Figure 2.

Regions A^Dam1p and B^Ask1p support load-bearing interactions with the Ndc80 complex on growing microtubule tips. (A) Method for phosphorylating specific interaction regions within the Dam1 complex. In the A^Dam1p complex, alanine substitutions at Ask1p^S200 and Spc34p^T199 block regions B^Ask1p and C^Spc34p, so treatment of A^Dam1p complex with Ipl1p kinase and ATP led to phosphorylation specifically of the region A^Dam1p sites. Analogous approaches were also used to specifically phosphorylate regions B^Ask1p or C^Spc34p or various combinations of regions A^Dam1p, B^Ask1p, and C^Spc34p. (B) Rupture strengths for Ndc80-decorated beads measured in the presence of Dam1 complex, phosphorylated at indicated sites (red P’s) and carrying phospho-blocking alanine substitutions (yellow A’s), as diagrammed at left. Interaction regions that were disrupted by phosphorylation are shown in the diagram as dashed outlines. The stacked bar graph in the middle shows the fraction of tested beads that exhibited each of the following three behaviors: (i) attached to the microtubule but did not hold the 1-pN preload force (gold), (ii) ruptured at a force >1 pN (gray), or (iii) right-censored, when the bead reached the maximum trap force before rupturing (purple). The graph on the right shows the measured rupture forces. Each colored circle represents a single rupture event. Each colored triangle represents right-censored data, when a bead reached the maximum trap force before rupturing. The total number of measurements for each condition, including ruptures and right-censored events, for each condition are indicated by n values below the schematics. Black circles represent median rupture forces, with bars showing 95% CIs. Numbers below the black circles indicate median values.