Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein

α-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant α-...

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Autores principales:	Roberti, M.J., Bertoncini, C.W., Klement, R., Jares-Erijman, E.A., Jovin, T.M.
Formato:	JOUR
Materias:	alpha synuclein amyloid cysteine fluorescent dye hybrid protein reactive oxygen metabolite article cell stress fluorescence analysis fluorescence resonance energy transfer oxidative stress Parkinson disease priority journal protein aggregation protein protein interaction chemistry confocal microscopy Escherichia coli fluorescence microscopy gene vector genetic procedures genetic transfection genetics human metabolism methodology tumor cell line alpha-Synuclein Amyloid Biosensing Techniques Cell Line, Tumor Cysteine Fluorescence Resonance Energy Transfer Fluorescent Dyes Genetic Vectors Humans Microscopy, Confocal Microscopy, Fluorescence Oxidative Stress Reactive Oxygen Species Recombinant Fusion Proteins Transfection
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_15487091_v4_n4_p345_Roberti
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	todo:paper_15487091_v4_n4_p345_Roberti
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spelling	todo:paper_15487091_v4_n4_p345_Roberti2023-10-03T16:23:12Z Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein Roberti, M.J. Bertoncini, C.W. Klement, R. Jares-Erijman, E.A. Jovin, T.M. alpha synuclein amyloid amyloid cysteine fluorescent dye hybrid protein reactive oxygen metabolite article cell stress fluorescence analysis fluorescence resonance energy transfer oxidative stress Parkinson disease priority journal protein aggregation protein protein interaction chemistry confocal microscopy Escherichia coli fluorescence microscopy fluorescence resonance energy transfer gene vector genetic procedures genetic transfection genetics human metabolism methodology tumor cell line alpha-Synuclein Amyloid Biosensing Techniques Cell Line, Tumor Cysteine Escherichia coli Fluorescence Resonance Energy Transfer Fluorescent Dyes Genetic Vectors Humans Microscopy, Confocal Microscopy, Fluorescence Oxidative Stress Reactive Oxygen Species Recombinant Fusion Proteins Transfection α-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant α-synuclein (α-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of α-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of α-synuclein-C4 transfected or microinjected into cells, particularly under oxidative stress conditions. Fluorescence resonance energy transfer (FRET) between FlAsH and ReAsH confirmed the close association of fibrillized α-synuclein-C4 molecules. α-synuclein-C4 offers the means for directly probing amyloid formation and interactions of α-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease. Fil:Roberti, M.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Jares-Erijman, E.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_15487091_v4_n4_p345_Roberti
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	alpha synuclein amyloid amyloid cysteine fluorescent dye hybrid protein reactive oxygen metabolite article cell stress fluorescence analysis fluorescence resonance energy transfer oxidative stress Parkinson disease priority journal protein aggregation protein protein interaction chemistry confocal microscopy Escherichia coli fluorescence microscopy fluorescence resonance energy transfer gene vector genetic procedures genetic transfection genetics human metabolism methodology tumor cell line alpha-Synuclein Amyloid Biosensing Techniques Cell Line, Tumor Cysteine Escherichia coli Fluorescence Resonance Energy Transfer Fluorescent Dyes Genetic Vectors Humans Microscopy, Confocal Microscopy, Fluorescence Oxidative Stress Reactive Oxygen Species Recombinant Fusion Proteins Transfection
spellingShingle	alpha synuclein amyloid amyloid cysteine fluorescent dye hybrid protein reactive oxygen metabolite article cell stress fluorescence analysis fluorescence resonance energy transfer oxidative stress Parkinson disease priority journal protein aggregation protein protein interaction chemistry confocal microscopy Escherichia coli fluorescence microscopy fluorescence resonance energy transfer gene vector genetic procedures genetic transfection genetics human metabolism methodology tumor cell line alpha-Synuclein Amyloid Biosensing Techniques Cell Line, Tumor Cysteine Escherichia coli Fluorescence Resonance Energy Transfer Fluorescent Dyes Genetic Vectors Humans Microscopy, Confocal Microscopy, Fluorescence Oxidative Stress Reactive Oxygen Species Recombinant Fusion Proteins Transfection Roberti, M.J. Bertoncini, C.W. Klement, R. Jares-Erijman, E.A. Jovin, T.M. Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein
topic_facet	alpha synuclein amyloid amyloid cysteine fluorescent dye hybrid protein reactive oxygen metabolite article cell stress fluorescence analysis fluorescence resonance energy transfer oxidative stress Parkinson disease priority journal protein aggregation protein protein interaction chemistry confocal microscopy Escherichia coli fluorescence microscopy fluorescence resonance energy transfer gene vector genetic procedures genetic transfection genetics human metabolism methodology tumor cell line alpha-Synuclein Amyloid Biosensing Techniques Cell Line, Tumor Cysteine Escherichia coli Fluorescence Resonance Energy Transfer Fluorescent Dyes Genetic Vectors Humans Microscopy, Confocal Microscopy, Fluorescence Oxidative Stress Reactive Oxygen Species Recombinant Fusion Proteins Transfection
description	α-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant α-synuclein (α-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of α-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of α-synuclein-C4 transfected or microinjected into cells, particularly under oxidative stress conditions. Fluorescence resonance energy transfer (FRET) between FlAsH and ReAsH confirmed the close association of fibrillized α-synuclein-C4 molecules. α-synuclein-C4 offers the means for directly probing amyloid formation and interactions of α-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease.
format	JOUR
author	Roberti, M.J. Bertoncini, C.W. Klement, R. Jares-Erijman, E.A. Jovin, T.M.
author_facet	Roberti, M.J. Bertoncini, C.W. Klement, R. Jares-Erijman, E.A. Jovin, T.M.
author_sort	Roberti, M.J.
title	Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein
title_short	Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein
title_full	Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein
title_fullStr	Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein
title_full_unstemmed	Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein
title_sort	fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein
url	http://hdl.handle.net/20.500.12110/paper_15487091_v4_n4_p345_Roberti
work_keys_str_mv	AT robertimj fluorescenceimagingofamyloidformationinlivingcellsbyafunctionaltetracysteinetaggedasynuclein AT bertoncinicw fluorescenceimagingofamyloidformationinlivingcellsbyafunctionaltetracysteinetaggedasynuclein AT klementr fluorescenceimagingofamyloidformationinlivingcellsbyafunctionaltetracysteinetaggedasynuclein AT jareserijmanea fluorescenceimagingofamyloidformationinlivingcellsbyafunctionaltetracysteinetaggedasynuclein AT jovintm fluorescenceimagingofamyloidformationinlivingcellsbyafunctionaltetracysteinetaggedasynuclein
_version_	1807323311722463232

Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein

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