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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

Perturbation of MT growth in vitro by full-length KIF21B(A, B) Kymographs illustrating perturbation of MT growth in vitro by 0.5 nM KIF21B-FL-GFP in the presence of 15 and 30 µM tubulin with 3% Rhodamine-tubulin and 20 nM mCherry-EB3. Time lapse images on the right illustrate MT plus tip curling after the binding of KIF21B-FL-GFP to the MT plus end. The position of the kinesin on the MT is indicated by arrows. Asterisks show the position of growing MT tips extending beyond the point of KIF21B attachment. Boxed area is zoomed. See also Supplemental Video 2. Kymographs were generated from 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.24746.021
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fig4s1: Perturbation of MT growth in vitro by full-length KIF21B(A, B) Kymographs illustrating perturbation of MT growth in vitro by 0.5 nM KIF21B-FL-GFP in the presence of 15 and 30 µM tubulin with 3% Rhodamine-tubulin and 20 nM mCherry-EB3. Time lapse images on the right illustrate MT plus tip curling after the binding of KIF21B-FL-GFP to the MT plus end. The position of the kinesin on the MT is indicated by arrows. Asterisks show the position of growing MT tips extending beyond the point of KIF21B attachment. Boxed area is zoomed. See also Supplemental Video 2. Kymographs were generated from 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.24746.021

Mentions: How can relatively infrequent arrivals of KIF21B to growing MT tips significantly affect MT elongation rate? We noticed that, even when the polymerization of a MT plus end was not fully suppressed by the incoming KIF21B molecule, in cases when the kinesin did not immediately detach from the MT, MT elongation was typically strongly perturbed (Figure 4, Figure 4—figure supplement 1A). We observed many events where a MT tip was undergoing short repeated growth and shortening excursions from the point of KIF21B stalling (Figure 4A,B, Supplemental Video 2). Such MT behavior indicates that KIF21B immobilized at a MT tip prevented both its normal elongation and also its depolymerization. At 30 μM tubulin, the KIF21B stalling events typically led to very irregular growth, which ended in catastrophe (Figure 4—figure supplement 1B). Importantly, after the point where KIF21B was stalled, the path of the growing MT often became curved (Figure 4, Figure 4—figure supplement 1, Supplemental Video 2), while control MTs always grew straight in our assays. Taken together, these data suggest that KIF21B attached to the plus end might be blocking growth of a few protofilaments, leading to the extension of an incomplete and thus more flexible tube, which is more prone to undergo a catastrophe.10.7554/eLife.24746.019Figure 4.KIF21B molecules persisting on a MT tip can perturb MT growth.


Kinesin-4 KIF21B is a potent microtubule pausing factor
Perturbation of MT growth in vitro by full-length KIF21B(A, B) Kymographs illustrating perturbation of MT growth in vitro by 0.5 nM KIF21B-FL-GFP in the presence of 15 and 30 µM tubulin with 3% Rhodamine-tubulin and 20 nM mCherry-EB3. Time lapse images on the right illustrate MT plus tip curling after the binding of KIF21B-FL-GFP to the MT plus end. The position of the kinesin on the MT is indicated by arrows. Asterisks show the position of growing MT tips extending beyond the point of KIF21B attachment. Boxed area is zoomed. See also Supplemental Video 2. Kymographs were generated from 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.24746.021
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Related In: Results  -  Collection

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fig4s1: Perturbation of MT growth in vitro by full-length KIF21B(A, B) Kymographs illustrating perturbation of MT growth in vitro by 0.5 nM KIF21B-FL-GFP in the presence of 15 and 30 µM tubulin with 3% Rhodamine-tubulin and 20 nM mCherry-EB3. Time lapse images on the right illustrate MT plus tip curling after the binding of KIF21B-FL-GFP to the MT plus end. The position of the kinesin on the MT is indicated by arrows. Asterisks show the position of growing MT tips extending beyond the point of KIF21B attachment. Boxed area is zoomed. See also Supplemental Video 2. Kymographs were generated from 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.24746.021
Mentions: How can relatively infrequent arrivals of KIF21B to growing MT tips significantly affect MT elongation rate? We noticed that, even when the polymerization of a MT plus end was not fully suppressed by the incoming KIF21B molecule, in cases when the kinesin did not immediately detach from the MT, MT elongation was typically strongly perturbed (Figure 4, Figure 4—figure supplement 1A). We observed many events where a MT tip was undergoing short repeated growth and shortening excursions from the point of KIF21B stalling (Figure 4A,B, Supplemental Video 2). Such MT behavior indicates that KIF21B immobilized at a MT tip prevented both its normal elongation and also its depolymerization. At 30 μM tubulin, the KIF21B stalling events typically led to very irregular growth, which ended in catastrophe (Figure 4—figure supplement 1B). Importantly, after the point where KIF21B was stalled, the path of the growing MT often became curved (Figure 4, Figure 4—figure supplement 1, Supplemental Video 2), while control MTs always grew straight in our assays. Taken together, these data suggest that KIF21B attached to the plus end might be blocking growth of a few protofilaments, leading to the extension of an incomplete and thus more flexible tube, which is more prone to undergo a catastrophe.10.7554/eLife.24746.019Figure 4.KIF21B molecules persisting on a MT tip can perturb MT growth.

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