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dc.provenanceFacultad de Ciencias Exactas y Naturales de la UBA-
dc.contributorRoberti, M.J.-
dc.contributorJovin, T.M.-
dc.contributor<div class="autor_fcen" id="4466">Jares-Erijman, E.</div>-
dc.creatorRoberti, M.J.-
dc.creatorJovin, T.M.-
dc.creator<div class="autor_fcen" id="4466">Jares-Erijman, E.</div>-
dc.date.accessioned2018-05-04T21:57:30Z-
dc.date.accessioned2018-05-28T15:49:07Z-
dc.date.available2018-05-04T21:57:30Z-
dc.date.available2018-05-28T15:49:07Z-
dc.date.issued2011-
dc.identifier.urihttp://10.0.0.11:8080/jspui/handle/bnmm/68608-
dc.descriptionWe assessed the intracellular association states of the Parkinson's disease related protein α-synuclein (AS) in living cells by transfection with a functional recombinant mutant protein (AS-C4) bearing a tetracysteine tag binding the fluorogenic biarsenical ligands FlAsH and ReAsH, The aggregation states of AS-C4 were assessed by in situ microscopy of molecular translational mobility with FRAP (fluorescence recovery after photobleaching) and of local molecular density with confocal fluorescence anisotropy (CFA). FRAP recovery was quantitative and rapid in regions of free protein, whereas AS in larger aggregates was&gt;80% immobile. A small 16% recovery characterized by an apparent diffusion constant of 0.03-0.04 μm 2/s was attributed to the dynamics of smaller, associated forms of AS-C4 and the exchange of mobile species with the larger immobile aggregates. By CFA, the larger aggregates exhibited high brightness and very low anisotropy, consistent with homoFRET between closely packed AS, for which a Förster distance (R o) of 5.3 nm was calculated. Other bright regions had high anisotropy values, close to that of monomeric AS, and indicative of membrane-associated protein with both low mobility and low degree of association. The anisotropy-fluorescence intensity correlations also revealed regions of free protein or of small aggregates, undetectable by conventional fluorescence imaging alone. The combined strategy (FRAP+CFA) provides a highly sensitive means for elucidating both the dynamics and structural features of protein aggregates and other intracellular complexes in living cells, and can be extended to other amyloid systems and to drug screening protocols. © 2011 Roberti et al.-
dc.descriptionFil:Jares-Erijman, E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.-
dc.formatapplication/pdf-
dc.languageeng-
dc.rightsinfo:eu-repo/semantics/openAccess-
dc.rightshttp://creativecommons.org/licenses/by/2.5/ar-
dc.sourcePLoS ONE 2011;6(8)-
dc.source.urihttp://digital.bl.fcen.uba.ar/Download/paper/paper_19326203_v6_n8_p_Roberti.pdf-
dc.subjectalpha synuclein-
dc.subjectamyloid-
dc.subjectcysteine-
dc.subjectFIAsH ligand-
dc.subjectligand-
dc.subjectmembrane protein-
dc.subjectmonomer-
dc.subjectREAsH ligand-
dc.subjectrecombinant mutant protein-
dc.subjectrecombinant protein-
dc.subjecttetracysteine-
dc.subjectunclassified drug-
dc.subjectalpha synuclein-
dc.subjectamyloid-
dc.subjectcysteine-
dc.subjectfluorescent dye-
dc.subjecthybrid protein-
dc.subjectanisotropy-
dc.subjectarticle-
dc.subjectbrightness-
dc.subjectchemical structure-
dc.subjectconfocal fluorescence anisotropy-
dc.subjectcontrolled study-
dc.subjectdiffusion coefficient-
dc.subjectfluorescence microscopy-
dc.subjectfluorescence recovery after photobleaching-
dc.subjectgenetic transfection-
dc.subjecthuman-
dc.subjecthuman cell-
dc.subjecthuman tissue-
dc.subjectin situ hybridization-
dc.subjectmolecular density-
dc.subjectmolecular dynamics-
dc.subjectParkinson disease-
dc.subjectprotein aggregation-
dc.subjectprotein structure-
dc.subjectquantitative analysis-
dc.subjecttranslation regulation-
dc.subjectalgorithm-
dc.subjectchemistry-
dc.subjectconfocal microscopy-
dc.subjectfluorescence-
dc.subjectfluorescence polarization-
dc.subjectgenetics-
dc.subjectkinetics-
dc.subjectmetabolism-
dc.subjectmethodology-
dc.subjectmutation-
dc.subjectsingle cell analysis-
dc.subjecttumor cell line-
dc.subjectAlgorithms-
dc.subjectalpha-Synuclein-
dc.subjectAmyloid-
dc.subjectCell Line, Tumor-
dc.subjectCysteine-
dc.subjectFluorescence-
dc.subjectFluorescence Polarization-
dc.subjectFluorescence Recovery After Photobleaching-
dc.subjectFluorescent Dyes-
dc.subjectHumans-
dc.subjectKinetics-
dc.subjectMicroscopy, Confocal-
dc.subjectMutation-
dc.subjectRecombinant Fusion Proteins-
dc.subjectSingle-Cell Analysis-
dc.subjectTransfection-
dc.titleConfocal fluorescence anisotropy and FRAP imaging of α-synuclein amyloid aggregates in living cells-
dc.typeinfo:eu-repo/semantics/article-
dc.typeinfo:ar-repo/semantics/artículo-
dc.typeinfo:eu-repo/semantics/publishedVersion-
Aparece en las colecciones: FCEN - Facultad de Ciencias Exactas y Naturales. UBA

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