultured hippocampal neuronsPriming reactionPresynaptic CaVesiclesFundamental processesProtein densityProtein massRelease of neurotransmittersNeurotransmitter releaseMachineryPresynaptic terminalsReleasable poolHippocampal neuronsVariable number
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 Research
2019
Structural basis for the clamping and Ca2+ activation of SNARE-mediated fusion by synaptotagmin
Grushin K, Wang J, Coleman J, Rothman JE, Sindelar CV, Krishnakumar SS. Structural basis for the clamping and Ca2+ activation of SNARE-mediated fusion by synaptotagmin. Nature Communications 2019, 10: 2413. PMID: 31160571, PMCID: PMC6546687, DOI: 10.1038/s41467-019-10391-x.Peer-Reviewed Original ResearchConceptsCryo-electron microscopy structureActivation of SNAREsDependent membrane interactionsAnionic lipid headgroupsFusion clampActivator functionSNARE bundleSNARE proteinsMicroscopy structureC2B domainStructural basisSynaptotagmin-1SNAREpinsAliphatic loopsMembrane interactionsComplete assemblyLipid headgroupsLipid membranesNeurotransmitter releaseMembraneKey determinantSynaptotagminSyt1Calcium influxPartial insertionMechanisms of Neurological Dysfunction in GOSR2 Progressive Myoclonus Epilepsy, a Golgi SNAREopathy
Jepson JEC, Praschberger R, Krishnakumar SS. Mechanisms of Neurological Dysfunction in GOSR2 Progressive Myoclonus Epilepsy, a Golgi SNAREopathy. Neuroscience 2019, 420: 41-49. PMID: 30954670, DOI: 10.1016/j.neuroscience.2019.03.057.Peer-Reviewed Original ResearchConceptsEndoplasmic reticulumSNARE proteinsProgressive myoclonus epilepsySecretory trafficking pathwaysCis-Golgi membranesMis-sense mutationsTransport vesiclesGolgi transportTrafficking pathwaysVesicles budSecretory pathwaySuccessive fusion eventsTarget membraneFusion eventsEssential functionsDevelopmental defectsMolecular mechanismsMyoclonus epilepsyProteinFusion stepSevere neurological disordersMutationsMembranePathwayInitial stepMutations in the Neuronal Vesicular SNARE VAMP2 Affect Synaptic Membrane Fusion and Impair Human Neurodevelopment
Salpietro V, Malintan NT, Llano-Rivas I, Spaeth CG, Efthymiou S, Striano P, Vandrovcova J, Cutrupi MC, Chimenz R, David E, Di Rosa G, Marce-Grau A, Raspall-Chaure M, Martin-Hernandez E, Zara F, Minetti C, Study D, Group S, Salpietro V, Efthymiou S, Kriouile Y, Khorassani M, Aguennouz M, Karashova B, Avdjieva D, Kathom H, Tincheva R, Van Maldergem L, Nachbauer W, Boesch S, Arning L, Timmann D, Cormand B, Pérez-Dueñas B, Di Rosa G, Pironti E, Goraya J, Sultan T, Kirmani S, Ibrahim S, Jan F, Mine J, Banu S, Veggiotti P, Ferrari M, Verrotti A, Marseglia G, Savasta S, Garavaglia B, Scuderi C, Borgione E, Dipasquale V, Cutrupi M, Portaro S, Sanchez B, Pineda-Marfa’ M, Munell F, Macaya A, Boles R, Heimer G, Papacostas S, Manole A, Malintan N, Zanetti M, Hanna M, Rothman J, Kullmann D, Houlden H, Bello O, De Zorzi R, Fortuna S, Dauber A, Alkhawaja M, Sultan T, Mankad K, Vitobello A, Thomas Q, Mau-Them F, Faivre L, Martinez-Azorin F, Prada C, Macaya A, Kullmann D, Rothman J, Krishnakumar S, Houlden H. Mutations in the Neuronal Vesicular SNARE VAMP2 Affect Synaptic Membrane Fusion and Impair Human Neurodevelopment. American Journal Of Human Genetics 2019, 104: 721-730. PMID: 30929742, PMCID: PMC6451933, DOI: 10.1016/j.ajhg.2019.02.016.Peer-Reviewed Original ResearchMeSH KeywordsAdolescentAutistic DisorderBrainChildChild, PreschoolEpilepsyExocytosisFemaleHeterozygoteHumansIntellectual DisabilityLipidsMagnetic Resonance ImagingMaleMembrane FusionMovement DisordersMuscle HypotoniaMutationNeurodevelopmental DisordersNeuronsNeurotransmitter AgentsPhenotypeProtein DomainsR-SNARE ProteinsSynapsesVesicle-Associated Membrane Protein 2ConceptsNon-synonymous variantsDe novo mutationsSNARE protein VAMP2Synaptic membrane fusionC-terminal regionNovo mutationsSNARE motifSynaptosomal-associated protein 25C-terminusMembrane fusionVAMP2Vesicle fusionHuman brain developmentAcid deletionSynaptic vesiclesVesicular exocytosisHeterozygous de novo mutationsProtein 25Hyperkinetic movement disordersAdditional neurological featuresHuman neurodevelopmentCentral visual impairmentDisease mechanismsUnrelated individualsMutationsSynaptotagmin oligomers are necessary and can be sufficient to form a Ca2+‐sensitive fusion clamp
Ramakrishnan S, Bera M, Coleman J, Krishnakumar SS, Pincet F, Rothman JE. Synaptotagmin oligomers are necessary and can be sufficient to form a Ca2+‐sensitive fusion clamp. FEBS Letters 2019, 593: 154-162. PMID: 30570144, PMCID: PMC6349546, DOI: 10.1002/1873-3468.13317.Peer-Reviewed Original ResearchSymmetrical organization of proteins under docked synaptic vesicles
Li X, Radhakrishnan A, Grushin K, Kasula R, Chaudhuri A, Gomathinayagam S, Krishnakumar SS, Liu J, Rothman JE. Symmetrical organization of proteins under docked synaptic vesicles. FEBS Letters 2019, 593: 144-153. PMID: 30561792, PMCID: PMC6353562, DOI: 10.1002/1873-3468.13316.Peer-Reviewed Original ResearchConceptsCryo-electron tomography analysisSymmetrical organizationCalcium-regulated exocytosisMunc18 proteinsProtein machineryFusion machinerySingle SNAREpinCircular oligomersMutational analysisRadial positioningSynaptic vesiclesRelease machineryMachinerySynaptotagminProteinRing hypothesisVesiclesObserved arrangementUnderlying mechanismSNAREpinsComplexinNerve growthExocytosisGrowth
2018
Using Nanodiscs to Probe Ca2+-Dependent Membrane Interaction of Synaptotagmin-1
Stroeva E, Krishnakumar SS. Using Nanodiscs to Probe Ca2+-Dependent Membrane Interaction of Synaptotagmin-1. Methods In Molecular Biology 2018, 1860: 221-236. PMID: 30317508, DOI: 10.1007/978-1-4939-8760-3_14.BooksSynaptotagmin oligomerization is essential for calcium control of regulated exocytosis
Bello OD, Jouannot O, Chaudhuri A, Stroeva E, Coleman J, Volynski KE, Rothman JE, Krishnakumar SS. Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis. Proceedings Of The National Academy Of Sciences Of The United States Of America 2018, 115: e7624-e7631. PMID: 30038018, PMCID: PMC6094142, DOI: 10.1073/pnas.1808792115.Peer-Reviewed Original ResearchConceptsRegulated exocytosisFusion machineryC2 domain proteinsCore fusion machinerySingle vesicle exocytosisConstitutive exocytosisPrincipal CaVesicular releaseMolecular mechanismsSensitive oligomersExocytosisPheochromocytoma cellsSelective disruptionSpontaneous fusionCritical roleMachineryOligomerizationDirect activationCentral componentStructural featuresConsiderable insightCalcium controlPHluorinSyt1SYT