We 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>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.
Author affiliation: Jares-Erijman, E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Keywords: alpha synuclein; amyloid; cysteine; FIAsH ligand; ligand; membrane protein; monomer; REAsH ligand; recombinant mutant protein; recombinant protein; tetracysteine; unclassified drug; alpha synuclein; amyloid; cysteine; fluorescent dye; hybrid protein; anisotropy; article; brightness; chemical structure; confocal fluorescence anisotropy; controlled study; diffusion coefficient; fluorescence microscopy; fluorescence recovery after photobleaching; genetic transfection; human; human cell; human tissue; in situ hybridization; molecular density; molecular dynamics; Parkinson disease; protein aggregation; protein structure; quantitative analysis; translation regulation; algorithm; chemistry; confocal microscopy; fluorescence; fluorescence polarization; genetics; kinetics; metabolism; methodology; mutation; single cell analysis; tumor cell line; Algorithms; alpha-Synuclein; Amyloid; Cell Line, Tumor; Cysteine; Fluorescence; Fluorescence Polarization; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Humans; Kinetics; Microscopy, Confocal; Mutation; Recombinant Fusion Proteins; Single-Cell Analysis; Transfection.