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Kinesin-4 KIF21B is a potent microtubule pausing factor

View Article: PubMed Central - PubMed

ABSTRACT

Microtubules are dynamic polymers that in cells can grow, shrink or pause, but the factors that promote pausing are poorly understood. Here, we show that the mammalian kinesin-4 KIF21B is a processive motor that can accumulate at microtubule plus ends and induce pausing. A few KIF21B molecules are sufficient to induce strong growth inhibition of a microtubule plus end in vitro. This property depends on non-motor microtubule-binding domains located in the stalk region and the C-terminal WD40 domain. The WD40-containing KIF21B tail displays preference for a GTP-type over a GDP-type microtubule lattice and contributes to the interaction of KIF21B with microtubule plus ends. KIF21B also contains a motor-inhibiting domain that does not fully block the interaction of the protein with microtubules, but rather enhances its pause-inducing activity by preventing KIF21B detachment from microtubule tips. Thus, KIF21B combines microtubule-binding and regulatory activities that together constitute an autonomous microtubule pausing factor.

Doi:: http://dx.doi.org/10.7554/eLife.24746.001

No MeSH data available.


Related in: MedlinePlus

Characterization of full-length KIF21B in vitro(A) Histograms of fluorescence intensities at the initial moment of observation of single molecules of the indicated proteins immobilized on coverslips (symbols) and the corresponding fits with lognormal distributions (lines). n = 5063, 8830 and 12230 molecules; fluorophore density was 0.23, 0.4 and 0.45 µm−2 for GFP, GFP-EB3 and KIF21B-FL-GFP proteins. (B) Representative photobleaching time traces of GFP, EB3-GFP and KIF21B-FL-GFP individual molecules (background subtracted). (C) Kymographs illustrating MT dynamics in vitro in the presence of 15 µM tubulin with 3% Rhodamine-tubulin and 0.5 nM KIF21B-FL-GFP. Kymographs were generated from the movies acquired using CoolSNAP HQ2 CCD camera (Roper Scientific) with a 1.2-s interval between frames and an exposure time of 100 ms.DOI:http://dx.doi.org/10.7554/eLife.24746.01410.7554/eLife.24746.015Figure 3—figure supplements 1—source data 1.An excel sheet with numerical data on the quantification of the KIF21B-FL dimer and photobleaching step analysis represented as plots in Figure 3—figure supplement 1A,B.DOI:http://dx.doi.org/10.7554/eLife.24746.015
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fig3s1: Characterization of full-length KIF21B in vitro(A) Histograms of fluorescence intensities at the initial moment of observation of single molecules of the indicated proteins immobilized on coverslips (symbols) and the corresponding fits with lognormal distributions (lines). n = 5063, 8830 and 12230 molecules; fluorophore density was 0.23, 0.4 and 0.45 µm−2 for GFP, GFP-EB3 and KIF21B-FL-GFP proteins. (B) Representative photobleaching time traces of GFP, EB3-GFP and KIF21B-FL-GFP individual molecules (background subtracted). (C) Kymographs illustrating MT dynamics in vitro in the presence of 15 µM tubulin with 3% Rhodamine-tubulin and 0.5 nM KIF21B-FL-GFP. Kymographs were generated from the movies acquired using CoolSNAP HQ2 CCD camera (Roper Scientific) with a 1.2-s interval between frames and an exposure time of 100 ms.DOI:http://dx.doi.org/10.7554/eLife.24746.01410.7554/eLife.24746.015Figure 3—figure supplements 1—source data 1.An excel sheet with numerical data on the quantification of the KIF21B-FL dimer and photobleaching step analysis represented as plots in Figure 3—figure supplement 1A,B.DOI:http://dx.doi.org/10.7554/eLife.24746.015

Mentions: Next, we purified the full-length KIF21B-GFP from HEK293T cells (Figure 2—figure supplement 1) and confirmed by single molecule analysis that it is a dimer (Figure 3—figure supplement 1A,B, Supplementary file 2). Mass spectrometry analysis of this purified protein revealed no known MT regulators (Supplementary file 1). Next, we assayed the activity of KIF21B-GFP on MTs in vitro (Figure 3A, Figure 3—figure supplement 1C). Strikingly, the full-length protein showed a strong preference for GMPCPP-stabilized MT seeds, on which it landed and moved in the direction of the plus-end, while hardly any motor landing events were observed on the dynamic (presumably GDP) MT lattice (Figure 3A,C). KIF21B-GFP motors accumulated at the tips of the seeds, and these accumulations could prevent MT outgrowth (Figure 3A). Both the enrichment of KIF21B-GFP at the tip of seeds and the inhibition of MT outgrowth were more prominent for longer seeds (Figure 3A,B). This indicates that GMPCPP-seeds act as ‘antennae’ that accumulate the kinesin motor at their ends in a length-dependent manner, similar to what has been previously described for the yeast kinesins Kip3 and Kip2 (Hibbel et al., 2015; Su et al., 2012; Varga et al., 2006). Significant blocking of growth from MT seeds, especially the longer ones, was observed already with 3 nM KIF21B-GFP, while complete inhibition of MT outgrowth from all seeds was seen at higher KIF21B-GFP concentrations. At lower concentrations of KIF21B (0.5 nM) growth of some seeds was still blocked, but some MTs were growing, and the effect of KIF21B on MT plus-end dynamics could be analyzed.10.7554/eLife.24746.012Figure 3.KIF21B can induce MT pausing or catastrophe in vitro.


Kinesin-4 KIF21B is a potent microtubule pausing factor
Characterization of full-length KIF21B in vitro(A) Histograms of fluorescence intensities at the initial moment of observation of single molecules of the indicated proteins immobilized on coverslips (symbols) and the corresponding fits with lognormal distributions (lines). n = 5063, 8830 and 12230 molecules; fluorophore density was 0.23, 0.4 and 0.45 µm−2 for GFP, GFP-EB3 and KIF21B-FL-GFP proteins. (B) Representative photobleaching time traces of GFP, EB3-GFP and KIF21B-FL-GFP individual molecules (background subtracted). (C) Kymographs illustrating MT dynamics in vitro in the presence of 15 µM tubulin with 3% Rhodamine-tubulin and 0.5 nM KIF21B-FL-GFP. Kymographs were generated from the movies acquired using CoolSNAP HQ2 CCD camera (Roper Scientific) with a 1.2-s interval between frames and an exposure time of 100 ms.DOI:http://dx.doi.org/10.7554/eLife.24746.01410.7554/eLife.24746.015Figure 3—figure supplements 1—source data 1.An excel sheet with numerical data on the quantification of the KIF21B-FL dimer and photobleaching step analysis represented as plots in Figure 3—figure supplement 1A,B.DOI:http://dx.doi.org/10.7554/eLife.24746.015
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5383399&req=5

fig3s1: Characterization of full-length KIF21B in vitro(A) Histograms of fluorescence intensities at the initial moment of observation of single molecules of the indicated proteins immobilized on coverslips (symbols) and the corresponding fits with lognormal distributions (lines). n = 5063, 8830 and 12230 molecules; fluorophore density was 0.23, 0.4 and 0.45 µm−2 for GFP, GFP-EB3 and KIF21B-FL-GFP proteins. (B) Representative photobleaching time traces of GFP, EB3-GFP and KIF21B-FL-GFP individual molecules (background subtracted). (C) Kymographs illustrating MT dynamics in vitro in the presence of 15 µM tubulin with 3% Rhodamine-tubulin and 0.5 nM KIF21B-FL-GFP. Kymographs were generated from the movies acquired using CoolSNAP HQ2 CCD camera (Roper Scientific) with a 1.2-s interval between frames and an exposure time of 100 ms.DOI:http://dx.doi.org/10.7554/eLife.24746.01410.7554/eLife.24746.015Figure 3—figure supplements 1—source data 1.An excel sheet with numerical data on the quantification of the KIF21B-FL dimer and photobleaching step analysis represented as plots in Figure 3—figure supplement 1A,B.DOI:http://dx.doi.org/10.7554/eLife.24746.015
Mentions: Next, we purified the full-length KIF21B-GFP from HEK293T cells (Figure 2—figure supplement 1) and confirmed by single molecule analysis that it is a dimer (Figure 3—figure supplement 1A,B, Supplementary file 2). Mass spectrometry analysis of this purified protein revealed no known MT regulators (Supplementary file 1). Next, we assayed the activity of KIF21B-GFP on MTs in vitro (Figure 3A, Figure 3—figure supplement 1C). Strikingly, the full-length protein showed a strong preference for GMPCPP-stabilized MT seeds, on which it landed and moved in the direction of the plus-end, while hardly any motor landing events were observed on the dynamic (presumably GDP) MT lattice (Figure 3A,C). KIF21B-GFP motors accumulated at the tips of the seeds, and these accumulations could prevent MT outgrowth (Figure 3A). Both the enrichment of KIF21B-GFP at the tip of seeds and the inhibition of MT outgrowth were more prominent for longer seeds (Figure 3A,B). This indicates that GMPCPP-seeds act as ‘antennae’ that accumulate the kinesin motor at their ends in a length-dependent manner, similar to what has been previously described for the yeast kinesins Kip3 and Kip2 (Hibbel et al., 2015; Su et al., 2012; Varga et al., 2006). Significant blocking of growth from MT seeds, especially the longer ones, was observed already with 3 nM KIF21B-GFP, while complete inhibition of MT outgrowth from all seeds was seen at higher KIF21B-GFP concentrations. At lower concentrations of KIF21B (0.5 nM) growth of some seeds was still blocked, but some MTs were growing, and the effect of KIF21B on MT plus-end dynamics could be analyzed.10.7554/eLife.24746.012Figure 3.KIF21B can induce MT pausing or catastrophe in vitro.

View Article: PubMed Central - PubMed

ABSTRACT

Microtubules are dynamic polymers that in cells can grow, shrink or pause, but the factors that promote pausing are poorly understood. Here, we show that the mammalian kinesin-4 KIF21B is a processive motor that can accumulate at microtubule plus ends and induce pausing. A few KIF21B molecules are sufficient to induce strong growth inhibition of a microtubule plus end in vitro. This property depends on non-motor microtubule-binding domains located in the stalk region and the C-terminal WD40 domain. The WD40-containing KIF21B tail displays preference for a GTP-type over a GDP-type microtubule lattice and contributes to the interaction of KIF21B with microtubule plus ends. KIF21B also contains a motor-inhibiting domain that does not fully block the interaction of the protein with microtubules, but rather enhances its pause-inducing activity by preventing KIF21B detachment from microtubule tips. Thus, KIF21B combines microtubule-binding and regulatory activities that together constitute an autonomous microtubule pausing factor.

Doi:: http://dx.doi.org/10.7554/eLife.24746.001

No MeSH data available.


Related in: MedlinePlus