Supplementary Figure 5

CET of microtubules assembled in the presence of NPnf or EB1-gold (a) Control experiments of microtubules assembled in the presence of GTPγS and BeF3- (related to Fig. 1c, d). Left: CET of GTPγS-microtubules nucleated by isolated centrosomes in the presence of non-functionalized gold nanoparticles (NPnf) at 200 nM concentration (Supplementary Table 2a). Right: CET of GDP-BeF3–microtubules nucleated by isolated centrosomes in the presence of non-functionalized gold nanoparticles (NPnf) at 200 nM concentration (Supplementary Table 2b). Each figure shows a projection of a few slices in the centre of the tomogram (top) and cross-sections (bottom) corresponding to the boxed region in the upper image. Scale bars: 100 nm. (b) CET of the microtubule shown in Fig. 2a–a′. The top panel (left) shows a Z-projection that encompasses the two air-water interfaces. Cross-sections (top right) of the regions labeled 1–3 show that most NPs stick to the air-water interfaces, except those that interact with the tip of the microtubule. Sections at different levels of the tomogram are shown below: (4) first air-water interface, (5) and (6) sections inside the tomogram at the level of the microtubule, (7) second air-water interface. The ice thickness (distance between layers 4 and 7) is ∼90 nm. Scale bars: 50 nm. (c) Protofilament number of microtubules with EB1-gold attached to their ends (n = 33). N.d.: not determined. (d) Size-exclusion chromatography of EB1-gold (red) and non-functionalized nanoparticles (NPnf, blue) on a Sephacryl S-300 column. The elution profile of EB1-gold shows an additional peak (arrow), probably corresponding to EB1 dimers conjugated to 2 gold NPs as observed by CET. The star shows a small peak on the NPnf elution profile corresponding to aggregates, and which elute in front of the main peak. No aggregates can be detected in the elution profile of the conjugate. (e) NPs of 6.5 nm (left) and 9.8 nm (right) diameter fully trapped in the lumen of microtubules. Scale bars: 25 nm.