Figure 3.
Kip2–Bik1 interaction is required to retain Kip2 on cytoplasmic microtubule plus-ends efficiently. (A) Representative images of preanaphase cells expressing Spc42-mCherry (magenta) and Kip2-3xsfGFP (green) or Kip2-ΔT-3xsfGFP (green) in the presence or absence of Bik1. (B–D) Representative images (left) and quantifications (right) of fluorescence intensities (a.u.) from endogenous Kip2-∆T-3xsfGFP (C) and Kip2-3xsfGFP in the presence (B) or absence (D) of Bik1 along preanaphase cytoplasmic microtubules (boxed areas). Signals were aligned to SPBs using the Spc42-mCherry (magenta) intensity peak and binned by microtubule length (2 pixel = 266.7 nm bin size). Colored lines show mean Kip2-3xsfGFP fluorescence per bin and shaded areas represent 95% confidence intervals for the mean. Gray dashed lines denote weighted linear regressions for the mean GFP fluorescence on plus-ends over all bins. Scale bars, 2 µm. 53 ≤ n ≤ 180 per bin. These graphs have consistent scales for direct comparison and per-bin comparisons are shown in Fig. S3. B has been published as Fig. 1 B in Chen et al. (2019b). (E) Scheme of the mathematically estimated parameters (Chen et al., 2019b). Free Kip2 (with concentration [Kip2]free<[Kip2]total) binds to the microtubule minus-end anchored at the SPB with rate rin=kin[Kip2]free if the minus-end site is free and to any free lattice site with rate ron=kon[Kip2]free. A bound motor can detach with rate koff, and it can advance with rate if the following site toward the plus-end is free. At the plus-end, the motor detaches with a different rate, kout. (F) Likelihood of total Kip2 concentration [Kip2]total estimated from fit in B–D. (G) Likelihood of on rate constant kon and in rate constant kin estimated from fit in B–D. Statistical significance for differences determined by sampling from the likelihood (see supporting information), the difference in the median as indicated. (H) Likelihood of out rate kout constant estimated from fit in B–D. For F–H, median values are indicated as circles, interquartile range (IQR) by thick gray bars, and 1.5×IQR by thin gray bars. Kernel density estimates are computed from 20,000 samples from the likelihood function. Dashed black lines indicate sampled parameter ranges.

Kip2–Bik1 interaction is required to retain Kip2 on cytoplasmic microtubule plus-ends efficiently. (A) Representative images of preanaphase cells expressing Spc42-mCherry (magenta) and Kip2-3xsfGFP (green) or Kip2-ΔT-3xsfGFP (green) in the presence or absence of Bik1. (B–D) Representative images (left) and quantifications (right) of fluorescence intensities (a.u.) from endogenous Kip2-∆T-3xsfGFP (C) and Kip2-3xsfGFP in the presence (B) or absence (D) of Bik1 along preanaphase cytoplasmic microtubules (boxed areas). Signals were aligned to SPBs using the Spc42-mCherry (magenta) intensity peak and binned by microtubule length (2 pixel = 266.7 nm bin size). Colored lines show mean Kip2-3xsfGFP fluorescence per bin and shaded areas represent 95% confidence intervals for the mean. Gray dashed lines denote weighted linear regressions for the mean GFP fluorescence on plus-ends over all bins. Scale bars, 2 µm. 53 ≤ n ≤ 180 per bin. These graphs have consistent scales for direct comparison and per-bin comparisons are shown in Fig. S3. B has been published as Fig. 1 B in Chen et al. (2019b). (E) Scheme of the mathematically estimated parameters (Chen et al., 2019b). Free Kip2 (with concentration [Kip2]free<[Kip2]total) binds to the microtubule minus-end anchored at the SPB with rate rin=kin[Kip2]free if the minus-end site is free and to any free lattice site with rate ron=kon[Kip2]free. A bound motor can detach with rate koff, and it can advance with rate if the following site toward the plus-end is free. At the plus-end, the motor detaches with a different rate, kout. (F) Likelihood of total Kip2 concentration [Kip2]total estimated from fit in B–D. (G) Likelihood of on rate constant kon and in rate constant kin estimated from fit in B–D. Statistical significance for differences determined by sampling from the likelihood (see supporting information), the difference in the median as indicated. (H) Likelihood of out rate kout constant estimated from fit in B–D. For F–H, median values are indicated as circles, interquartile range (IQR) by thick gray bars, and 1.5×IQR by thin gray bars. Kernel density estimates are computed from 20,000 samples from the likelihood function. Dashed black lines indicate sampled parameter ranges.

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