Supplementary Figure 7: Validation of CKK–MT contact sites by using CKK mutants.
From: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins
(a) CD spectra (left) and thermal unfolding profiles (right) of CAMSAP3 CKK variants. The spectra were recorded at 5 °C in PBS at a protein concentration of 0.25 mg/ml. The thermal unfolding profiles were recorded at 206 nm.
(b,c) TIRFM images and quantification of GFP-CAMSAP3 CKK and N1130A mutant binding to GMPCPP-stabilized MTs. The intensity is normalized to the average minus-end intensity of WT. Similar to CAMSAP1 N1492A mutant, CAMSAP3 N1130A mutant shows increased MT lattice binding. n=80 MTs.
(d-f) TIRFM images and quantifications of the binding of GFP-CAMSAP1mini N1492A and N1492T mutants to the minus ends and lattice of dynamic MTs at different concentrations. Both mutants show minus-end specificity at lower concentrations (see Fig. 4a,d for images at 50 nM). The intensity is normalized to the average minus-end intensity of wild type. n=30-50 MTs. The data for N1492A and N1492T mutants at 50 nM was replotted from Fig. 4d.
(g,h) TIRFM images and quantification of binding of GFP-CAMSAP1mini and mutants to the minus ends and lattice of dynamic MTs. The intensity is normalized to the average minus-end intensity of wild type. n=30 MTs.
Scale bars, 1 μm. Data represent mean ± SD.
See also Supplementary Table 2.
