Motility and microtubule depolymerization mechanisms of the Kinesin-8 motor, KIF19A
- Graduate School of Medicine, The University of Tokyo, Japan
- RIKEN Center for Life Science Technologies, Japan
- King Abdulaziz University, Saudi Arabia
Figures
Figure 1
Characteristics of the dual function mouse KIF19A motor domain.
(A) Schematic of mouse KIF19A motor domain constructs. The KIF19A monomer KIF19A-353 (referred to as 353WT) was used in this study. NL, neck-linker. (B) MT gliding assays of 353WT on …
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Figure 1—source data 1
The data and analysis for 353WT.
Sheet F1B: Motility Velocity of 353WT. Sheet F1E: The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT.
- https://doi.org/10.7554/eLife.18101.004
Figure 2 with 2 supplements
Crystal structure of the dual function mouse KIF19A motor domain.
(A, B) Crystal structure of the KIF19A motor domain in the ADP-state seen from the MT binding side (A) and 90 degree rotation around the ordinate axis (B). L2 (yellow), L8-α3-L9 (green), …
Figure 2—figure supplement 1
The unmodeled helix determination by the long wavelength X-ray diffraction.
(A) The 2Fo-Fc electron density map contoured at 1.0 σ (blue) is shown. The dashed line rectangle shows the common α4 position for all reported kinesin structures where no corresponding density was …
Figure 2—figure supplement 2
Sequence alignment between representative kinesin members.
(A) Sequence Alignment around β8-L14-α6 between representative kinesin members.
Figure 3
Basic and hydrophobic residues in L2 contribute to MT-depolymerizing activity.
(A) Top, sequence alignment of L2 between KIF19A and KIF2C. Bottom, the L2 swap mutation and other point mutation strategies are shown. (B) β1b-L2-β1c structure diagram of KIF2C (gold) and KIF19A …
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Figure 3—source data 1
The data and analysis for 353WT and L2 mutants.
Sheet F3D: The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT and L2 mutants.
- https://doi.org/10.7554/eLife.18101.010
Figure 4 with 2 supplements
L2 also affects KIF19A kinetics and motility.
(A) Steady state ATPase kinetics of 100 nM 353WT and its L2 mutants. Data are presented as the mean ± SD (n = 3). (B) Graph of the bound fraction of 353WT and L2 mutants plotted against the …
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Figure 4—source data 1
The data and analysis for 353WT, L2 and swap mutants.
Sheet F4A_F4FS1: The data and analysis of ATPase activity of 353WT and L2 mutants. Sheet F4B: The data and analysis of MT binding assay of 353WT and L2 mutants. Sheet F4C: Motility Velocity of 353WT and L55A mutant. Sheet F4E: Motility Velocity of KIF8A WT and KIF18A swap. Sheet F4G Motility Velocity of KIF5C WT and KIF5C swap.
- https://doi.org/10.7554/eLife.18101.012
Figure 4—figure supplement 1
ATPase kinetics of 353WT and Its L2 mutants.
(A) The basal ATPase kinetics of 353WT and its L2 mutants. (B) The max basal ATPase rate of 353WT and L2 mutants. (C) Microtubule-stimulated ATPase activity of 353WT and its L2 mutants. (D) The max …
Figure 4—figure supplement 2
Binding affinity difference between 353WT and PC2A.
(A) Incubate 10 ng/μl MTs with 353WT and different concentrations of PC2A. (B) Incubate 4 ng/μl MTs with 353WT and different concentrations of PC2A. (C) Incubate 2 ng/μl MTs with 353WT and different …
Figure 5 with 1 supplement
Basic residues in L12 assist KIF19A to effectively depolymerize microtubules.
(A) Top, sequence alignment of KIF19A, KIF5C and KIF18A. Bottom, the L12 swap mutation and other point mutation strategies are shown. (B) L11-α4-L12-α5 structure of KIF18A (light blue) and KIF19A …
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Figure 5—source data 1
The data and analysis for 353WT and L12 mutants.
Sheet F5E: The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT and L12 mutants. Sheet F5H_F5FS1: The data and analysis of ATPase activity of 353WT and L12 mutants. Sheet F5I: The data and analysis of MT binding assay of 353WT and L12 mutants.
- https://doi.org/10.7554/eLife.18101.016
Figure 5—figure supplement 1
Steady state ATPase kinetics of 353WT and Its L12 mutants.
(A) The basal ATPase kinetics of 353WT and its L12 mutants. (B) The max basal ATPase rate of 353WT and L12 mutants. (C) Microtubule-stimulated ATPase activity of 353WT and its L12 mutants. (D) The …
Figure 6 with 2 supplements
Contribution of KIF19A-Specific Asn297.
(A) Sequence alignment of KIF19A and other typical kinesins for the α4-L12-α5 region. For more kinesin member sequence alignments see Figure 6—figure supplement 1A. (B) 1.5 μM GMPCPP-stabilized MTs …
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Figure 6—source data 1
The data and analysis for 353WT and N297P mutant.
Sheet F6C The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT and N297P mutant. Sheet F6F: Motility Velocity of 353WT and N297P mutant. Sheet F6H_F6FS2: The data and analysis of ATPase activity of 353WT and N297P mutant.
- https://doi.org/10.7554/eLife.18101.019
Figure 6—figure supplement 1
Sequence alignment between representative kinesin members.
(A) Sequence Alignment around L12-α5 between representative kinesin members.
Figure 6—figure supplement 2
Steady state ATPase kinetics of 353WT and N297P mutant.
(A) The Basal ATPase kinetics of 353WT and N297P mutant. (B) The Basal ATPase rate of 353WT and N297P mutant. (C) Microtubule-stimulated ATPase activity of 353WT and N297P mutant. (D) The max …
Figure 7 with 1 supplement
Cryo-EM reconstruction of KIF19A on a straight MT.
(A–D) Cryo-EM reconstruction of KIF19A-nucleotide-free complexed with GDP-taxol-MT with three different contour levels (grey, blue, cyan), and atomic models of KIF19A-ADP solved in this study …
Figure 7—figure supplement 1
Cryo-EM reconstruction of KIF19A-MT complex.
(A) FSC curves for standard FSC (black), FSC noise (high resolution noise substitution cutoff 10 Å, gray, Chen et al., 2013) and FSC true (cyan). The resolutions of FSC true according to the 0.143 …
Figure 8
Model of KIF19A motor domain function.
(A) Schematic diagram of the dual functions of KIF19A. (B) Simulated 7 Å maps of straight and curved MTs created from the atomic models of 1JFF and 3RYC, respectively. The two structures were …
Videos
Video 1
Polarity-marked microtubules sliding on 353WT.
353WT was fixed on the coverslip. The strongly-labeled MT minus-ends lead the MT gliding, indicating that KIF19A motor proteins move toward the plus end. 10 seconds intervals, total tracking time 15 …
Additional files
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Supplementary file 1
Crystal structure statistics for KIF19A motor domain 353WT.
Data collection and refinement statistic table.
- https://doi.org/10.7554/eLife.18101.025
