Kinesin-4 KIF21B is a potent microtubule pausing factor
- Utrecht University, Netherlands
- Paul Scherrer Institut, Switzerland
Figures
Figure 1 with 1 supplement
KIF21B inhibits MT growth in cells.
(A) COS-7 cells were transiently transfected with KIF21B-FL-GFP and EB3-TagRFP-T and imaged using TIRF microscopy. Represented are a single-frame, maximum intensity projection of 500 frames for the …
-
Figure 1—source data 1
An excel sheet with numerical data on the quantification of kinesin velocities and MT growth rate in COS-7 cells represented as plots in Figure 1B,C,E,I.
- https://doi.org/10.7554/eLife.24746.004
Figure 1—figure supplement 1
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 …
Figure 2 with 3 supplements
Dimeric motor domain of KIF21B slows down MT polymerization in vitro.
(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 …
-
Figure 2—source data 1
An excel sheet with numerical data on the quantification of KIF21B-MD-CC1-GFP dimer analysis, photobleaching-step analysis, velocities, run length, effects on MT growth rate and distribution of EB3 fluorescence intensity represented as plots in Figures 2A,B,D,E–H,J.
- https://doi.org/10.7554/eLife.24746.007
Figure 2—figure supplement 1
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.
Figure 2—figure supplement 2
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).
Figure 2—figure supplement 3
Effects of the dimeric motor domain of KIF21B on MT polymerization in vitro
(A) Kymographs illustrating the in vitro dynamics of MTs grown in the presence of different concentrations of tubulin along with 20 nM EB3 in the absence and presence of 2 nM KIF21B-MD-CC1-GFP. …
-
Figure 2—Figure Supplement 3—Source Data 1
An excel sheet with numerical data on the quantification of the MT minus end growth rates represented as plot in Figure 2—figure supplement 3B.
- https://doi.org/10.7554/eLife.24746.011
Figure 3 with 2 supplements
KIF21B can induce MT pausing or catastrophe in vitro.
(A) Kymographs showing the behavior of KIF21B in in vitro reconstitution assays on dynamic MTs grown from Rhodamine-tubulin-labeled seeds in the presence of 15 µM tubulin, 100 nM mCherry-EB3 (red) …
-
Figure 3—source data 1
An excel sheet with numerical data on the quantification of KIF21B-FL seed blocking activity, pause induction, effects on MT growth rate and catastrophe frequency and outcomes of KIF21B-FL-GFP arrival at MT plus ends represented as plots in Figure 3B,C,E,H,I.
- https://doi.org/10.7554/eLife.24746.013
Figure 3—figure supplement 1
Characterization of full-length KIF21B in vitro
(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 …
-
Figure 3—figure supplements 1—source data 1
An excel sheet with numerical data on the quantification of the KIF21B-FL dimer and photobleaching step analysis represented as plots in Figure 3—figure supplement 1A,B.
- https://doi.org/10.7554/eLife.24746.015
Figure 3—figure supplement 2
KIF21B-FL-GFP induces pausing of a depolymerizing MT.
The rightmost panel shows tracked positions of the kinesins and the MT tip together with the fluorescence intensities of the kinesins over time (white boxed area in kymograph). See also Supplemental …
-
Figure 3—Figure Supplement 2—Source Data 1
An excel sheet with numerical data on the quantification of tracked positions of the kinesins and the MT tip together with the fluorescence intensities of the kinesins over time represented as plot in Figure 3—figure supplement 2.
- https://doi.org/10.7554/eLife.24746.017
Figure 4 with 1 supplement
KIF21B molecules persisting on a MT tip can perturb MT growth.
(A, B) Kymographs illustrating perturbation of MT growth in vitro by 0.5 nM KIF21B-FL-GFP in the presence of 15 µM tubulin with 3% Rhodamine-tubulin and 20 nM mCherry-EB3. Kymographs were generated …
-
Figure 4—source data 1
An excel sheet with numerical data on the quantification of tracking of kinesins and MT tips over time represented as plots in Figure 4A,B.
- https://doi.org/10.7554/eLife.24746.020
Figure 4—figure supplement 1
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 …
Figure 5 with 2 supplements
Single events of kinesin arrival to the MT plus end can induce MT pausing.
(A) GFP intensity analysis of kinesins during MT pausing events. Values are normalized to the GFP intensity of proteins immobilized on the same coverslip in areas devoid of MTs. Data are from two …
-
Figure 5—source data 1
An excel sheet with numerical data on the quantification of KIF21B-FL intensity during MT pausing events, KIF5B-560 dimer analysis and comparison of fluorescence intensities of KIF5B-560 with KIF21B-FL represented as plots in Figure 5A,C,D–I.
- https://doi.org/10.7554/eLife.24746.024
-
Figure 5—figure Supplement 2—Source data 1
An excel sheet with numerical data on the quantification of photobleaching traces of KIF21B-FL-GFP represented as plots in Figure 5—figure supplement 2.
- https://doi.org/10.7554/eLife.24746.025
Figure 5—figure supplement 1
Kymographs illustrating KIF21B-FL-GFP (0.5 nM) moving on seeds and dynamic MTs in in vitro reconstitution assays, histograms of the corresponding fluorescence intensities (symbols) and the corresponding fits with lognormal distributions (lines).
Median values are also indicated.
-
Figure 5—figure supplements 1—Source data 1
An excel sheet with numerical data on the quantification of KIF21B-FL fluorescence intensities represented as plots in Figure 5—figure supplement 1.
- https://doi.org/10.7554/eLife.24746.027
Figure 5—figure supplement 2
Characteristic photobleaching traces of KIF21B-FL-GFP under two different imaging conditions.
KIF21B-FL-GFP immobilized on coverslip was exposed to low laser power (used for imaging shown in Figures 3–5 and 7) with 100 ms/stream acquisition or 100 ms exposure time, 1 frame per 1.2 s. Curves …
Figure 6 with 4 supplements
Mapping of the MT-binding domains in the tail of KIF21B.
(A) Overview of deletion mutants used in this study. Colocalization of the GFP-tagged KIF21B deletion mutants with MTs in transiently transfected COS-7 cells is indicated. +, localization to MTs, -, …
-
Figure 6—figure supplement 4—source data 1
An excel sheet with numerical data on the quantification of far-UV CD spectra (inset) and thermal unfolding profile of recombinant KIF21B rCC1 represented as plots in Figure 6—figure supplement 4A.
- https://doi.org/10.7554/eLife.24746.030
Figure 6—figure supplement 1
In vitro reconstitution of MT growth in the presence of 20 nM mCherry-EB3, 3% Rhodamine-tubulin and 0.5 nM KIF21B-FL-GFP.
KIF21B-FL-GFP is attached to one MT and walks along another one, causing MT bending. Arrow indicates position of KIF21B-FL-GFP, yellow arrowheads trace the MT that bends.
Figure 6—figure supplement 2
(A) COS-7 cells transiently transfected with the indicated KIF21B-GFP deletion constructs and stained for α-tubulin. (B) MT pelleting assay of taxol-stabilized MTs incubated with KIF21B-FL-GFP. …
Figure 6—figure supplement 3
Alignment of human KIF21A and KIF21B sequences.
Different protein domains and the autoinhibitory region in the coiled coil domain described for KIF21A (van der Vaart et al., 2013) are indicated, and CFEOM1-associated mutations found in KIF21A are …
Figure 6—figure supplement 4
(A) Far-UV CD spectra (inset) and thermal unfolding profile of recombinant KIF21B rCC. CD measurements, performed in PBS at a protein concentration of 0.166 mg/ml. (B) Oligomerization state of …
Figure 7 with 1 supplement
The WD40 domain and the autoinhibitory coiled coil region contribute to the pause-promoting activity of KIF21B.
(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 …
-
Figure 7—source data 1
An excel sheet with numerical data on the quantification of KIF21B mutants dimer analysis, photobleaching step analysis, velocities on seeds and MT lattices, MT growth rate in vitro and outcomes of the arrival of KIF21B mutants at MT plus ends, represented as plots in Figure 7A,C,F–I.
- https://doi.org/10.7554/eLife.24746.036
Figure 7—figure supplement 1
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 …
-
Figure 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.
- https://doi.org/10.7554/eLife.24746.038
Author response image 1
Author response image 2
Author response image 3
Example image of KIF21B-FL-GFP density used for quantification.
It can be seen that the density of single molecules is in the optimal range
Videos
Video 1
KIF21B induces pausing of a depolymerizing MT.
The movie shows the arrival of KIF21B-FL-GFP at the end of a depolymerizing MT and a subsequent pausing event. The arrival of additional KIF21B-FL-GFP molecules results in a long pause. The …
Video 2
KIF21B perturbs MT growth and induces MT bending.
The combined movie shows the two different events illustrated in Figure 4—figure supplement 1A and Figure 4B. The movie shows bending of an MT growing beyond the point where KIF21B-FL-GFP was …
Additional files
-
Supplementary file 1
Analysis of purified KIF21B and its deletion mutants used in this study by mass spectrometry.
Samples of purified KIF21B proteins were loaded on SDS-PAGE, isolated from the gel after in-gel digestion and subsequently analyzed by mass spectrometry to test for purity. All identified proteins are included in Supplementary file 1in alphabetical order. Indicated are the molecular weight and the number of unique peptides found for identified proteins in the different KIF21B samples. In total, 121 proteins were identified for KIF21B-FL-GFP, 183 for KIF21B-FL-ΔrCC-GFP, 107 for KIF21B-MD-CCΔrCC-GFP, 92 for GFP-L-WD40 and 63 for KIF21B-MD-CC1-GFP.
- https://doi.org/10.7554/eLife.24746.039
-
Supplementary file 2
Lognormal (best fit) values for the fluorescence intensity measurements.
- https://doi.org/10.7554/eLife.24746.040
