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. 2022 Sep 6;11:e77877. doi: 10.7554/eLife.77877

Figure 2. KLP-12 is a plus-end directed motor that represses microtubule polymerization.

(A) Schematic presentation of the KLP-12 constructs. KLP-12(FL): full-length KLP-12, KLP-12–LZ–GFP: KLP-12 (1-393) with GFP connected with a leucine zipper, KLP-12(M): KLP-12 motor domain (1-365), KLP-12–DARPin: KLP-12(M) with DARPin connected with a flexible linker. (B) A representative kymograph showing the motility of KLP-12–LZ–GFP on taxol-stabilized microtubules. Horizontal and vertical bars show 10 μm and 10 s, respectively. (C) Histogram showing the velocity of KLP-12–LZ–GFP on taxol-stabilized (green) or dynamic (magenta) microtubules. 0.81±0.32 µm/s (n=407) and 0.82±0.31 µm/s (n=215) on taxol-stabilized and dynamic microtubules, respectively. Mean ± standard deviation. No statistically significant differences were detected by Student’s t-test. (D) Histogram showing the run length of KLP-12–LZ–GFP on taxol-stabilized (green) or dynamic (magenta) microtubules. n=407 molecules. 1.30±0.89 µm (n=407) and 1.11±0.57 µm (n=215) on taxol-stabilized and dynamic microtubules, respectively. Mean ± standard deviation. No statistically significant differences were detected by Student’s t-test. (E) Representative kymographs showing the microtubule dynamics and the motility of KLP-12–LZ–GFP. 10 μM of fluorescently labeled microtubules were polymerized from GMPCPP stabilized microtubule seeds fixed on the cover glass in the presence of 0, 60, or 600 nM KLP-12–LZ–GFP at 30 °C. Horizontal and vertical bars show 5 μm and 60 s, respectively. (F–I) The effect of KLP-12–LZ–GFP on microtubule dynamics. 10 μM of fluorescently labeled microtubules were observed in the presence of indicated concentrations of KLP-12–LZ–GFP at 30 °C. (F) Microtubule growth rate in vitro in the presence of KLP-12–LZ–GFP. Green bars show mean ± standard deviation. **, Adjusted p=0.0022, ***, Adjusted p=0.0001, ****, Adjusted p<0.0001, compared with control (0 nM). One-way ANOVA followed by Dunnett’s multiple comparisons test. n=52 microtubules. (G) Frequency of microtubule catastrophe events. The number of microtubule catastrophe in vitro was normalized by minute. mean ± standard deviation. ****, Adjusted p<0.0001, compared with control (0 nM). One-way ANOVA followed by Dunnett’s multiple comparisons test. n=101 microtubules. (H) Microtubule depolymerization rate in vitro in the presence of KLP-12–LZ–GFP. Green bars show mean ± standard deviation. No statistically significant differences were detected by One-Way ANOVA. n=30 microtubules. (I) Frequency of microtubule rescue events. The number of microtubule rescue events in vitro was normalized by minute. ****, Adjusted p<0.0001, compared with control (0 nM). One-way ANOVA followed by Dunnett’s multiple comparisons test. n=99 microtubules. The effect of microtubule growth rate by kinesin-4 family motors is available in Figure 2—figure supplement 1.

Figure 2—source data 1. Source data of microtubule growth rate in vitro in the presence of KLP-12–LZ–GFP (Figure 2C, D, F, G, H and I).

Figure 2.

Figure 2—figure supplement 1. The effect of microtubule growth rate by kinesin-4 family motors.

Figure 2—figure supplement 1.

The growth rates of the microtubules were normalized by those of microtubules without kinesin, which was set to the value of 1.0. The vertical axis indicates the ratio of the microtubule growth, and the horizontal axis indicates the kinesin concentration. The current study measures the values of the KLP-12, and the other kinesins were plotted based on the previous studies (Bieling et al., 2010; van der Vaart et al., 2013; Yue et al., 2018). Note that Bieling et al performed the experiment at 28°C ± 1°C (Xklp1), van der Vaart et al (KIF21A), Yue et al (KIF7 and KIF27), and our study was performed at 37 °C.
Figure 2—figure supplement 1—source data 1. The ratio of the microtubule growth by kinesin-4 family motors.