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Fluorescence lifetime-assisted probing of protein aggregation with sub-Organellar resolution

2024-10-05Journal article Open access
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dc.contributor.authorGupta, Karnika
dc.contributor.authorMaddison, Daniel C
dc.contributor.authorPinho Melo, Eduardo
dc.contributor.authorRosa da Costa, Ana M
dc.contributor.authorAvezov, Edward
dc.date.accessioned2024-12-05T10:33:58Z
dc.date.available2024-12-05T10:33:58Z
dc.date.issued2024-10-05
dc.description.abstractProtein misfolding fuels multiple neurodegenerative diseases, but existing techniques lack the resolution to pinpoint the location and physical properties of aggregates within living cells. Our protocol describes high-resolution confocal and fluorescent lifetime microscopy (Fast 3D FLIM) of an aggregation probing system. This system involves a metastable HaloTag protein (HT-aggr) labeled with P1 solvatochromic fluorophore, which can be targeted to subcellular compartments. This strategy allows to distinguish between aggregated and folded probe species, since P1 fluorophore changes its lifetime depending on the hydrophobicity of its microenvironment. The probe is not fluorescence intensity-dependent, overcoming issues related to intensity-based measurements of labeled proteins, such as control of probe quantity due to differences in expression or photobleaching of a proportion of the fluorophore population. Our approach reports on the performance of the machinery dealing with aggregation-prone substrates and thus opens doors to studying proteostasis and its role in neurodegenerative diseases. Key features • Aggregation state: Tracks aggregate formation and disaggregation with pulse-chase experiments • Sub-organellar resolution: Pinpoints and allows control of aggregate location within the cell, exceeding traditional techniques • Quantitative analysis: Measures aggregate load through image analysis • Methodology: • Metastable HaloTag variant labeling with a solvatochromic small-molecule reporter ligand • High-resolution confocal microscopy coupled with FLIM for aggregate identification and localization • Image analysis for aggregate quantification and distribution within the ER • Pulse-chase experiments to track aggregates.eng
dc.description.sponsorshipUK Dementia Research Institute through UK DRI Ltd, - Medical Research Council Evelyn Trust Alzheimer's Society Cambridge Indian Ramanujan Scholarship
dc.identifier.doi10.21769/BioProtoc.5080
dc.identifier.other39399587
dc.identifier.urihttp://hdl.handle.net/10400.1/26397
dc.language.isoeng
dc.peerreviewedyes
dc.publisherBIO-PROTOCOL
dc.relationAlgarve Centre for Marine Sciences
dc.relationAlgarve Centre for Marine Sciences
dc.relationCentre for Marine and Environmental Research
dc.rights.urihttp://creativecommons.org/licenses/by-nc/4.0/
dc.subject3D-FLIM
dc.subjectDisaggregation
dc.subjectER
dc.subjectFLIM
dc.subjectHT-aggrER
dc.titleFluorescence lifetime-assisted probing of protein aggregation with sub-Organellar resolutioneng
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleAlgarve Centre for Marine Sciences
oaire.awardTitleAlgarve Centre for Marine Sciences
oaire.awardTitleCentre for Marine and Environmental Research
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04326%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F04326%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/LA%2FP%2F0101%2F2020/PT
oaire.citation.issue19
oaire.citation.startPagee5080
oaire.citation.titleBio-protocol
oaire.citation.volume14
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.versionhttp://purl.org/coar/version/c_970fb48d4fbd8a85
person.familyNamePinho Melo
person.familyNameRosa da Costa
person.givenNameEduardo
person.givenNameAna M
person.identifier1443188
person.identifier305029
person.identifier.ciencia-id3C1C-C10C-1510
person.identifier.ciencia-idA418-A85E-5DB1
person.identifier.orcid0000-0002-0974-8977
person.identifier.orcid0000-0003-0225-9537
person.identifier.ridE-2165-2012
person.identifier.scopus-author-id35566177900
person.identifier.scopus-author-id53986075100
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
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