Figure 2: SAXS and TEM show that Tau-assembled microtubules in active bundles recapitulate key in vivo features of microtubule spacing and linear bundles.
(a) Azimuthally averaged SAXS data of WT Tau and microtubules registers Bragg peak positions consistent with hexagonal lattices for all six isoforms, as opposed to just microtubule form factor for no Tau (bottom profile). (b–e) Line-shape analysis of the SAXS data (resultant fits in red in a) yields the ensemble-averaged microtubule inner radius <rin> (b), hexagonal lattice parameter aH (c), wall-to-wall distance Dw–w (d) and Dw–w normalized by the calculated projection domain radius of gyration, RGPD (e; see Methods). Parameters plotted are the result of line-shape analyses of two representative data measurements after 12 h to ensure equilibration, with samples made from independent tubulin purifications and Tau expressions/purifications. (f,g) The average Dw–w and Dw–w/RGPD as a function of Tau net charge (QTau) shows a monotonic decrease in Dw–w and a nearly constant Dw–w/RGPD≈8–11, respectively. (3RM and 4RL, coincidentally, have the same charge by AA sequence and thus 4RL has been specially labelled.) (h) Electron microscopy of microtubules assembled with Tau (Φ3RM=1/20) at low magnification show distinct bundled domains, demonstrating phase separation. (i) Domains of hexagonally ordered arrays of microtubules (identified in white outlines, Φ3RL=1/20) with vacancies likely resulting from the suppressed (but still occurring) dynamic instability. (j) Linear bundles of microtubules (Φ3RL=1/20), a result of extensive vacancy introduction and mimicking string-like microtubule bundles in the AIS. In i and j, the staining process exaggerates the microtubule wall thickness. Scale bars, 1 μm (h); 500 nm (i,j).
