Kinesin-1, -2, and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport
- Department of Biomedical Engineering, Pennsylvania State University, United States
- Department of Biological Chemistry and the Life Sciences Institute, University of Michigan-Ann Arbor, United States
Figures
Figure 1 with 4 supplements
Experimental setup and visualization of dynein-dynactin-BicD2 (DDB)-Kin complexes.
(A) Schematic of kinesin constructs containing SNAP tags functionalized with an Alexa Fluor 647 dye and a single-stranded DNA oligo. (B) DDB and kinesin motors connected via complementary DNA oligos …
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Figure 1—source data 1
Position vs. time data for the plots in Figure 1D.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Purification gels and shift assays.
(A) Shift assay for K560SNAP, showing the oligo-labeled monomer concentration is 3.25 μM. (B) SDS-PAGE for the purification of K560SNAP showing 63% oligo-labeled monomer. (C) Shift assay for …
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Figure 1—figure supplement 1—source data 1
Purification and shift-assay SDS-PAGE gel images.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
Unloaded run length and velocity for Kin1/2/3.
(A) Velocity distributions for kin1, kin2, and kin3. Values represent mean and standard deviation. (B) Cumulative distributions of the run length for Kin1, Kin2, and Kin3. Values represent mean of a …
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Figure 1—figure supplement 2—source data 1
Velocity and run length data for Kin1, Kin2, and Kin3.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
Unloaded run length and velocity for dynein-dynactin-BicD2 (DDB).
(A) Velocity distributions for DDB alone. Values represent mean and standard deviation. (B) Cumulative distributions of the run length for DDB alone. Values represent mean of a single exponential …
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Figure 1—figure supplement 3—source data 1
Velocity and run length data for DDB.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig1-figsupp3-data1-v2.xlsx
Figure 1—figure supplement 4
Sample traces for dynein-dynactin-BicD2 (DDB)-kin1/2/3 pairs.
(A) Sample fast (v>vel threshold) traces for DDB-Kin1 pairs. (B) Sample fast (v>vel threshold) traces for DDB-Kin2 pairs. (C) Sample fast (v>vel threshold) traces for DDB-Kin3 pairs. (D) Sample slow …
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Figure 1—figure supplement 4—source data 1
Position vs time data for the sample plots.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig1-figsupp4-data1-v2.xlsx
Figure 2
Dynein-dynactin-BicD2 (DDB)-Kin1 pairs move faster, and more frequently, to the plus-end than DDB-Kin2/3 pairs.
(A) Scatter plots showing whole trace velocities of the kinesin alone (gray/top), DDB alone (green/middle), and the DDB-Kin1/2/3 pair (blue/orange/yellow/bottom). Error bars represent median values …
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Figure 2—source data 1
Velocity data for DDB-Kin1, DDB-Kin2, and DDB-Kin3.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig2-data1-v2.xlsx
Figure 3
Fast, plus-end events represent a diffusive or weakly bound dynein-dynactin-BicD2 (DDB).
(A) Run length distributions of the fast traces and unloaded kinesin. Error bars represent median values and quartiles. (B) Velocity distributions of the fast DDB-Kin traces and unloaded kinesin. …
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Figure 3—source data 1
Run length, velocity, and trace duration data for Kin1, Kin2, Kin3, and the DDB-Kin pairs.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig3-data1-v2.xlsx
Figure 4 with 2 supplements
Pauses are due to a DDB ‘stuck’ state.
(A) Distributions of segment velocities for the slow traces. Less than 10% of the data is excluded to zoom in on the peak at zero. (B) Fraction of segments that are paused (defined as moving <1 …
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Figure 4—source data 1
Segment velocity and duration data for Kin1, Kin2, Kin3, and the DDB-Kin pairs.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Slow segment velocity distributions.
Cumulative distributions of the slow segment (v<threshold) velocities for all three motor pairs.
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Figure 4—figure supplement 1—source data 1
Cumulative distribution of the segment velocity data for the DDB-Kin pairs.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Sample traces for dynein-dynactin-BicD2 (DDB) alone.
(A) Sample x-t plots for DDB alone. (B) Fraction of paused and moving segments for DDB alone.
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Figure 4—figure supplement 2—source data 1
Position vs. time data for DDB alone.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig4-figsupp2-data1-v2.xlsx
Figure 5
Dynein-dynactin-BicD2 (DDB)-Kin1/2/3 all compete effectively against DDB.
(A) Distribution of instantaneous velocities calculated over 1 s time windows for the moving segments (excluding pauses). Dashed line represents v=0 nm/s. Less than 13% of data are not shown to zoom …
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Figure 5—source data 1
Instantaneous velocity distributions for the DDB-Kin pairs.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig5-data1-v2.xlsx
Figure 6 with 1 supplement
Dynein-dynactin-BicD2 (DDB)-Kinesin stepping simulations can recapitulate experimental velocities.
(A) Schematic of the stochastic stepping model used in simulations. (B) Probability density functions (PDF) of the instantaneous velocity distributions of the experimental (blue, orange, and yellow …
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Figure 6—source code 1
DDB-Kin bidirectional transport simulation.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig6-code1-v2.zip
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Figure 6—source data 1
Simulated instantaneous velocity data.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Simulation results for slower reattachment rates.
(A) Raw simulation traces using the best-fit model for dynein-dynactin-BicD2 (DDB)-Kin1 (top/blue), DDB-Kin2 (middle/orange), and DDB-Kin3 (bottom/yellow). (B) Simulation velocity distributions for …
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Figure 6—figure supplement 1—source data 1
Simulated position vs. time and instantaneous velocity data.
- https://cdn.elifesciences.org/articles/82228/elife-82228-fig6-figsupp1-data1-v2.xlsx
Tables
Table 1
Model parameters.
| Kin1 | Kin2 | Kin3 | DDB | |
|---|---|---|---|---|
| V0 (nm/s) | 586 | 307 | 910 | 360 |
| kforward (s–1) | 76 | 41 | 117 | 60 |
| (s–1) | 0.96 | 0.76 | 0.16 | 0.1 |
| Fs (pN) | 6 | 6 | 6 | 3.6 |
| Fdetach (pN) | 6.8 | 3 | 1.3 | (Ideal bond) |
| kreattach (s–1) | 100 | 300 | 990 | 5 |
| kbackstep (s–1) | 3 | 3 | 3 | 5 |
| κmotor (pN/nm) | 0.2 | 0.2 | 0.2 | 0.2 |
