Figure 1.

Behavior of Ndc80C nanobeads in vitro on dynamic MTs loaded with Dam1C. (A) Diagram shows the model of an error-correction process proposed previously (Kalantzaki et al., 2015). Each step is explained in the text. (B) Coomassie Blue–stained gels showing purified Ndc80C-GFP (with GFP at the C terminus of Spc24), Dam1C, and Dam1C-GFP (with GFP at the C terminus of Dad1). (C) Diagram shows that Ndc80C-GFP was attached to a streptavidin-coated nanobead through a biotinylated anti-GFP nanobody (top). Dynamic MTs were grown from stable MT seeds on coverslips in the presence of Dam1C and Ndc80C-GFP–coated nanobeads (Ndc80C nanobeads) and observed by TIRF microscopy (bottom). (D) Images in time sequence (top) show that an Ndc80C nanobead formed MT lateral attachment (0–16 s time points) and end-on attachment (24–68 s) in the presence of Dam1C. Time 0 s was set arbitrarily. Scale bar, 5 µm. Also refer to diagrams (right). Kymograph was obtained for the same MT (bottom). Scale bars, 5 µm (horizontal) and 60 s (vertical). (E) Images in time sequence (top) show that an Ndc80C nanobead formed a MT lateral attachment (0–8 s) and an end-on attachment (14–20 s) and subsequently detached from the MT end (end-on drop-off; 22 s), in the absence of Dam1C. After the nanobead had detached from the MT end, it was not visible by TIRF microscopy, because it was no longer close to the coverslip. Also refer to diagrams (right). Kymograph was obtained for the same MT (bottom). Scale bars are as in D. (F) Graph shows the percentage of events (rescue at nanobead, end-on drop-off, and continuous end-on attachment) observed in the absence (n = 54) and presence (n = 101) of Dam1C. The difference between the two groups is significant (****, P < 0.0001).