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. 2019 Sep 6;8:e48627. doi: 10.7554/eLife.48627

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© 2019, Chen et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 5. — (a) Representative images of Kip2-G374A-3xsfGFP (green) in preanaphase cells of indicated genotype. Spindles are visualized with Spc42-mCherry (magenta). See Figure 3c for control. (b) Normalized Kip2-G374A-3xsfGFP fluorescence (%) associated with b- (blue) and m-SPBs (yellow) in cells shown in (a) (n > 3 independent clones or technical replicates,>701 cells per genotype). (c) Quantification of asymmetry index for Kip2-G374A-3xsfGFP distribution (fluorescence intensity: (FI_b-SPB – FI_m-SPB) / FI_both-SPBs) between SPBs in cells in (b) with correctly orientated (blue) and inverted (red) SPBs. For cells with inverted SPBs, statistical significances of difference from zero were tested with one-way ANOVA. (d) Representative images (left) and quantifications (right) of fluorescence intensities (a.u.) from endogenous Kip2-3xsfGFP along preanaphase aMTs (boxed areas) in bfa1∆bub2∆ cells. The graph format is the same as in Figure 1. 45 ≤ n ≤ 99 per bin. The pink dashed line denotes the weighted linear regressions for the mean GFP fluorescence on plus-ends in wild-type cells. See Figure 5—figure supplement 1 for more details. (e) In silico likelihood of on rate constant $k_{on}$ and in rate constant $k_{in}$ estimated from model fits to Kip2-3xsfGFP distribution in wt and bfa1∆bub2∆ cells. Statistical significance for $k_{in, b f a 1 Δ b u b 2 Δ} < k_{in,wt}$ (***, p=8∙10⁻⁴, was determined by sampling from the likelihood for ${[Kip2]}_{total} \geq 35 nM$ (see Appendix 1), difference in median as indicated. Graph as in Figure 2d. (f) Normalized Kip2-3xsfGFP fluorescence (%) associated with microtubule plus-ends in preanaphase cells carrying both b- (blue) and m- (yellow) microtubules (n > 3 independent clones or technical replicates,>275 cells per genotype). See Figure 5—figure supplement 1a for representative images. (g) Quantification of asymmetry index for Kip2-3xsfGFP accumulation at m- and b-microtubule plus-ends (fluorescence intensity: (FI_b-MT – FI_m-MT) / FI_both-MTs) in preanaphase cells for data in (f) with correctly orientated (blue) and inverted (red) SPBs. Graphs and statistical analysis are as in Figure 4 (b,c,e,f,g and i) unless otherwise indicated. Statistical significances within each genotype are marked at the bottom. Scale bars, 2 µm. See also Figure 5—figure supplement 1.

Figure 5—figure supplement 1. — (a) Representative images of Kip2-G374A-3xsfGFP (green) in preanaphase cells of indicated genotype. Spindles are visualized with Spc42-mCherry (magenta). See Figure 3c for control. (b) Normalized Kip2-G374A-3xsfGFP fluorescence (%) associated with b- (blue) and m-SPBs (yellow) in cells shown in (a) (n > 3 independent clones or technical replicates,>701 cells per genotype). (c) Quantification of asymmetry index for Kip2-G374A-3xsfGFP distribution (fluorescence intensity: (FI_b-SPB – FI_m-SPB) / FI_both-SPBs) between SPBs in cells in (b) with correctly orientated (blue) and inverted (red) SPBs. For cells with inverted SPBs, statistical significances of difference from zero were tested with one-way ANOVA. (d) Representative images (left) and quantifications (right) of fluorescence intensities (a.u.) from endogenous Kip2-3xsfGFP along preanaphase aMTs (boxed areas) in bfa1∆bub2∆ cells. The graph format is the same as in Figure 1. 45 ≤ n ≤ 99 per bin. The pink dashed line denotes the weighted linear regressions for the mean GFP fluorescence on plus-ends in wild-type cells. See Figure 5—figure supplement 1 for more details. (e) In silico likelihood of on rate constant $k_{on}$ and in rate constant $k_{in}$ estimated from model fits to Kip2-3xsfGFP distribution in wt and bfa1∆bub2∆ cells. Statistical significance for $k_{in, b f a 1 Δ b u b 2 Δ} < k_{in,wt}$ (***, p=8∙10⁻⁴, was determined by sampling from the likelihood for ${[Kip2]}_{total} \geq 35 nM$ (see Appendix 1), difference in median as indicated. Graph as in Figure 2d. (f) Normalized Kip2-3xsfGFP fluorescence (%) associated with microtubule plus-ends in preanaphase cells carrying both b- (blue) and m- (yellow) microtubules (n > 3 independent clones or technical replicates,>275 cells per genotype). See Figure 5—figure supplement 1a for representative images. (g) Quantification of asymmetry index for Kip2-3xsfGFP accumulation at m- and b-microtubule plus-ends (fluorescence intensity: (FI_b-MT – FI_m-MT) / FI_both-MTs) in preanaphase cells for data in (f) with correctly orientated (blue) and inverted (red) SPBs. Graphs and statistical analysis are as in Figure 4 (b,c,e,f,g and i) unless otherwise indicated. Statistical significances within each genotype are marked at the bottom. Scale bars, 2 µm. See also Figure 5—figure supplement 1.