Figure 10.
Processive end-tracking activity to the tripartite Ndc80-Cdt1-Ska1 complex is characterized by enhanced diffusive behavior of the complex. (A) Single-molecule diffusion kymograph for Ndc80alexa647 alone (top), and Ndc80alexa647 in the presence of GFP-Cdt1 and SKA1/2 (bottom). (B) Histogram of residence times for Ndc80alexa647 alone (left) and Ndc80alexa647 in the presence of GFP-Cdt1 and untagged-SKA1/2 (right). Optimized decay constants from the single exponential fits to the data (in red) represent the respective residence times (τres) for each condition; data from at least 189 individual Ndc80alexa647 tracks and N ≥ 2 independent trials. (C) Mean Squared Displacement (MSD) vs. time for Ndc80alexa647 alone (black plot) and Ndc80alexa647 in the presence of GFP-Cdt1 and untagged-SKA1/2 (red plot); tracks from at least 22 individual Ndc80alexa647 complexes were averaged; symbols are experimental; data lines are linear fits to the data up to 0.22 s, the slope of which is the diffusion coefficient; error bars are SEM. (D) Same as A but in this case for GFP-Cdt1; tracks from at least 250 individual GFP-Cdt1 complexes were averaged. (E) Same as D but in this case for GFP-Cdt1. (F) Same as C but in this case for GFP-Cdt1; data from at least 125 individual GFP-Cdt1 tracks and N ≥ 2 independent trials. (G) Mutual interactions between the Ndc80 complex, Cdt1, and the Ska1 complex to form a super-complex and the binding of the resulting super-complex furnishes the load bearing attachment at kinetochore. (H) Schematics (not to scale) show multi-molecular lawn of the Ndc80 complex molecules interacting with a kinetochore microtubule bundle. Propensity of Cdt1 (orange) and Ska1 (purple) proteins toward binding to curved microtubule protofilaments in conjunction with the intermolecular binding between Ndc80-Cdt1-Ska1 enables the formation of standalone microtubule tip-tracking units. Green microtubules segments showing nascent GTP-tubulin cap. (I) Zoomed in section of k-microtubule attachment shown the Ndc80-Cdt1-Ska1 trio contacting the microtubules at two point of attachments one) on the microtubule lattice primarily by the CH-domains of Ndc80 proteins and second) Ska1-Cdt1-mediated interaction with the curved protofilaments flanked by Ndc80.

Processive end-tracking activity to the tripartite Ndc80-Cdt1-Ska1 complex is characterized by enhanced diffusive behavior of the complex. (A) Single-molecule diffusion kymograph for Ndc80alexa647 alone (top), and Ndc80alexa647 in the presence of GFP-Cdt1 and SKA1/2 (bottom). (B) Histogram of residence times for Ndc80alexa647 alone (left) and Ndc80alexa647 in the presence of GFP-Cdt1 and untagged-SKA1/2 (right). Optimized decay constants from the single exponential fits to the data (in red) represent the respective residence times (τres) for each condition; data from at least 189 individual Ndc80alexa647 tracks and N ≥ 2 independent trials. (C) Mean Squared Displacement (MSD) vs. time for Ndc80alexa647 alone (black plot) and Ndc80alexa647 in the presence of GFP-Cdt1 and untagged-SKA1/2 (red plot); tracks from at least 22 individual Ndc80alexa647 complexes were averaged; symbols are experimental; data lines are linear fits to the data up to 0.22 s, the slope of which is the diffusion coefficient; error bars are SEM. (D) Same as A but in this case for GFP-Cdt1; tracks from at least 250 individual GFP-Cdt1 complexes were averaged. (E) Same as D but in this case for GFP-Cdt1. (F) Same as C but in this case for GFP-Cdt1; data from at least 125 individual GFP-Cdt1 tracks and N ≥ 2 independent trials. (G) Mutual interactions between the Ndc80 complex, Cdt1, and the Ska1 complex to form a super-complex and the binding of the resulting super-complex furnishes the load bearing attachment at kinetochore. (H) Schematics (not to scale) show multi-molecular lawn of the Ndc80 complex molecules interacting with a kinetochore microtubule bundle. Propensity of Cdt1 (orange) and Ska1 (purple) proteins toward binding to curved microtubule protofilaments in conjunction with the intermolecular binding between Ndc80-Cdt1-Ska1 enables the formation of standalone microtubule tip-tracking units. Green microtubules segments showing nascent GTP-tubulin cap. (I) Zoomed in section of k-microtubule attachment shown the Ndc80-Cdt1-Ska1 trio contacting the microtubules at two point of attachments one) on the microtubule lattice primarily by the CH-domains of Ndc80 proteins and second) Ska1-Cdt1-mediated interaction with the curved protofilaments flanked by Ndc80.

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