Figure 5.
The motor–tubulin interactions underlie the microtubule polymerase activity of Kip2. (A) SEC-MALS analysis of a mixture of apo Kip2-MD (calculated molecular mass of the monomer: 44 kD) and tubulin (calculated molecular mass of the dimer: 110 kD). (B) Homology model of the Kip2-MD (blue) in complex with the αβ-tubulin heterodimer (gray). The three positively charged Kip2-MD surface residue patches crucial for tubulin or microtubule-binding are highlighted in different colors (see corresponding legend). (C) SEC-MALS analysis of a mixture of MBP-Kip2-MD-mCherry (calculated molecular mass of the monomer: 125.8 kD) and tubulin. (D) SEC-MALS analysis of a mixture of MBP-Kip2-MD-P1−-mCherry (same calculated molecular mass as MBP-Kip2-MD-mCherry) and tubulin. (E) Representative images of metaphase yeast cells expressing Spc42-mCherry (magenta) and wild-type Kip2 or Kip2 containing mutations in tubulin-binding patches C-terminally fused with 3xsfGFP (green). (F) Quantification of GFP fluorescence intensities at aMT plus-ends from cells shown in E, error bars represent mean ± SD (n = 3 independent clones, with a total of >100 cells per genotype analyzed). For P2 and P3 mutant cells, the GFP signal was only weakly associated with SPBs; therefore, in both cases, the intensities on aMT plus-ends were not determined (n.d.). The statistical significance (n.s.: not significant) was test with two-tailed Student’s t test. (G and H) Quantification of 3D length of aMTs. Kip2-wt represents the control of inserting the selection marker TRP1 downstream of the KIP2 gene; see the Materials and methods section for more details. Representative images of metaphase cells of indicated genotype are shown in G; red arrowheads mark the plus-ends of aMTs. Quantification results are shown in H, the dashed (solid) bar represents the median (mean), the box marks the interquartile range, and the vertical line covers a 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. Scale bars, 2 μm. Source data for F and H are available in Data S1.

The motor–tubulin interactions underlie the microtubule polymerase activity of Kip2. (A) SEC-MALS analysis of a mixture of apo Kip2-MD (calculated molecular mass of the monomer: 44 kD) and tubulin (calculated molecular mass of the dimer: 110 kD). (B) Homology model of the Kip2-MD (blue) in complex with the αβ-tubulin heterodimer (gray). The three positively charged Kip2-MD surface residue patches crucial for tubulin or microtubule-binding are highlighted in different colors (see corresponding legend). (C) SEC-MALS analysis of a mixture of MBP-Kip2-MD-mCherry (calculated molecular mass of the monomer: 125.8 kD) and tubulin. (D) SEC-MALS analysis of a mixture of MBP-Kip2-MD-P1−-mCherry (same calculated molecular mass as MBP-Kip2-MD-mCherry) and tubulin. (E) Representative images of metaphase yeast cells expressing Spc42-mCherry (magenta) and wild-type Kip2 or Kip2 containing mutations in tubulin-binding patches C-terminally fused with 3xsfGFP (green). (F) Quantification of GFP fluorescence intensities at aMT plus-ends from cells shown in E, error bars represent mean ± SD (n = 3 independent clones, with a total of >100 cells per genotype analyzed). For P2 and P3 mutant cells, the GFP signal was only weakly associated with SPBs; therefore, in both cases, the intensities on aMT plus-ends were not determined (n.d.). The statistical significance (n.s.: not significant) was test with two-tailed Student’s t test. (G and H) Quantification of 3D length of aMTs. Kip2-wt represents the control of inserting the selection marker TRP1 downstream of the KIP2 gene; see the Materials and methods section for more details. Representative images of metaphase cells of indicated genotype are shown in G; red arrowheads mark the plus-ends of aMTs. Quantification results are shown in H, the dashed (solid) bar represents the median (mean), the box marks the interquartile range, and the vertical line covers a 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. Scale bars, 2 μm. Source data for F and H are available in Data S1.

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