Kinesin and dynein use distinct mechanisms to bypass obstacles
- University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
Single dynein motors, but not kinesin motors, bypass QD obstacles.
(A) Schematic of single-molecule motility assays on surface-immobilized MTs decorated with streptavidin-coated QD585 obstacles. Human kinesin-1, yeast dynein, and mammalian DDB are labeled with …
Figure 1—figure supplement 1
Analysis of single-molecule trajectories on surface-immobilized MTs without normalization.
(A) Mobile fraction, (B) velocity and (C) run length of single motors on surface-immobilized MTs in the presence of QD obstacles (mean ± SD). From left to right, n = 271, 423, 405 for kinesin, 315, …
Figure 2 with 2 supplements
Kinesin pauses more frequently than dynein when encountering QD obstacles.
(A) (Left) Representative traces of yeast dynein, DDB, and kinesin in the absence of QD obstacles on surface-immobilized MTs. (Right) Residence times of the motors in each section of the traces. (B) …
Figure 2—figure supplement 1
Kinesin pauses in the presence of QD obstacles.
Representative kymographs reveal frequent pauses in kinesin motility in the presence of 1 QD µm−1 (top row) or 2 QD µm−1 (bottom row). Most pauses were permanent throughout recording. Processive …
Figure 2—figure supplement 2
Simulations for the pause analysis.
(A) An example trajectory simulated with a pause density of 0.8 µm−1 in the absence of tracking noise (see Materials and methods for the parameters used to generate these trajectories). (B) An …
Figure 3 with 1 supplement
Kinesin detaches from MTs when encountering antibody obstacles.
(A) Kinesin and dynein were labeled with organic dyes and their motility was tested in the presence and absence of anti-tubulin antibody on MTs. (B) Kymographs of TMR-kinesin and LD555-DDB walking …
Figure 3—figure supplement 1
Larger obstacles block access to more protofilaments on an MT.
The schematics represent the cross-sectional view of a motor bypassing an obstacle by taking sideways steps on an MT. Increasing the size of the obstacle or the cargo attached to the motor is …
Figure 4 with 2 supplements
Suspending MTs from the surface does not aid kinesin in avoiding obstacles.
(A) Schematic of a single-molecule motility assay on MT bridges coated with QD obstacles (not to scale). (B) An example image of Cy5-labeled MT bridges in the microfabricated chamber. PDMS ridges …
Figure 4—figure supplement 1
Chamber design and raw data of single-molecule motility along MT bridges.
(A) Workflow for bridge microfabrication. The photoresist is spun and patterned on a silicon wafer. PDMS is then cast on top of the photoresist and silanized to produce a reactive surface. (B) …
Figure 4—figure supplement 2
Kinesin pauses in the presence of QD obstacles on suspended MTs.
Representative kymographs reveal frequent pauses in kinesin motility in the presence of 1 QD µm−1 on suspended MT bridges. Most pauses were permanent throughout recording.
Figure 5 with 1 supplement
Cargos driven by multiple kinesins successfully bypass obstacles.
(A) Schematic of bead motility driven by multiple motors along surface-immobilized MTs decorated with QD obstacles (not to scale). (B) Kymographs reveal the motility of beads coated with kinesin or …
Figure 5—figure supplement 1
The analysis of beads driven by multiple motors on surface-immobilized MTs without normalization.
(A) Mobile fraction and (B) velocity of beads driven by multiple motors on surface-immobilized MTs in the presence of QD obstacles (mean ± SD, three independent experiments). The mobile fraction of …
Figure 6
Cargos driven by multiple motors bypass large obstacles by rotating around the MT.
(A) Schematic of multi-motor bead motility on MT bridges. The position of the bead in the z-axis is determined from changes in bead intensity under brightfield illumination. If a bead is positioned …
Videos
Video 1
Motility of DDB motors is not strongly affected by the QD obstacles.
DDB motors were labeled with QD655 at their N-termini. Single-molecule motility of DDB in the presence of no obstacles (top) or 25 nM QD585 obstacles (bottom) on surface-immobilized MTs. The …
Video 2
Kinesin motors walk processively in the absence of QD obstacles, but motility was completely impaired in the presence of 25 nM QDs.
Kinesin motors were labeled with QD655 at their C-termini. Single-molecule motility of kinesin in the presence of no obstacles (top) or 25 nM QD585 obstacles (bottom) on surface-immobilized MTs. The …
Video 3
Kinesin motors labeled with an organic dye are inhibited by antibody obstacles.
Kinesin motors labeled with TMR walk on MTs in the presence (right) and absence (left) of antibody obstacles. MT fluorescence was recorded in a separate channel (not shown). There was a notable …
Video 4
DDB motors labeled with an organic dye are not strongly inhibited by antibody obstacles.
DDB motors labeled with LD555 walk on MTs in the presence (right) and absence (left) of tubulin antibody obstacles. MT fluorescence was recorded in a separate channel (not shown). DDB motors were …
Video 5
Beads driven by multiple kinesins move processively at high QD obstacle concentrations.
500 nm diameter beads were labeled with 1.5 µM kinesin. Beads move along surface-immobilized MTs in the presence of no obstacles (left) or 166 nM obstacles (right). Boxes highlight the processive …
Video 6
Beads driven by multiple kinesin motors walk processively on MTs coated with antibody obstacle.
Beads coated with 1.5 µM kinesin motors walk on MTs in the presence (right) and absence (left) of antibody obstacles. MT fluorescence was recorded in a separate channel (not shown). Images were …
Video 7
Beads driven by multiple kinesins bypass the PDMS wall.
The box highlights the processive motility of a bead driven by multiple kinesins on an MT bridge (unlabeled) suspended over the PDMS ridges. The valley (dark) is in the center of the movie while the …
Video 8
Beads driven by multiple DDBs bypass the PDMS wall.
The box highlights the processive motility of a bead driven by multiple DDBs on an MT bridge (unlabeled) suspended over the PDMS ridges. The valley (dark) is in the center of the movie while the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Other | Amino quantum dot (655) | ThermoFisher | Q21521MP | |
| Other | Streptavidin quantum dot (585) | ThermoFisher | Q10111MP | |
| Chemical | APTES | Sigma | 440140 | |
| Antibody | Anti-tubulin antibody (mouse monoclonal) | Sigma, Tub 2.1 | T5201 | Dilution range 0–20 µg/mL |
| Peptide, recombinant protein | Human Kinesin-1 | Belyy et al., 2016 | N/A | |
| Peptide, recombinant protein | Yeast dynein heavy chain | Reck-Peterson et al., 2006 | N/A | |
| Peptide, recombinant protein | BicD2 (amino acids 1–400) | Schlager et al., 2014 | Addgene 111862 | |
| Peptide, recombinant protein | Human cytoplasmic dynein complex | Zhang et al., 2017 | N/A | |
| Chemical | Acetone | Sigma | 270725 | |
| Chemical | Ethanol | Sigma | 459828 | |
| Other | IgG Sepharose Beads | GE Healthcare | 17096902 | |
| Chemical | Glutaraldehyde | Fisher Scientific | G1511 | |
| Other | PDMS | Sylgard 184 Silicone Elastomer | N/A | |
| Other | Glucose oxidase | Sigma | G2133 | |
| Other | Catalase | Sigma | C3155 | |
| Chemical | Taxol | Sigma | T7191 | |
| Peptide, recombinant protein | Pig Brain Dynactin | Schlager et al., 2014 | N/A | |
| Peptide, recombinant protein | Pig Brain Tubulin | Castoldi and Popov, 2003 | N/A | |
| Chemical | ATP | Sigma | A3377 | |
| Other | Ni-NTA beads | Thermo Scientific | 88221 | |
| Chemical | Fugene HD transfection reagent | Promega | E2311 | |
| Chemical | HaloTag Ligand succinimidyl ester | Promega | P6751 | |
| Chemical | SU-8 2010 photoresist | Microchem | N/A | |
| Other | Super Active Latex Beads | Thermo Fisher | C37481 | |
| Chemical | Sulfo-NHS | Thermo Fisher | 24510 | |
| Chemical | EDC | Thermo Fisher | 22980 | |
| Software | U-track | Jaqaman et al., 2008 | N/A | |
| Antibody | Anti-GFP (anti-rabbit polyclonal) | Covance | N/A | Used at0.4 mg/mL |
| Peptide, recombinant protein | Mouse BicDR1 (full length) | Urnavicius et al., 2018 | Adapted from Addgene 111585 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.48629.023
