Figure 3: Cooperative envelope formation is a divergent property within the tau-family.

a. Schematics of the MAP proteins analyzed, highlighting the conserved microtubule-binding regions (green), proline-rich region (blue), and pseudo-repeat (yellow). Below: Coomassie stained gels showing purity of the MAP2c and MAP4 proteins used. See Source Data for the uncropped gels. b. Multi-channel fluorescence micrographs showing the binding of 0.5 nM GFP-MAP2c or GFP-MAP4 (green) to either taxol-lattice (blue) or GMPCPP-lattice microtubules (red). Note the clear formation of envelopes by MAP2c on taxol-lattice, but not on GMPCPP-lattice microtubules. Below: quantification of the fluorescence intensity of MAPs on the indicated lattices (mean ± s.d., n = 108, 134, 116, 106, 150 microtubule segments in 3 chambers each.). ‘Total’ refers to the intensity on the entire lattice including regions outside and inside envelopes for MAP2c. Scale bar: 2 μm. One-way ANOVA, **** indicates p<0.001 c. Fluorescence images of 0.25 nM GFP-MAP proteins on taxol-lattice microtubules in the absence or presence of 10% 1,6-hexanediol or 2,5-hexanediol (HD). Below: quantification of the fluorescence intensity of MAPs in the indicated conditions (mean ± s.d., n=108, 134, 199, 146, 125 microtubule segments, respectively, 2 experiments each). One-way ANOVA, **** indicates p<0.001. d. Example fluorescence images showing GFP-MAP (green) and SiR-tubulin (magenta) signals along microtubules. Right: quantification of average SiR-tubulin fluorescence intensity (mean ± s.d., n=65, 74, 72, 70 microtubule segments, respectively, 2 experiments each). One-way ANOVA, **** indicates p<0.001. e. Compaction of the microtubule lattice measured on speckled microtubules (same method as data presented in Fig. 1h) after the addition of MAPs. For MAP2c, compaction was 3.0 ± 1.1 % in the envelope regions and 0.0 ± 0.6 % on the lattice outside the envelopes (mean ± s.d., n= 78 envelopes, n=95 lattices, in 5 experiments). For MAP4, compaction was 0.0 ± 0.4 % (n = 105 microtubules in 11 experiments). f. Quantification of the tubulin monomer spacing from cryo-EM images of taxol-lattice microtubules in the absence or presence of the indicated MAPs (n=46, 36, 44, 61, 30 microtubules, respectively). Red lines denote previously reported tubulin spacing for the indicated lattices⁵. g. Quantification of the enrichment of GFP-MAPs, based on fluorescence intensity, within mScarlet-2N4R tau envelopes (mean ± s.d., n=97, 115, 94, 112, 206, and 183 tau envelopes, respectively, 2 experiments each). 0.5 nM MAP protein was used for each condition. Note that MAP4 values below 1 indicates the protein is excluded from tau envelopes (red asterisks). One-way ANOVA, **** indicates p<0.001. h. Multi-channel fluorescence micrograph showing all three orthogonally-labeled MAPs mixed together on microtubules. Arrows denote the exclusion of MAP4 from the regions enriched with both tau and MAP2c. Scale bar: 5 μm. Right: TIRF image showing a single microtubule coated in tau/MAP2c envelopes. Kymograph below shows the behavior of single MAP4 molecules (green) visualized at a lower concentration, revealing that they diffuse outside (yellow bracket) but not inside tau/MAP2c envelopes (cyan & magenta). N = 2 experiments. Kymograph scale bars: 5 μm, 10 sec.