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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 KIF21B tail fragments in vitro(A) In vitro reconstitution of MT dynamics in the presence of 100 nM GFP-EB3 and the extracts of HEK293T cells expressing mCherry-CC2 or mCherry-L-WD40. GMPCPP-stabilized MT seeds were labeled with HiLyte Fluor 488-tubulin (lines below kymographs). (B) Quantification of the intensity of purified GFP-L-WD40 on GMPCPP- and taxol-stabilized seeds or dynamic MTs grown from GMPCCP or taxol-stabilized seeds. (C) Overview of the interactions of GFP-tagged KIF21B and its deletion mutants with MTs in vitro.DOI:http://dx.doi.org/10.7554/eLife.24746.03710.7554/eLife.24746.038Figure 7—figure supplements 1—source data 1.An excel sheet with numerical data on the quantification of the intensity of KIF21B-L-WD40 on seeds and dynamic MTs represented as plot in Figure 7—figure supplement 1B.DOI:http://dx.doi.org/10.7554/eLife.24746.038
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fig7s1: Characterization of KIF21B tail fragments in vitro(A) In vitro reconstitution of MT dynamics in the presence of 100 nM GFP-EB3 and the extracts of HEK293T cells expressing mCherry-CC2 or mCherry-L-WD40. GMPCPP-stabilized MT seeds were labeled with HiLyte Fluor 488-tubulin (lines below kymographs). (B) Quantification of the intensity of purified GFP-L-WD40 on GMPCPP- and taxol-stabilized seeds or dynamic MTs grown from GMPCCP or taxol-stabilized seeds. (C) Overview of the interactions of GFP-tagged KIF21B and its deletion mutants with MTs in vitro.DOI:http://dx.doi.org/10.7554/eLife.24746.03710.7554/eLife.24746.038Figure 7—figure supplements 1—source data 1.An excel sheet with numerical data on the quantification of the intensity of KIF21B-L-WD40 on seeds and dynamic MTs represented as plot in Figure 7—figure supplement 1B.DOI:http://dx.doi.org/10.7554/eLife.24746.038

Mentions: If the rCC does not fully inhibit the full-length KIF21B motor, what is the function of this region? To address this question, we have purified the KIF21B protein lacking the rCC (KIF21B-FL-ΔrCC), and a shorter version of this protein, which also lacked the WD40 domain (KIF21B-MD-CCΔrCC) (Figure 2—figure supplement 1). Mass spectrometry analysis demonstrated that the contaminants present in these two KIF21B preparations were essentially the same as in the isolated full-length KIF21B (Supplementary file 1). Analysis of fluorescence intensity and photobleaching confirmed that both deletion mutants are dimers, similar to the full-length molecule (Figure 7A, Supplementary file 2). In vitro assays showed that unlike the full-length protein, both kinesins lacking the rCC could land not only on GMPCPP-seeds but also on newly polymerized MT lattices (Figure 7B). Both full-length KIF21B and KIF21B-FL-ΔrCC exhibited slower motility on seeds compared to fresh GDP-MT lattices (Figure 7C). In contrast, the KIF21B-MD-CCΔrCC protein showed no reduced velocity on the MT lattice (Figure 7B,C), suggesting that the C-terminal WD40-containing region creates friction on GMPCPP-seeds. Consistent with this notion, we found that the L-WD40-GFP fragment displayed high preference for GMPCPP-seeds in vitro, both when present in cell extracts and in purified form, while the CC2 fragment showed no such preference (Figure 7D, Figure 7—figure supplement 1A–C). The preference for the GMPCPP seeds was not due to their attachment to glass or inclusion of biotinylated tubulin, because L-WD40-GFP showed no preference for taxol-stabilized MT seeds prepared in the same way as the GMPCPP seeds (Figure 7D, Figure 7—figure supplement 1B,C). However, we did not observe any accumulation of L-WD40-GFP at the growing MT plus ends, indicating that the preference for a specific nucleotide state is not sufficient to induce plus-end tracking of this protein fragment.10.7554/eLife.24746.035Figure 7.The WD40 domain and the autoinhibitory coiled coil region contribute to the pause-promoting activity of KIF21B.


Kinesin-4 KIF21B is a potent microtubule pausing factor
Characterization of KIF21B tail fragments in vitro(A) In vitro reconstitution of MT dynamics in the presence of 100 nM GFP-EB3 and the extracts of HEK293T cells expressing mCherry-CC2 or mCherry-L-WD40. GMPCPP-stabilized MT seeds were labeled with HiLyte Fluor 488-tubulin (lines below kymographs). (B) Quantification of the intensity of purified GFP-L-WD40 on GMPCPP- and taxol-stabilized seeds or dynamic MTs grown from GMPCCP or taxol-stabilized seeds. (C) Overview of the interactions of GFP-tagged KIF21B and its deletion mutants with MTs in vitro.DOI:http://dx.doi.org/10.7554/eLife.24746.03710.7554/eLife.24746.038Figure 7—figure supplements 1—source data 1.An excel sheet with numerical data on the quantification of the intensity of KIF21B-L-WD40 on seeds and dynamic MTs represented as plot in Figure 7—figure supplement 1B.DOI:http://dx.doi.org/10.7554/eLife.24746.038
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fig7s1: Characterization of KIF21B tail fragments in vitro(A) In vitro reconstitution of MT dynamics in the presence of 100 nM GFP-EB3 and the extracts of HEK293T cells expressing mCherry-CC2 or mCherry-L-WD40. GMPCPP-stabilized MT seeds were labeled with HiLyte Fluor 488-tubulin (lines below kymographs). (B) Quantification of the intensity of purified GFP-L-WD40 on GMPCPP- and taxol-stabilized seeds or dynamic MTs grown from GMPCCP or taxol-stabilized seeds. (C) Overview of the interactions of GFP-tagged KIF21B and its deletion mutants with MTs in vitro.DOI:http://dx.doi.org/10.7554/eLife.24746.03710.7554/eLife.24746.038Figure 7—figure supplements 1—source data 1.An excel sheet with numerical data on the quantification of the intensity of KIF21B-L-WD40 on seeds and dynamic MTs represented as plot in Figure 7—figure supplement 1B.DOI:http://dx.doi.org/10.7554/eLife.24746.038
Mentions: If the rCC does not fully inhibit the full-length KIF21B motor, what is the function of this region? To address this question, we have purified the KIF21B protein lacking the rCC (KIF21B-FL-ΔrCC), and a shorter version of this protein, which also lacked the WD40 domain (KIF21B-MD-CCΔrCC) (Figure 2—figure supplement 1). Mass spectrometry analysis demonstrated that the contaminants present in these two KIF21B preparations were essentially the same as in the isolated full-length KIF21B (Supplementary file 1). Analysis of fluorescence intensity and photobleaching confirmed that both deletion mutants are dimers, similar to the full-length molecule (Figure 7A, Supplementary file 2). In vitro assays showed that unlike the full-length protein, both kinesins lacking the rCC could land not only on GMPCPP-seeds but also on newly polymerized MT lattices (Figure 7B). Both full-length KIF21B and KIF21B-FL-ΔrCC exhibited slower motility on seeds compared to fresh GDP-MT lattices (Figure 7C). In contrast, the KIF21B-MD-CCΔrCC protein showed no reduced velocity on the MT lattice (Figure 7B,C), suggesting that the C-terminal WD40-containing region creates friction on GMPCPP-seeds. Consistent with this notion, we found that the L-WD40-GFP fragment displayed high preference for GMPCPP-seeds in vitro, both when present in cell extracts and in purified form, while the CC2 fragment showed no such preference (Figure 7D, Figure 7—figure supplement 1A–C). The preference for the GMPCPP seeds was not due to their attachment to glass or inclusion of biotinylated tubulin, because L-WD40-GFP showed no preference for taxol-stabilized MT seeds prepared in the same way as the GMPCPP seeds (Figure 7D, Figure 7—figure supplement 1B,C). However, we did not observe any accumulation of L-WD40-GFP at the growing MT plus ends, indicating that the preference for a specific nucleotide state is not sufficient to induce plus-end tracking of this protein fragment.10.7554/eLife.24746.035Figure 7.The WD40 domain and the autoinhibitory coiled coil region contribute to the pause-promoting activity of KIF21B.

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