Split GFP complementation assay: a novel approach to quantitatively measure aggregation of tau in situ: effects of GSK3beta activation and caspase 3 cleavage.
To quantitatively measure tau aggregation in situ, we established a cell model system using a split green fluorescence protein (GFP) complementation assay. In this assay the more aggregated the protein of interest the lower the GFP fluorescence. Tau microtubule-binding domain constructs, whose aggregation characteristics have been described previously (Khlistunova et al. 2006), were used to validate the assay. The aggregation-prone construct exhibited the lowest GFP intensity whereas the aggregation-resistant construct showed the highest GFP intensity. To examine the role of glycogen synthase kinase 3beta (GSK3beta) activity and caspase 3 cleavage on tau aggregation, GFP complementation of full length (T4), caspase-cleaved (T4C3), and pseudophosphorylated at S396/S404 (T4-2EC) tau was examined in the presence of an active or a kinase-dead GSK3beta. Extensive phosphorylation of T4 by GSK3beta resulted in increased GFP intensity. T4C3 showed neither efficient phosphorylation nor a significant GFP intensity change by GSK3beta. The GFP intensity of T4-2EC was significantly reduced by GSK3beta accompanying its presence in the sarkosyl-insoluble fraction, thus demonstrating that T4-2EC was partitioning into aggregates. This indicates that if the majority of tau is phosphorylated at S396/S404, in combination with increased GSK3beta activity, tau aggregation is favored. These data demonstrate that split GFP complementation may be a valuable approach to determine the aggregation process in living cells.
Chun
Wanjoo
W
Department of Psychiatry, University of Alabama at Birmingham School of Medicine, Alabama, USA.
Waldo
Geoffrey S
GS
Johnson
Gail V W
GV
D001665:Binding Sites / Q000502:physiology; D001681:Biological Assay / Q000379:methods; D001921:Brain / Q000378:metabolism / Q000503:physiopathology; D053148:Caspase 3 / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D005453:Fluorescence; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D016874:Neurofibrillary Tangles / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests / Q000379:methods; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D012680:Sensitivity and Specificity; D012694:Serine / Q000378:metabolism; D024801:Tauopathies / Q000175:diagnosis / Q000378:metabolism / Q000503:physiopathology; D016875:tau Proteins / Q000737:chemistry / Q000378:metabolism*
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D016875:tau Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D053148:Caspase 3