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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.


Effects of KIF21B expression on MT organization and regrowth in cells(A) Time-lapse imaging of transiently transfected COS-7 cells expressing KIF21B-FL-GFP and TagRFP-tubulin. Yellow dashed lines in the overlay indicate the cell edge. (B) Nocodazole washout experiments of COS-7 cells expressing GFP or KIF21B-FL-GFP. Cells were transiently transfected with the indicated proteins and treated with 5 μM nocodazole for 2 hr. Subsequently, nocodazole was washed out and cells were fixed at the indicated time points. Antibodies against α-tubulin were used for cell staining. Yellow dashed lines in the overlay indicate the cell edge.DOI:http://dx.doi.org/10.7554/eLife.24746.005
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fig1s1: Effects of KIF21B expression on MT organization and regrowth in cells(A) Time-lapse imaging of transiently transfected COS-7 cells expressing KIF21B-FL-GFP and TagRFP-tubulin. Yellow dashed lines in the overlay indicate the cell edge. (B) Nocodazole washout experiments of COS-7 cells expressing GFP or KIF21B-FL-GFP. Cells were transiently transfected with the indicated proteins and treated with 5 μM nocodazole for 2 hr. Subsequently, nocodazole was washed out and cells were fixed at the indicated time points. Antibodies against α-tubulin were used for cell staining. Yellow dashed lines in the overlay indicate the cell edge.DOI:http://dx.doi.org/10.7554/eLife.24746.005

Mentions: To get insight into the ability of KIF21B to regulate MT dynamics, we have expressed the full-length protein with a C-terminal GFP tag in COS-7 cells, which do not express endogenous KIF21B. Unlike its paralogue KIF21A, which is largely diffuse when expressed in similar conditions (van der Vaart et al., 2013), KIF21B bound to MTs and accumulated at their ends at the cell periphery (Figure 1A). Live cell imaging showed that KIF21B processively moves along MTs with an average speed of 0.63 ± 0.22 µm/s (mean±SD) (Figure 1B); this velocity is three times faster than that recently described for HaloTag-labeled KIF21B in neurons (Ghiretti et al., 2016). In internal cell regions, where no clear accumulation of the motor at growing MT ends was observed, the expression of KIF21B led to a ~1.5 fold reduction in the MT growth rate measured with the MT plus-end marker EB3-TagRFP-T (Stepanova et al., 2003; van der Vaart et al., 2013) (Figure 1C). At the cell periphery, strong accumulation of KIF21B-GFP and stalling of MT growth were observed; however, the exact quantification of MT dynamics at the periphery of KIF21B-overexpressing cells was severely complicated by the frequent sliding of MT tips against each other. Interestingly, in cells with high expression levels of KIF21B, the MT network often strongly retracted, leaving significant portions of the cytoplasm largely devoid of MTs (Figure 1D). The remaining MT network in such cells was still dense and appeared to be ‘corralled’ by KIF21B accumulations. Time lapse imaging showed that the retraction of the MT network in KIF21B-expressing cells was a gradual process that could be detected during 1–2 hr of observation (Figure 1—figure supplement 1A). In addition, expression of KIF21B prevented full extension of MTs in experiments where the MT network recovered from treatment with the MT-depolymerizing drug nocodazole (Figure 1—figure supplement 1B). We conclude that at high expression levels, KIF21B can accumulate at MT plus ends, block their polymerization and cause their very slow shortening (Figure 1—figure supplement 1A).10.7554/eLife.24746.003Figure 1.KIF21B inhibits MT growth in cells.


Kinesin-4 KIF21B is a potent microtubule pausing factor
Effects of KIF21B expression on MT organization and regrowth in cells(A) Time-lapse imaging of transiently transfected COS-7 cells expressing KIF21B-FL-GFP and TagRFP-tubulin. Yellow dashed lines in the overlay indicate the cell edge. (B) Nocodazole washout experiments of COS-7 cells expressing GFP or KIF21B-FL-GFP. Cells were transiently transfected with the indicated proteins and treated with 5 μM nocodazole for 2 hr. Subsequently, nocodazole was washed out and cells were fixed at the indicated time points. Antibodies against α-tubulin were used for cell staining. Yellow dashed lines in the overlay indicate the cell edge.DOI:http://dx.doi.org/10.7554/eLife.24746.005
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fig1s1: Effects of KIF21B expression on MT organization and regrowth in cells(A) Time-lapse imaging of transiently transfected COS-7 cells expressing KIF21B-FL-GFP and TagRFP-tubulin. Yellow dashed lines in the overlay indicate the cell edge. (B) Nocodazole washout experiments of COS-7 cells expressing GFP or KIF21B-FL-GFP. Cells were transiently transfected with the indicated proteins and treated with 5 μM nocodazole for 2 hr. Subsequently, nocodazole was washed out and cells were fixed at the indicated time points. Antibodies against α-tubulin were used for cell staining. Yellow dashed lines in the overlay indicate the cell edge.DOI:http://dx.doi.org/10.7554/eLife.24746.005
Mentions: To get insight into the ability of KIF21B to regulate MT dynamics, we have expressed the full-length protein with a C-terminal GFP tag in COS-7 cells, which do not express endogenous KIF21B. Unlike its paralogue KIF21A, which is largely diffuse when expressed in similar conditions (van der Vaart et al., 2013), KIF21B bound to MTs and accumulated at their ends at the cell periphery (Figure 1A). Live cell imaging showed that KIF21B processively moves along MTs with an average speed of 0.63 ± 0.22 µm/s (mean±SD) (Figure 1B); this velocity is three times faster than that recently described for HaloTag-labeled KIF21B in neurons (Ghiretti et al., 2016). In internal cell regions, where no clear accumulation of the motor at growing MT ends was observed, the expression of KIF21B led to a ~1.5 fold reduction in the MT growth rate measured with the MT plus-end marker EB3-TagRFP-T (Stepanova et al., 2003; van der Vaart et al., 2013) (Figure 1C). At the cell periphery, strong accumulation of KIF21B-GFP and stalling of MT growth were observed; however, the exact quantification of MT dynamics at the periphery of KIF21B-overexpressing cells was severely complicated by the frequent sliding of MT tips against each other. Interestingly, in cells with high expression levels of KIF21B, the MT network often strongly retracted, leaving significant portions of the cytoplasm largely devoid of MTs (Figure 1D). The remaining MT network in such cells was still dense and appeared to be ‘corralled’ by KIF21B accumulations. Time lapse imaging showed that the retraction of the MT network in KIF21B-expressing cells was a gradual process that could be detected during 1–2 hr of observation (Figure 1—figure supplement 1A). In addition, expression of KIF21B prevented full extension of MTs in experiments where the MT network recovered from treatment with the MT-depolymerizing drug nocodazole (Figure 1—figure supplement 1B). We conclude that at high expression levels, KIF21B can accumulate at MT plus ends, block their polymerization and cause their very slow shortening (Figure 1—figure supplement 1A).10.7554/eLife.24746.003Figure 1.KIF21B inhibits MT growth in cells.

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.