Figure 1.
Kip2 motor domain and the C-terminal tail domain are essential for microtubule growth in vivo. (A) Schematic representations of protein constructs used. (B) Analysis of the Kip2-NMD amino acid sequence using the MultiCoil-prediction server with a cutoff of 0.5. (C) Representative images of preanaphase cells of indicated genotype expressing Bik1-3xGFP and Spc72-GFP. Scale bars, 2 µm. Magenta arrowheads mark the plus-ends of cytoplasmic microtubules. (D) The maximum intensity z-projection of time point one of a representative preanaphase cell. The 3D coordinates of aMT plus-end, proximal SPB, and distal SPB were tracked throughout the 80 time points. Their kymograph traces are colored in green, magenta, and blue, respectively. (E) The extracted 3D aMT length of each time point was plotted as a function of time, shown as gray dots. For this particular aMT, the maximum aMT length was 2.3 µm, and the lifetime was the full image acquisition window of 85.6 s. Two growth phases (green line over gray dots) and one shrinkage phase (magenta line over gray dots) were observed. The speeds of growth and shrinkage were calculated for each phase by dividing the corresponding ∆length by the ∆time. (F–I) Quantification results are shown. (F) All maximum lengths of microtubules below the detection limit (666.7 nm owing to the microscope resolution) were marked as 0 µm. See Materials and methods for details. The dashed (solid) bar represents the median (mean), the box marks the interquartile range, and the vertical line covers the 95% confidence interval (n = 3 independent clones with a total of >400 cells per genotype analyzed). Statistical significances were calculated using one-way ANOVA, **** P < 0.0001, n.s., not significant. (G) Maximum aMT length shown as histogram, mean of length shown in red (mean ± SD of mean). (H) The lifetime of aMTs shown as cumulative distribution, statistical significance was assessed with the Kolmogorov-Smirnov test. (I) Speeds of microtubule growth and shrinkage. Statistical significances were calculated using two-tailed Student's t test unless otherwise specified. **** P < 0.0001; * P < 0.05; n.s., not significant. Source data are available in Data S1.

Kip2 motor domain and the C-terminal tail domain are essential for microtubule growth in vivo. (A) Schematic representations of protein constructs used. (B) Analysis of the Kip2-NMD amino acid sequence using the MultiCoil-prediction server with a cutoff of 0.5. (C) Representative images of preanaphase cells of indicated genotype expressing Bik1-3xGFP and Spc72-GFP. Scale bars, 2 µm. Magenta arrowheads mark the plus-ends of cytoplasmic microtubules. (D) The maximum intensity z-projection of time point one of a representative preanaphase cell. The 3D coordinates of aMT plus-end, proximal SPB, and distal SPB were tracked throughout the 80 time points. Their kymograph traces are colored in green, magenta, and blue, respectively. (E) The extracted 3D aMT length of each time point was plotted as a function of time, shown as gray dots. For this particular aMT, the maximum aMT length was 2.3 µm, and the lifetime was the full image acquisition window of 85.6 s. Two growth phases (green line over gray dots) and one shrinkage phase (magenta line over gray dots) were observed. The speeds of growth and shrinkage were calculated for each phase by dividing the corresponding ∆length by the ∆time. (F–I) Quantification results are shown. (F) All maximum lengths of microtubules below the detection limit (666.7 nm owing to the microscope resolution) were marked as 0 µm. See Materials and methods for details. The dashed (solid) bar represents the median (mean), the box marks the interquartile range, and the vertical line covers the 95% confidence interval (n = 3 independent clones with a total of >400 cells per genotype analyzed). Statistical significances were calculated using one-way ANOVA, **** P < 0.0001, n.s., not significant. (G) Maximum aMT length shown as histogram, mean of length shown in red (mean ± SD of mean). (H) The lifetime of aMTs shown as cumulative distribution, statistical significance was assessed with the Kolmogorov-Smirnov test. (I) Speeds of microtubule growth and shrinkage. Statistical significances were calculated using two-tailed Student's t test unless otherwise specified. **** P < 0.0001; * P < 0.05; n.s., not significant. Source data are available in Data S1.

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