Figure 3.

Dam1C-4D disrupts the interaction with Ndc80C during the transfer of an Ndc80C nanobead between MTs. (A) Coomassie Blue–stained gel showing purified Ndc80C-His (without GFP and with His tag at the C terminus of Spc24). (B) Diagram explains various outcomes in the MT crossing assay regarding the position of an Ndc80C nanobead and the location of Dam1C-GFP (WT or 4D) signals. (C) Images in time sequence show that the MT end-on attachment to an Ndc80C (WT) nanobead continued after it had passed across the side of another MT in the presence of Dam1C (WT)-GFP. Dam1C (WT)-GFP signals were at the end of the depolymerizing MT with nanobead throughout the end-on attachment. Time 0 s was set arbitrarily. Scale bar, 5 µm. Also refer to diagrams (right). Asterisk indicates a crossing MT. (D) Images in time sequence show that an Ndc80C (WT) nanobead was transferred from the end of one MT to the side of another MT in the presence of Dam1C-4D-GFP. Dam1C-4D-GFP signals tracked the end of depolymerizing MT, moving away from the Ndc80C nanobead. Time 0 s was set arbitrarily. Scale bar, 5 µm. Also refer to diagrams (right). Keys are the same as in C.(E) Frequency of events in the MT crossing assay. The cases where Ndc80C nanobeads were transferred to the lateral side of another MT, with Ndc80C (WT, no GFP) and Dam1C-4D-GFP or with Ndc80C (WT)-GFP and Dam1C-4D (no GFP), were investigated. After transfer of the nanobead to the lateral side of another MT, Dam1C-4D-GFP always tracked the end of the original MT (9 out of 9 events), while Ndc80C-GFP always located at the nanobead (18 out of 18 events). The difference between Dam1C-4D-GFP and Ndc80C (WT)-GFP is significant (P < 0.0001).