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In eukaryotic cells, cytoplasmic dynein is the molecular motor responsible for nearly all minus-end directed microtubule-based transport. Cytoplasmic dynein transports numerous cargo including organelles, proteins and RNAs and is required for cell division. Compared to kinesins and myosins, the molecular mechanism of dynein-based microtubule transport is much less well understood. Dynein has been challenging primarily because of its size: cytoplasmic dynein is a homodimer of two ~500 kDa proteins (Figure 1).
In the Vale lab, we have developed tools using the model system, S. cerevisiae, to probe the molecular mechanism of cytoplasmic dynein (Reck-Peterson et al, 2006).
Yeast cytoplasmic dynein is a processive motor
Processive motors can take multiple steps along their track without detaching. To study cytoplasmic dynein processivity we placed a tag after the COOH-terminus of the dynein motor domain (after AAA 6) that we could label with the fluorescent dye, TMR, in vitro. Using total internal reflection (TIRF) microscopy we showed that single TMR-labeled dynein molecules moved processively on microtubules (Movie 1). We also found that dimerization was required for processivity, as dynein monomers are not processive, but artificially dimerized dynein dimers are processive. Despite having over 11 associated subunits that are all essential for dynein function in vivo, none of these subunits were required for processive movement in vitro.
Cytoplasmic dynein takes predominately 8 nm steps
In order to observe single steps taken by individual dynein molecules we labeled dynein dimers on either a single motor domain or on the tail domain with a bright quantum dot. We found that dynein takes predominately 8 nm steps- the minimal distance between binding sites on the microtubule lattice. However, we also found that dynein’s step size is quite variable: it can take longer steps as well as backwards and sideways steps, indicating that its step has a considerable diffusive element. Labeling of individual motor domains (instead of the centroid position of the molecule) produced longer steps, leading us to suggest that dynein’s two motor domains coordinately alternate their forward and rear position. Based on these studies we proposed the first model for how dimeric cytoplasmic dynein moves processively along microtubules (Figure 2).
Fig 1 - The domain structure of cytoplasmic dynein. Dynein is a dimer of two ~ 500 kDa heavy chains. Dynein is a member of the AAA+ family of ATPases- its motor domains contains 6 AAA domains, 4 of which can bind ATP.
Fig 2 - Model for processive movement of dimeric cytoplasmic dynein. The head (COOH-terminus)- labeled dynein (H) is represented by the red tag and the center-of-mass labeled dynein (T) is represented by the yellow tags (the AAA ring is shown schematically from this top view as a rectangle with rounded edges; the AAA rings and tubulin dimers are shown approximately to scale). (A) In this model, the two dynein motor domains alternate between forward and rear positions, the tail labeled dynein takes predominantly successive 8 nm steps. In contrast, a single dynein motor domain takes a 16 nm step and then remains stationary (a 0 nm step) as its partner ring advances. Although dynein is shown here moving along a single protofilament, we have shown that dynein can also move on neighboring protofilaments.
Movie 1 - Yeast cytoplasmic dynein is a processive motor. Single TMR-labeled dynein molecules can be seen moving processively along axonemal microtubules. The data was collected at 0.5 frames/ sec and the movies shown are sped up 100X. The size of the movie window is 7.4 x 18.3 um.
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