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. 2021 Apr 14;220(6):e202011117. doi: 10.1083/jcb.202011117

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© 2021 Doodhi et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 4. — Behaviors of purified kinetochore particles (KCps) on dynamic MTs in vitro. (A) i: Western blots of yeast cell lysates from dam1-aid strains with NDC80-WT (WT; left) and -7D (right). Cells were harvested before (lane 1) and after (lane 2) NAA treatment. The blots were probed with anti-Dam1 antibody. Arrows indicate Dam1-AID protein. Right: protein size markers (kDa). ii: Purified KCp from dam1-aid strains with NDC80 -WT (left) and -7D (right). Proteins were separated by SDS-PAGE and stained with SyproRuby. Proteins were identified by mass spectrometry (Table S1) and are shown at predicted sizes. Left: protein size markers (kDa). (B) Images in time sequence in a MT crossing assay shows that the MT end-on attachment to the KCp (purified from Dam1-depleted cells) continued after it had passed over the lateral side of another MT, in the presence of recombinant Dam1C. Also refer to diagrams (right). Asterisk indicates a crossing MT. Scale bar, 5 µm. In this example, KCp contained Ndc80-7D, and recombinant Dam1C-WT was added to system. (C) Images in time sequence show that the KCp (with Ndc80-WT, purified from Dam1-depleted cells) was transferred from the end of one MT to the lateral side of another MT (∼34 s) in the absence of recombinant Dam1C. Keys are the same as in B. (D) Images in time sequence show that the KCp (with Ndc80-7D, purified from Dam1-depleted cells) detached from the end of a shrinking MT while passing over the lateral side of another MT (between 18 and 20 s) in the absence of recombinant Dam1C. After the KCp had detached from the MT end, it was not visible by TIRF microscopy, because it was no longer close to the coverslip. Keys are the same as in B. (E) Percentage of various outcomes in the MT crossing assay for the KCp with Ndc80-WT (left) or Ndc80-7D (right). The KCp was purified from Dam1-depleted cells. The MT crossing assay was conducted in the absence of recombinant Dam1C or in the presence of recombinant Dam1C-WT or Dam1C-4D (from left to right: n = 45, 44, and 36 for the KCp with Ndc80-WT and n = 37, 30, and 39 for the KCp with Ndc80-7D). Outcomes include (1) continued end-on attachment (blue), (2) transfer to the lateral side of another MT (orange), or (3) end-on drop-off during crossing of another MT (green). Also refer to the diagram (right). Comparisons between two Dam1 conditions give the following: Ndc80-WT, ***, P = 0.0005; ns, P = 0.53; **, P = 0.0086; Ndc80-7D, **, P = 0.0085; ns1, P = 0.080’ ns2, P = 0.39. (F) Diagram shows the behaviors of Ndc80C nanobeads and the purified KCp in the MT crossing assay. The Ndc80C nanobead was often transferred to the lateral side of another MT in the presence of Dam1C-4D. By contrast, most KCps continued to track the end of a shrinking MT while passing over the other MT in the presence of Dam1C-4D. This difference may be explained by the difference in distribution and orientation of Ndc80Cs. The Ndc80Cs are randomly distributed around the 100-nm nanobeads and orient in all directions, whereas Ndc80Cs on the KCp may have a smaller footprint and orient mostly in one direction toward a MT (Dimitrova et al., 2016; Gonen et al., 2012).