Fig. 4

Aggregation of Tau proteins with GFP tags monitored by light scattering and electron microscopy. (a-b) Attachment of GFP to Tau repeat domain severely inhibits aggregation, but full length Tau+GFP remains aggregation competent. Aggregation of Tau to PHF-like fibers was monitored by light scattering at 350 nm. 50 μM of Tau^FL-wt or Tau^RDΔK with or without GFP on either end was incubated in the presence of heparin 16,000 (12.5 μM) in BES buffer, pH 7.0, at 37 °C at different time intervals. (a) Unlabeled Tau^FL-wt (green) aggregates mostly into PHF-like fibers with t_1/2 ~ 3 h. GFP–Tau^FL-wt (blue, t_1/2 ~ 2 h) and Tau^FL-wt–GFP (magenta, t_1/2 ~ 12 h) aggregate with distinct rates and reach higher final levels, consistent with the higher mass of the subunits and variations in elongation. (b) Unlabeled Tau^RDΔK (green curve) shows aggregation with t_1/2 ~ 6 h into PHF-like fibers. By contrast, GFP-Tau^RDΔK (blue) shows no increase in light scattering, and Tau^RDΔK–GFP (magenta) reaches only a low level of light scattering saturating at ~ 2 h. Thus in both cases the aggregation of GFP-labeled protein is severely inhibited. (c-h) Electron microscopy reveals heterogeneous aggregation forms of Tau + GFP Samples of Tau^FL-wt, Tau^RDΔK with and without GFP aggregated for 24 h at 37 °C were placed on carbon grids and imaged by negative stain electron microscopy. Top row, Tau^FL-wt proteins with or without GFP forms typical long filaments (c-e), whereas repeat domain Tau^RDΔK does not (f-h, bottom row). (c) Tau^FL-wt aggregates into typical PHF-like twisted filaments (diameter ~ 28 nm). (d) GFP-Tau^FL-wt forms long filaments with increased diameter (~ 40 nm). (e) Tau^FL-wt-GFP forms long filaments and bundles (~ 28 nm). (f) Repeat domain Tau^RDΔK without GFP forms filaments of typical PHF-like morphology but smaller diameter (~ 21 nm). (g) GFP-Tau^RDΔK does not form filaments but only amorphous small aggregates. (h) Tau^RDΔK-GFP forms short filaments and oligomers with the length < 100 nm; average diameter ~ 37 nm