Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains
- Howard Hughes Medical Institute, University of California, San Francisco, United States
Figures
Figure 1
Dynein crosslinks and slides MTs in bundles.
(A) Schematic overview of the dynein constructs used in this study. The N-terminal tail is shown in gray, the linker in purple, the six numbered AAA+ domains are in light blue and the stalk and MT …
Figure 2
Dynein crosslinks and slides single MT overlaps.
(A) Example of single MT-MT sliding driven by GST-Dyn1331kDa. Successive frames, separated by 52 s, from the video and corresponding kymograph show the sliding. The transport MT is captured and …
Figure 3 with 1 supplement
Natively dimerized dynein can drive MT-MT sliding in the absence of associated factors.
(A) Schematic overview of the Dyn1387kD construct compared to Dyn1 and GST-Dyn331kDa, with labels as in Figure 1A. (B) Gel-filtered Dyn1387kD was run on a denaturing gel and proteins were visualized …
Figure 3—figure supplement 1
Further characterization of GFP-Dyn1387kD.
(A) Dyn1387kD induces MT bundling in the absence of ATP. Scale bar is 5 μm. (B) Successive frames from Video 5 showing sliding of red MTs in the presence of ATP within a red- and green-labeled MT …
Figure 4 with 1 supplement
Dynein can bind two MTs simultaneously.
(A–E) Biotinylated Cy5-MTs were incubated with 2 nM GFP-Ase1 or with buffer for 5 min. MTs were then bound to glass slides using surface-bound streptavidin. After washing unbound MTs, TMR-labeled …
Figure 4—figure supplement 1
Analysis of single Dyn1387kD molecules in MT overlaps.
Biotinylated Cy5-MTs were incubated with 2 nM GFP-Ase1 for 5 min. MTs were then bound to glass and GFP-Dyn1387kDa was introduced into the flow cell. A kymograph of GFP-Dyn1387kDa within the MT …
Figure 5 with 1 supplement
Dynein’s motor domains are sufficient to produce an inward force in the spindle.
(A) HEK293 cells were either mock transfected or transfected with siRNA targeting the dynein heavy chain. After 24 hr cells were re-transfected with siRNA. 72 hr after initial transfection, cells …
Figure 5—figure supplement 1
Analysis of GST-hDyn activity in vivo.
(A) HEK293 cells were transfected with GST-hDyn. 24 hr after transfection cells were lysed and pulldowns were performed with either beads alone (1), an HA antibody (2) or a GFP antibody (3) to …
Figure 6
Mechanisms of MT-MT sliding.
(A) Kinesin-5 forms tetrameric molecules, allowing them to crosslink and slide MTs using motor domains at opposite ends of the tetramer. (B) Kinesin-1 and kinesin-14 utilize a secondary, non-motor, …
Videos
Video 1
Rat brain cytoplasmic dynein bundles and slides MTs.
Red-labeled MTs are slid and extruded from a bundle of red- and green-labled MT bundles by rat brain cytoplasmic dynein upon addition of ATP. Total time of the video is 551 s. Playback is 30 fps.
Video 2
GST-Dyn1331kDa slides MTs within bundles.
Red-labeled MTs are extruded from a bundle of red- and green-labeled MTs by GST-Dyn1331kDa upon addition of ATP, indicating the dynein motor domains alone are sufficient for this activity. Total …
Video 3
Rat brain cytoplasmic dynein slides single MT overlaps.
A red-labeled ‘transport’ MT is slid over a green-labeled ‘track’ MT by rat brain cytoplasmic dynein. The transport MT pauses before reaching the end of the track MT. Total time of the video is 271 …
Video 4
GST-Dyn1331kDa slides single MT overlaps.
A red-labeled ‘transport’ MT is slid over a green-labeled ‘track’ MT by GST-Dyn1331kDa, indicating that the dynein motor domains alone are sufficient for this activity. The transport MT slides to …
Video 5
Dyn1387kDa slides MTs within bundles.
Red-labeled MTs are extruded from a bundle of red- and green-labeled MTs by Dyn1387kDa upon addition of ATP. Total time of the video is 250 s. Playback is 12 fps.
Video 6
Dyn1387kDa slides single MT overlaps.
A red-labeled ‘transport’ MT is slid over a green-labeled ‘track’ MT by Dyn1387kDa. Total time of the video is 198 s. Playback is 12 fps.
Video 7
Observation of single Dyn1387kDa molecules in sliding MT-MT overlaps.
Single GFP-labeled Dyn1387kDa molecules visualized during a MT-MT sliding event show homogenous fluorescence, further ruling out protein aggregation as a cause of the sliding behavior. Track MT is …
