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Kinesin 2 (Osm3)

 

We were interested the regulation of OSM-3 motility, a kinesin motor protein involved in cilia formation in sensory neurons. Using a single molecule fluorescent assay, we showed that OSM-3 GFP does not move processively (take multiple steps along a microtubule without dissociation) and displayed low microtubule-stimulated ATPase activity. This result was surprising since in vivo, intraflagellar transport particles (IFT's) transported by OSM-3 move at 1.3 µM/s for distances up to 2 µM. We found that a chimera of the OSM-3 motor domain attached to a portion of the stable coiled-coil of conventional kinesin, a processive Kinesin-1 family member, did move processively at velocities similar to those measured in vivo (Movie 1). This surprising gain of function mutation suggested that the C-terminal region of OSM-3 (consisting of the coiled-coil and a tail domain) was involved in regulating processivity. We hypothesized that a hinge region in the coiled-coil domain of OSM-3 could be responsible for stimulating head/tail interactions that inhibited processivity. Consistent with this notion, we found that a single point mutant (G444E) in a predicted hinge region, along with a deletion of the predicted hinge, could also activate the processivity and ATPase activity of OSM-3 (Movie 2). Subsequent sucrose gradient analysis revealed that these hinge mutants cause a conformational change in OSM-3, which leads these mutants to adopt a more extended conformation. We found that the processivity of the wild type OSM-3 could be activated by attaching the motor to beads in an optical trap, a situation which may mimic attachment of IFT cargo in vivo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this auto-inhibition in vivo (Fig 1). Interestingly, the G444E allele in C. Elegans produces similar ciliary defects to an Osm-3 null suggesting that auto-inhibition is important for the biological function of OSM-3.

Fig. 1

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Fig. 2
   

Movie 1 - Wild type Osm-3 (0.5) was introduced into a flow cell with Cy5 labeled axonemes bound to the cover slip. Cy5 Axonemes can be visualized in the first 10 frames (white lines), the rest of the frames OSM-3 (white dots) can be observed transiently interacting with the axonemes and the glass cover slip. The dimensions of the movie are 10.6 X 24.1 µM. The video was taken and is displayed at 30 frames per secondMovie 1 Wild type Osm-3 (0.5) was introduced into a flow cell with Cy5 labeled axonemes bound to the cover slip. Cy5 Axonemes can be visualized in the first 10 frames (white lines), the rest of the frames OSM-3 (white dots) can be observed transiently interacting with the axonemes and the glass cover slip. The dimensions of the movie are 10.6 X 24.1 µM. The video was taken and is displayed at 30 frames per second.

(Click on image above to see movie. If movie does not work, click here.)
   
Movie 2 - Osm-3-G444E (0.5nM) was introduce into flow cell with axonemes bound to the cover slip. Single Osm-3-G444E molecules (white dots) can be observed moving in a unidirection manner along the axoneme (not labeled in this video). The dimensions of the movie are 11.6 X 28.4 µM. The video was taken and is displayed at 30 frames per second.

 

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updated 4/9/07


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