The assay was performed as previously described with small modifications (Espeut et al., 2012 (link)). In brief, decoration of Spc105 proteins on polystyrene beads was achieved using streptavidin-biotin system. 100 nm each of WT and mutant Spc105 proteins was incubated with 10 µl of 0.1% (wt/vol) streptavidin polystyrene beads (Spherotech) conjugated with anti-penta-his biotin antibody (Qiagen) for 90 min at 4°C. Before imaging, the beads were sonicated for 3 min in a water bath containing ice cubes. Subsequently, the beads were introduced inside the flow chamber coated with taxol-stabilized microtubules. The images were acquired using a TIRF microscope.