Featured Publications
PRRT2 Regulates Synaptic Fusion by Directly Modulating SNARE Complex Assembly
Coleman J, Jouannot O, Ramakrishnan SK, Zanetti MN, Wang J, Salpietro V, Houlden H, Rothman JE, Krishnakumar SS. PRRT2 Regulates Synaptic Fusion by Directly Modulating SNARE Complex Assembly. Cell Reports 2018, 22: 820-831. PMID: 29346777, PMCID: PMC5792450, DOI: 10.1016/j.celrep.2017.12.056.Peer-Reviewed Original ResearchConceptsProline-rich transmembrane protein 2SNARE complex assemblyComplex assemblyTrans-SNARE complex assemblyTerminal proline-rich domainSynaptic SNARE proteinsProline-rich domainParoxysmal neurological disordersSynaptic vesicle primingLive-cell imagingTransmembrane protein 2Synaptic fusionSNARE proteinsVesicle primingSingle exocytotic eventsBiophysical analysisFusion assaysMolecular mechanismsFunction mutationsPhysiological roleExocytotic eventsPre-synaptic terminalsPC12 cellsProtein 2Single vesiclesSynergistic 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 roleFusionExocytosisMachineryProteinBindsMechanismMolecular 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 functionDomainHelixVesiclesFunctionMembraneInteractionNative 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 microscopyMembrane
2024
Photosensitive Nanoprobes for Rapid High Purity Isolation and Size‐Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations (Adv. Funct. Mater. 34/2024)
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 (Adv. Funct. Mater. 34/2024). Advanced Functional Materials 2024, 34 DOI: 10.1002/adfm.202470191.Peer-Reviewed Original ResearchVesicle biogenesisExtracellular vesicle subpopulationsLipid nanoprobesVesicle subpopulationsNative extracellular vesiclesVesicle populationsSeparate vesiclesPurity isolationExtracellular vesiclesVesiclesCellular originHydrophobic interactionsBiogenesisSize variabilitySubpopulationsIsolatesExtracellularPhotosensitive 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 mediaCleavageBreaking ground: A new model for dual-phase insulin exocytosis
Thoduvayil S, Weerakkody J, Ramakrishnan S. Breaking ground: A new model for dual-phase insulin exocytosis. Biophysical Journal 2024, 123: 66a. DOI: 10.1016/j.bpj.2023.11.476.Peer-Reviewed Original ResearchIntegrated suspended lipid membrane platform: Harnessing amperometric and single-molecule capabilities for deep insights into fusion dynamics
Weerakkody J, Thoduvayil S, Ramakrishnan S. Integrated suspended lipid membrane platform: Harnessing amperometric and single-molecule capabilities for deep insights into fusion dynamics. Biophysical Journal 2024, 123: 380a. DOI: 10.1016/j.bpj.2023.11.2323.Peer-Reviewed Original Research
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 actionProteinAssemblyChaperonesSynaptotagminExocytosisBilayersRoles 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
Suspended Lipid Bilayer: A Versatile Platform for Nextgen Drug Discovery and Biomedical Applications
Ramakrishnan S, Weerakkody J. Suspended Lipid Bilayer: A Versatile Platform for Nextgen Drug Discovery and Biomedical Applications. Accounts Of Materials Research 2022, 3: 996-998. DOI: 10.1021/accountsmr.2c00157.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 clustersCopies
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 setupRecapitulationSynaptotagmin 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 Research
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 compartment
2017
Ellipsometric study of peptide layers – island-like character, depolarization and quasi-absorption
Pápa Z, Ramakrishnan S, Martin M, Cloitre T, Zimányi L, Tóth Z, Gergely C, Budai J. Ellipsometric study of peptide layers – island-like character, depolarization and quasi-absorption. Applied Surface Science 2017, 421: 707-713. DOI: 10.1016/j.apsusc.2017.02.263.Peer-Reviewed Original ResearchCover Image, Volume 7, Issue 1
Ramakrishnan S, Zhu J, Gergely C. Cover Image, Volume 7, Issue 1. Wiley Interdisciplinary Reviews Computational Molecular Science 2017, 7 DOI: 10.1002/wcms.1302.Peer-Reviewed Original ResearchOrganic–inorganic interface simulation for new material discoveries
Ramakrishnan SK, Zhu J, Gergely C, Wiley Interdisciplinary Reviews: Computational Molecular Science 2017, 7, e1277.Peer-Reviewed Reviews, Practice Guidelines, Standards, and Consensus Statements