Skip to main content
. 2023 Jul 20;42(17):e114534. doi: 10.15252/embj.2023114534

Figure 2. Ndc10 stabilizes Cse4CENP‐A on CEN DNA.

Figure 2

  1. Example graph of total identified colocalization residences observed on CEN DNA per imaging sequence. Each row represents one identified CEN DNA with all identified residences shown over entire imaging sequence (2,700 s) for Ndc10 (magenta) and Cse4CENP‐A (green). Instances where Ndc10 and Cse4CENP‐A residence coincides (white‐merge) represent simultaneous observation of both proteins on single CEN DNA, termed ternary residences. Complete series shown in Fig EV1A.
  2. Estimated survival function plots of Kaplan–Meier analysis of CEN DNA residence lifetimes of Ndc10 (magenta—median lifetime of 102 s, n = 3,231 over three experiments of ~1,000 DNA molecules using different extracts) and Cse4CENP‐A (red—median lifetime of 79 s, n = 1,049 over three experiments of ~1,000 DNA molecules using different extracts). Significant difference between ternary Ndc10 residence lifetime survival plots (***) compared to Cse4CENP‐A (two‐tailed P‐value of 0 as determined by log‐rank test). 95% confidence intervals indicated (dashed lines), right‐censored lifetimes (plus icons) were included and unweighted in survival function estimates.
  3. Schematic for off‐rate estimate calculations with representative residence lifetime assay traces of Ndc10 and Cse4CENP‐A on CEN DNA. Example kymographs of Cse4CENP‐A (top‐488 nm) and Ndc10 (bottom‐568 nm) in relation to a single identified CEN DNA (arrow), with normalized intensity trace below (gray) as well as identified colocalization residences (blue). Ndc10 First example (left) illustrates Ndc10 colocalization that precedes Cse4CENP‐A, yielding only TernaryNdc10 Cse4CENP‐A residences. Ndc10 Last example (right) illustrates Cse4CENP‐A colocalization that precedes Ndc10, yielding both Non‐TernaryNdc10 Cse4CENP‐A residences and TernaryNdc10 Cse4CENP‐A residence after Ndc10 colocalization initiation. Images were acquired every 5 s with normalized fluorescence intensity shown in arbitrary units. Bottom panel includes formulas used to calculate the ternary Cse4CENP‐A off‐rate, kt, where Nt represents the total number of apparent Cse4‐GFP detachments recorded during ternary residences, and ∑T1+T2+T5 represents the total amount of ternary residence time observed. In addition, the formula for non‐ternary Cse4 off‐rate, kn, where Nn represents the total number of apparent Cse4‐GFP detachments recorded during non‐ternary residences, and ∑T3+T4 represents the total amount of non‐ternary residence time observed.
  4. Cse4CENP‐A has slower off‐rates after colocalization with Ndc10 on CEN DNA. Quantification of the estimated Cse4CENP‐A off‐rates of the Non‐TernaryNdc10 and TernaryNdc10 pools (29  ± 2 h−1 and 22  ± 1 h−1 respectively, avg ± s.d. n = 1,151 over three experiments of ~1,000 DNA molecules using different extracts). Significant difference between off‐rates (*) with a P‐value of 0.02 as determined by two‐tailed unpaired t‐test.
  5. Kaplan–Meier analysis of TernaryNdc10 residence lifetimes of Cse4CENP‐A on CEN DNA (purple—median lifetime of 88 s, n = 539 over three experiments of ~1,000 DNA molecules using different extracts) and of Non‐TernaryNdc10 Cse4CENP‐A residence lifetimes (teal—median lifetime of 69 s, n = 612 over three experiments of ~1,000 DNA molecules using different extracts). There is a significant difference (**) between TernaryNdc10 and Non‐TernaryNdc10 lifetime survival plots (two‐tailed P‐value of 7.4e‐4 as determined by log‐rank test). 95% confidence intervals indicated (dashed lines), right‐censored lifetimes (plus icons) were included and unweighted in survival function estimates.
  6. Example plot of total identified residences observed on CEN DNA per imaging sequence of Ndc10 (left) and Cse4CENP‐A (right) independently sorted by residence initiation time, starting at the top with initiation time of 0 s (arrow).
  7. Timing of all non‐simultaneous Ndc10 and Cse4CENP‐A ternary residence events. The proportion when Ndc10 precedes Cse4CENP‐A is 0.83 ± 0.06 and when Cse4CENP‐A precedes Ndc10 is 0.17 ± 0.06 (avg ± s.d., n = 504 over three experiments of ~1,000 DNA molecules using different extracts). Simultaneous arrival defined as residence initiation within 5 s of each other and comprise 0.07 ± 0.04 of all ternary residence events (avg ± s.d., n = 35 over three experiments of ~1,000 DNA molecules using different extracts).