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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

Kymographs illustrating in vitro dynamics of MTs grown in the presence of 15 µM tubulin in the absence and presence of 10 nM purified GFP or 2, 5 and 10 nM KIF21B-MD-CC1-GFP.Kymographs were generated from the movies of 600 frames (stream acquisition, exposure time 500 ms) using Photometrics Evolve 512 EMCCD camera (Roper Scientific).DOI:http://dx.doi.org/10.7554/eLife.24746.009
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fig2s2: Kymographs illustrating in vitro dynamics of MTs grown in the presence of 15 µM tubulin in the absence and presence of 10 nM purified GFP or 2, 5 and 10 nM KIF21B-MD-CC1-GFP.Kymographs were generated from the movies of 600 frames (stream acquisition, exposure time 500 ms) using Photometrics Evolve 512 EMCCD camera (Roper Scientific).DOI:http://dx.doi.org/10.7554/eLife.24746.009

Mentions: (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 = 3107, 5802 and 4674 molecules and fluorophore density was 0.15, 0.28 and 0.23 µm−2 for GFP, GFP-EB3 and KIF21B-MD-CC1-GFP proteins. (B) Representative photobleaching time traces of GFP, GFP-EB3 and KIF21B-MD-CC1-GFP individual molecules (background subtracted). (C) Kymographs illustrating the dynamics of MTs grown in vitro in the presence of 20 nM mCherry-EB3 alone, with 10 nM purified GFP or with 2 and 10 nM KIF21B-MD-CC1-GFP. Zooms of the boxed areas are shown on the right. Kymographs were generated from movies acquired using a Photometrics Evolve 512 EMCCD camera (Roper Scientific) (stream acquisition, exposure time 500 ms). (D) Histograms of fluorescence intensities of single GFP molecules immobilized on coverslips and KIF21B-MD-CC1-GFP moving on MTs in a separate chamber on the same coverslip (symbols) and the corresponding fits with lognormal distributions (lines). n = 4815 and 1381 molecules; fluorophore density was 0.16 and 0.09 µm−2 for GFP and KIF21B-MD-CC1-GFP proteins (for the latter, MT-containing regions were manually selected for analysis). Dashed lines show corresponding relative median values. (E) Histogram of KIF21B-MD-CC1-GFP velocities in vitro is shown with black bars. Red line shows fitting with a normal distribution. n = 675 in two independent experiments. (F) Histogram of KIF21B-MD-CC1-GFP run lengths in vitro is shown with black bars. Red line shows fitting with an exponential distribution. n = 675 in two independent experiments. (G) Upper panel - quantification of the MT growth rate illustrated in C. n = 71 for control, n = 65 for purified GFP, n = 71, 67 and 54 for 2, 5 and 10 nM KIF21B-MD-CC1-GFP, respectively. Lower panel shows quantification of the MT growth rate with 15 µM tubulin alone or with 10 nM purified GFP or with 2, 5 and 10 nM KIF21B-MD-CC1-GFP as illustrated in Figure 2—figure supplement 2. n = 67 for control, n = 57 for purified GFP, n = 71, 66 and 80 for 2, 5 and 10 nM KIF21B-MD-CC1-GFP, respectively, two independent experiments, p<0.0001, Mann-Whitney U test (indicated by asterisks). (H) Quantification of the MT growth rate with different concentrations of tubulin along with 20 nM EB3 in the absence and presence of 2 nM KIF21B-MD-CC1-GFP as illustrated in Figure 2—figure supplement 3. ND; not determined, n = 71 for all conditions. two independent experiments, p<0.0001, Mann-Whitney U test (indicated by asterisks). (I) Kymographs illustrating the dynamics of MTs grown in vitro in the presence of 20 nM EB3 and 1 nM KIF21B-MD-CC1-GFP. Zoom of the boxed area is shown on the right. Kymographs were generated from a movie acquired using Photometrics Evolve 512 EMCCD camera (Roper Scientific) (stream acquisition, exposure time 100 ms). (J) Distribution of EB3 fluorescence intensity fluctuations over time (normalized to its maximum value during a course of a growth event) at MT tip in the presence of 1 nM GFP or 1 nM KIF21B-MD-CC1-GFP (solid line) with Gaussian fit (dotted line). n = 25 in both cases. Thick dotted lines show the peak of the Gaussian fitting. MT dynamics assay was performed in the presence of 15 µM tubulin, 20 nM EB3 and 1 nM GFP or 1 nM KIF21B-MD-CC1-GFP in the separate chambers of the same coverslip. (I) Plot of the mean and SD values of Gaussian fits shown in Figure 2J.


Kinesin-4 KIF21B is a potent microtubule pausing factor
Kymographs illustrating in vitro dynamics of MTs grown in the presence of 15 µM tubulin in the absence and presence of 10 nM purified GFP or 2, 5 and 10 nM KIF21B-MD-CC1-GFP.Kymographs were generated from the movies of 600 frames (stream acquisition, exposure time 500 ms) using Photometrics Evolve 512 EMCCD camera (Roper Scientific).DOI:http://dx.doi.org/10.7554/eLife.24746.009
© Copyright Policy
Related In: Results  -  Collection

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fig2s2: Kymographs illustrating in vitro dynamics of MTs grown in the presence of 15 µM tubulin in the absence and presence of 10 nM purified GFP or 2, 5 and 10 nM KIF21B-MD-CC1-GFP.Kymographs were generated from the movies of 600 frames (stream acquisition, exposure time 500 ms) using Photometrics Evolve 512 EMCCD camera (Roper Scientific).DOI:http://dx.doi.org/10.7554/eLife.24746.009
Mentions: (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 = 3107, 5802 and 4674 molecules and fluorophore density was 0.15, 0.28 and 0.23 µm−2 for GFP, GFP-EB3 and KIF21B-MD-CC1-GFP proteins. (B) Representative photobleaching time traces of GFP, GFP-EB3 and KIF21B-MD-CC1-GFP individual molecules (background subtracted). (C) Kymographs illustrating the dynamics of MTs grown in vitro in the presence of 20 nM mCherry-EB3 alone, with 10 nM purified GFP or with 2 and 10 nM KIF21B-MD-CC1-GFP. Zooms of the boxed areas are shown on the right. Kymographs were generated from movies acquired using a Photometrics Evolve 512 EMCCD camera (Roper Scientific) (stream acquisition, exposure time 500 ms). (D) Histograms of fluorescence intensities of single GFP molecules immobilized on coverslips and KIF21B-MD-CC1-GFP moving on MTs in a separate chamber on the same coverslip (symbols) and the corresponding fits with lognormal distributions (lines). n = 4815 and 1381 molecules; fluorophore density was 0.16 and 0.09 µm−2 for GFP and KIF21B-MD-CC1-GFP proteins (for the latter, MT-containing regions were manually selected for analysis). Dashed lines show corresponding relative median values. (E) Histogram of KIF21B-MD-CC1-GFP velocities in vitro is shown with black bars. Red line shows fitting with a normal distribution. n = 675 in two independent experiments. (F) Histogram of KIF21B-MD-CC1-GFP run lengths in vitro is shown with black bars. Red line shows fitting with an exponential distribution. n = 675 in two independent experiments. (G) Upper panel - quantification of the MT growth rate illustrated in C. n = 71 for control, n = 65 for purified GFP, n = 71, 67 and 54 for 2, 5 and 10 nM KIF21B-MD-CC1-GFP, respectively. Lower panel shows quantification of the MT growth rate with 15 µM tubulin alone or with 10 nM purified GFP or with 2, 5 and 10 nM KIF21B-MD-CC1-GFP as illustrated in Figure 2—figure supplement 2. n = 67 for control, n = 57 for purified GFP, n = 71, 66 and 80 for 2, 5 and 10 nM KIF21B-MD-CC1-GFP, respectively, two independent experiments, p<0.0001, Mann-Whitney U test (indicated by asterisks). (H) Quantification of the MT growth rate with different concentrations of tubulin along with 20 nM EB3 in the absence and presence of 2 nM KIF21B-MD-CC1-GFP as illustrated in Figure 2—figure supplement 3. ND; not determined, n = 71 for all conditions. two independent experiments, p<0.0001, Mann-Whitney U test (indicated by asterisks). (I) Kymographs illustrating the dynamics of MTs grown in vitro in the presence of 20 nM EB3 and 1 nM KIF21B-MD-CC1-GFP. Zoom of the boxed area is shown on the right. Kymographs were generated from a movie acquired using Photometrics Evolve 512 EMCCD camera (Roper Scientific) (stream acquisition, exposure time 100 ms). (J) Distribution of EB3 fluorescence intensity fluctuations over time (normalized to its maximum value during a course of a growth event) at MT tip in the presence of 1 nM GFP or 1 nM KIF21B-MD-CC1-GFP (solid line) with Gaussian fit (dotted line). n = 25 in both cases. Thick dotted lines show the peak of the Gaussian fitting. MT dynamics assay was performed in the presence of 15 µM tubulin, 20 nM EB3 and 1 nM GFP or 1 nM KIF21B-MD-CC1-GFP in the separate chambers of the same coverslip. (I) Plot of the mean and SD values of Gaussian fits shown in Figure 2J.

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