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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.


Coomassie blue stained gels with purified GFP, KIF21B-FL-GFP and its deletion mutants.Protein purification was performed using TEV protease cleavage as described in the Materials and Methods section. Black arrows indicate isolated proteins; blue arrows indicate the TEV protease.DOI:http://dx.doi.org/10.7554/eLife.24746.008
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fig2s1: Coomassie blue stained gels with purified GFP, KIF21B-FL-GFP and its deletion mutants.Protein purification was performed using TEV protease cleavage as described in the Materials and Methods section. Black arrows indicate isolated proteins; blue arrows indicate the TEV protease.DOI:http://dx.doi.org/10.7554/eLife.24746.008

Mentions: To investigate whether the observed effect of KIF21B-MD-CC1 is direct, we next purified GFP alone and KIF21B-MD-CC1, which was C-terminally tagged with GFP, from HEK293T cells (Figure 2—figure supplement 1). Using mass spectrometry, we confirmed that this purification method did not result in co-isolation of known MT regulators (Supplementary file 1). Analysis of fluorescence intensity of single KIF21B-MD-CC1-GFP molecules in comparison to monomeric GFP and dimeric EB3-GFP indicated that they were dimers, as expected (Figure 2A, Supplementary file 2). This conclusion was confirmed by two-step photobleaching profiles (Figure 2B) and was in agreement with the published data obtained in HeLa cell lysates with a similar construct (Ghiretti et al., 2016).10.7554/eLife.24746.006Figure 2.Dimeric motor domain of KIF21B slows down MT polymerization in vitro.


Kinesin-4 KIF21B is a potent microtubule pausing factor
Coomassie blue stained gels with purified GFP, KIF21B-FL-GFP and its deletion mutants.Protein purification was performed using TEV protease cleavage as described in the Materials and Methods section. Black arrows indicate isolated proteins; blue arrows indicate the TEV protease.DOI:http://dx.doi.org/10.7554/eLife.24746.008
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383399&req=5

fig2s1: Coomassie blue stained gels with purified GFP, KIF21B-FL-GFP and its deletion mutants.Protein purification was performed using TEV protease cleavage as described in the Materials and Methods section. Black arrows indicate isolated proteins; blue arrows indicate the TEV protease.DOI:http://dx.doi.org/10.7554/eLife.24746.008
Mentions: To investigate whether the observed effect of KIF21B-MD-CC1 is direct, we next purified GFP alone and KIF21B-MD-CC1, which was C-terminally tagged with GFP, from HEK293T cells (Figure 2—figure supplement 1). Using mass spectrometry, we confirmed that this purification method did not result in co-isolation of known MT regulators (Supplementary file 1). Analysis of fluorescence intensity of single KIF21B-MD-CC1-GFP molecules in comparison to monomeric GFP and dimeric EB3-GFP indicated that they were dimers, as expected (Figure 2A, Supplementary file 2). This conclusion was confirmed by two-step photobleaching profiles (Figure 2B) and was in agreement with the published data obtained in HeLa cell lysates with a similar construct (Ghiretti et al., 2016).10.7554/eLife.24746.006Figure 2.Dimeric motor domain of KIF21B slows down MT polymerization 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.