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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 S3. — Supplemental data associated with Fig. 4. (A) Images in time sequence show that the KCp (purified from Dam1-depleted cells) is attached to the lateral side of a MT (white arrows, 0–28 s) and formed the end-on attachment (yellow arrows, 42–64 s) in the presence of recombinant Dam1C. Kymograph obtained for the same MT is shown on the right side. In this example, the KCp contained Ndc80C-WT, and recombinant Dam1C-WT was added to the system. (B) Images in time sequence show that the KCp (purified from Dam1-depleted cells) is attached to the lateral side of a MT (white arrows, 0–58 s), shrinking MT reaches KCp (yellow arrows, 76 s), and MT regrows (magenta arrows, 76–312 s). Kymograph obtained for the same MT is shown on the right side. In this example, the KCp contained Ndc80-WT and recombinant Dam1C-4D was added to the system. (C) Images in time sequence show that the KCp (purified from Dam1-depleted cells) is attached to the lateral side of a MT (white arrows, 0–10 s), formed the end-on attachment (yellow arrows, 14 s), and detached from the MT end (14–16 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. Kymograph obtained for the same MT is shown on the right side. In this example, the KCp contained Ndc80-7D. (D) Percentage of events (rescue at KCp [green], end-on drop-off [orange], and continuous end-on attachment (blue), observed for the KCp with Ndc80-WT (left) or Ndc80-7D [right]) soon after the end of shrinking MTs reached the KCp. The KCp was purified from Dam1-depleted cells. Experiments were conducted in the absence of recombinant Dam1C or in the presence of recombinant Dam1C-WT or Dam1C-4D (from left to right: n = 98, 91, and 99 for the KCp with Ndc80-WT; n = 61, 76, and 80 for the KCp with Ndc80-7D). ****, P < 0.0001; ns1, P = 0.97; ns2, P = 0.26. (E) MT depolymerization rates in the absence (left, n = 39) and presence (right, n = 40) of recombinant Dam1C-WT, when KCp (Nd80-WT) was tracking the end of depolymerizing MTs. Difference between the two groups is significant (P = 0.03). Error bars represent SD. Kymographs (right) show representative events of KCp tracking the end of a depolymerizing MT (white lines a and b). Scale bars, 5 µm (horizontal) and 60 s (vertical). (F) Number of Ndc80C (WT) nanobeads or KCp (with Ndc80-WT) that bind the lateral side of dynamic MTs in each microscope field of view (normalized to per millimeter length of MT) after equal numbers of Ndc80C nanobeads and KCp were added to the system. Difference between the two groups is not significant. ns, P = 0.40. Error bars represent SD.