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Split GFP complementation assay: a novel approach to quantitatively measure aggregation of tau in situ : effects of GSK3β activation and caspase 3 cleavage
Author(s) -
Chun Wanjoo,
Waldo Geoffrey S.,
Johnson Gail V. W.
Publication year - 2007
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2007.04941.x
Subject(s) - green fluorescent protein , gsk 3 , protein fragment complementation assay , complementation , chemistry , cleavage (geology) , phosphorylation , bimolecular fluorescence complementation , biophysics , microbiology and biotechnology , biochemistry , biology , phenotype , gene , paleontology , fracture (geology)
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 3β (GSK3β) 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 GSK3β. Extensive phosphorylation of T4 by GSK3β resulted in increased GFP intensity. T4C3 showed neither efficient phosphorylation nor a significant GFP intensity change by GSK3β. The GFP intensity of T4‐2EC was significantly reduced by GSK3β 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 GSK3β 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.