Supplementary Figure 4: Characterization of EB1-gold conjugates.

(a) Human EB1 carrying a 6x-histidine tag (6xHis) inserted into a loop region of its C-terminal cargo-binding domain, which is located remote from its microtubule-binding calponin homology (CH) domain (top left) was conjugated to gold NPs coated with a mixed HS-PEG-peptidol matrix functionalized with Ni-trisNTA functions (top right). The SAXS-based structure of EB134 is presented (bottom) with the 6.5 and 9.8 nm NPs (dotted circles) drawn to scale. The 6xHis tags are localized opposite to the microtubule-binding domains of the EB1 homodimer. Analysis of the functionalization ratio indicated that 95% of the 6.5 nm NPs had one Ni-trisNTA function, and 5% had two functions. Similarly, the 9.8 nm NPs were essentially mono-functionalized (83%), while the remaining possessed two (15%) or three (2%) functions. (b) Granulometric analysis of the commercial 5 and 10 nm NPs used in this study. NPs were centrifuged over an electron microscope grid and analyzed by transmission electron microscopy (top images, scale bars: 100 nm). The histograms show the size distributions of the 5 and 10 nm NPs (bottom graphs, data binned every 0.5 nm), with mean diameters (±SD) of 6.5 ± 1.1 nm and 9.8 ± 0.9 nm, respectively (n values represent the number of NPs). These are referred to 6.5 and 9.8 nm NPs in the manuscript. (c) SDS-PAGE gel of the purified EB1-HisLoop. Eleven μg of the protein was deposited on a 12.5% polyacrylamide gel. Left lane: molecular weight markers. (d) Estimation of the conjugation efficiency by SDS-PAGE analysis (representative results). EB1-HisLoop at 33 μM was incubated with 1 μM of 9.8 nm non-functionalized (NPnf) or functionalized (NPf) nanoparticles at 4 °C and room temperature (RT) for 75 min. Free protein was removed by centrifugation cycles before gel loading. The same quantity of NPs (3.30 pmol) was deposited in each lane. Three standards of EB1-HisLoop alone (1.65, 3.30 and 4.95 pmol) were used to estimate the quantity of EB1-HisLoop monomers associated with NPs, which provided values of 2.65 pmol and 2.50 pmol of EB1-HisLoop at 4 °C and room temperature, respectively (right graph). No EB1-HisLoop molecules could be observed after incubation in the presence of NPnf and their extensive washing by cycles of centrifugation and resuspension in buffer. Left lane: molecular weight markers. (e) TIRF microscopy of microtubules assembled in the presence of kinesin-1-SxIP-GFP (10 nM) and EB1-gold (43.5 nM, 6.5 nm NPs, left panels) or EB1 alone (100 nM, right panels). Tubulin concentration was 15 μM. Scale bars: horizontal 2 μm, vertical 60 s.