Figure 1. Dam1p, Ask1p, and Spc34p form cross-links to three distinct Ndc80p regions.

(A) Cross-links between the Dam1p, Ask1p, and Spc34p of the Dam1 complex and Ndc80p of the Ndc80 complex. Dam1 and Ndc80 complexes were cross-linked in the presence of microtubules. Horizontal black bars represent proteins and the six vertical yellow lines indicate Aurora B kinase phosphorylation sites on the Dam1 complex. Red and blue lines show cross-links formed with DSS and EDC cross-linkers, respectively. For clarity, only the cross-links between the Dam1 complex proteins and Ndc80p are displayed. Data are shown for peptides with Percolator (Käll et al., 2007) assigned q-values ≤ 0.05. Red bars on Ndc80p indicate regions where clusters of lethal mutations mapped (from Tien et al., 2013) to cross-linked regions. Grey bars on Ndc80p indicate clusters of lethal insertions outside of cross-linked regions. Green, blue, and orange trapezoids represent putative interactions (A, B, and C) between the Dam1 and Ndc80 complexes. (B) Bar diagram of Ndc80p with structural features. Green, blue and orange arrows indicate the positions of lethal mutations in interaction regions A^Ndc80p, B^Ndc80p, and C^Ndc80p used in this study. (CH: calponin homology; HP: hairpin) (C) Diagram showing the setup of TIRF microscopy experiments. Single molecule Ndc80-GFP complex binding on microtubules was visualized in the presence or absence of the Dam1 complex. (D) Lethal mutation in region A^Ndc80, B^Ndc80, or C^Ndc80 partially disrupts the Ndc80 complex’s interaction with Dam1 complex. Average residence time of Ndc80-GFP mutant and wild-type complexes on microtubules in the presence or absence of Dam1 complex. Bars represent average residence time ± error of the mean (estimated by bootstrapping analysis; see Materials and methods for additional details). Ndc80-GFP complex microtubule residence time raw data are included in Figure 1—source data 1. Refer to Supplementary file 1A for statistical analysis of data in part (D).

DOI: http://dx.doi.org/10.7554/eLife.21069.003

Figure 1—figure supplement 1. Dam1 and Ndc80 complexes robustly react with DSS and EDC cross-linking agents.

(A) Four types of peptides identified by mass spectrometry after cross-linking and trypsin digestion. Horizontal black bars represent protein sequence and vertical white lines represent residues able to react with the cross-linking agent. Unlinked: peptide that did not react with cross-linking agent; mono-link: only one end of the cross-linking reagent reacted with a residue; loop-link: intra-protein cross-link without a tryptic site in between the two cross-linked residues; self cross-link: intra-protein cross-link with a tryptic site in between the two-cross-linked residues. Dam1 and Ndc80 complexes were cross-linked in the presence of microtubules using DSS (panels B and C) or EDC (panels D and E). (B, D) Cross-links above and below the black bars represent self-crosslinks and loop-links, respectively. Data are shown for peptides with Percolator (Käll et al., 2007) assigned q-values ≤ 0.05. Highlighted regions above and below black bars represent sequence coverage from mass spectrometry. (C, E) Lines below the black bars represent mono-links.

DOI: http://dx.doi.org/10.7554/eLife.21069.005

Figure 1—figure supplement 2. Dam1p, Ask1p, and Spc34p cross-link to Nuf2p, in agreement to the coiled-coil structure of Ndc80p and Nuf2p.

(A) Cross-links between the Dam1p, Ask1p, and Spc34p of the Dam1 complex and Ndc80p and Nuf2p of the Ndc80 complex. For clarity, cross-links between the Dam1 complex proteins are not shown. Red lines represent cross-links between Ndc80p and Dam1p, Ask1p, or Spc34p. Blue lines represent cross-links between Nuf2p and Dam1p, Ask1p, or Spc34p. Purple lines represent cross-links between Ndc80p and Nuf2p. (B) Cross-links above and below the black bar represent self-crosslinks and loop-links, respectively, for Nuf2p. (C) Lines below the black bar represent mono-links for Nuf2p. Data from cross-linking experiments using DSS and EDC cross-linking agents are both shown. Data are shown for peptides with Percolator (Käll et al., 2007) assigned q-values ≤ 0.05.

DOI: http://dx.doi.org/10.7554/eLife.21069.006

Figure 1—figure supplement 3. Ndc80 mutations outside of regions A, B, and C do not disrupt the interaction between Dam1 and Ndc80 complexes.

(A), (B) Survival probability of the data shown in Figure 1D: microtubule residence times of Ndc80-GFP complex with a lethal mutation in region A^Ndc80p, B^Ndc80p, or C^Ndc80p in the absence (A) and presence (B) of wild-type Dam1 complex. Data here have a range of n = 420 to 2051. (C) Average microtubule residence times of Ndc80-GFP complexes with wild-type Ndc80p, or lethal five amino acid insertional mutation at position 219 or 652. Bars represent average residence time ± error of the mean (estimated by bootstrapping analysis; see Materials and methods for additional details). Ndc80-GFP complex microtubule residence time raw data are included in Figure 1—figure supplement 3—source data 1. Refer to Supplementary file 1B for statistical analysis. (D) Survival probability curves of the data shown in (C). (E) Superdex 200 size exclusion chromatography of the Ndc80 complex with wild-type Ndc80p or insertional mutation in A^Ndc80p, B^Ndc80p, or C^Ndc80p.

DOI: http://dx.doi.org/10.7554/eLife.21069.007