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Cytoplasmic dynein-associated structures move bidirectionally in vivo

Department of Cell and Molecular Biology, Robert H. Lurie Comprehensive Cancer Center, and Center for Genetic Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA

View larger version (19K): [in a new window]   Fig. 1. The two-step approach to generate cells expressing IC-GFP in an IC-null background. (a) Step 1, introduction of an IC-GFP expression construct into wild-type background. (b) Step 2, targeting the endogenous dic gene by homologous recombination in cells stably expressing IC-GFP using the Thy1 selectable marker. (c) The dic gene is replaced by thy1 and the only IC expressed is the IC-GFP fusion.

View larger version (69K): [in a new window]   Fig. 2. IC-GFP expression in an IC-null background. (a) IC expression. Western blot of cell lysates probed with IC antibody IC144 (Ma et al., 1999). Lane 1, wild type; lane 2, IC-GFP-expressing cell line; lane 3, cell line (from 2) with disrupted gene for IC. (b) IC-GFP associates with dynein complex. Western blots of cell lysate (lane 3) or immunoprecipitates (lanes 1 and 2) were probed with anti-IC. Dynein was immunoprecipitated using the dynein heavy chain antibody NW127 (lane 1) and dynactin immunoprecipitated using the capping protein ß antibody R18 (lane 2) (Ma et al., 1999). (c) IC-GFP shows a punctate localization along linear tracks in fixed cells. The nucleus appears as a darker round region in the center. (d) Linear tracks of dynein movement visualized by superimposing a time-lapse series of dynein-GFP images collected from a living cell.

View larger version (44K): [in a new window]   Fig. 3. Dynein-GFP colocalizes with microtubules. Immunofluorescence staining of IC-GFP-expressing cells with an anti-tubulin antibody followed by Lissamine-conjugated secondary antibody. (a) Dynein-GFP signal. (b) Tubulin staining. (c) Overlay of (a) and (b), with GFP in green and tubulin staining in red. Yellow indicates colocalization. The arrowhead points to an example of punctate dynein-GFP signals lining along a microtubule. The arrow points to an example of dynein-GFP signal associated with microtubule plus end.

View larger version (101K): [in a new window]   Fig. 4. Dynein exhibits unidirectional movement in both retrograde and anterograde directions. Time-lapse images of dynein-GFP in living cells. (a) Retrograde movement (see also Movie 1 at jcs.biologists.org ). (b) Anterograde movement (see also Movie 2 at jcs.biologists.org ). The arrowhead indicates the position of a single dynein-associated structure in each frame. The last panels of (a) and (b) show the track of the moving vesicle by superimposing dots marking the position of the indicated dynein-containing structure from each frame. It is worth noting that each of the video sequences shows a variety of movement types. A single example in each video has been highlighted for simplicity. The elapsed time is shown as `seconds.milliseconds'. Scale bar, 5 µm.

View larger version (31K): [in a new window]   Fig. 5. Rates of movement and travel distances observed for dynein-containing structures. (a) Velocity histogram of retrograde and anterograde dynein movements. Velocities were measured as an average of distance over time for each run. (b) Histogram of travel distance. Movements with travel distance less than 1 µm were excluded from the analysis. A total of 65 retrograde and 55 anterograde movements were analyzed for (a) and (b).

View larger version (62K): [in a new window]   Fig. 6. Dynein containing structures show rapid changes in direction. (a) Dynein-containing structures exhibit bidirectional movement along a given track (arrowhead) (see Movie 3 in jcs.biologists.org ). In the last panel, the positions at each time point are indicated by a colored dot, with anterograde movement in red and retrograde in green. The yellow color results from overlapping of red and green dots, suggesting that the vesicle was moving along the same track after switching direction. The elapsed time is shown as `seconds.milliseconds'. Scale bar, 5 µm. (b) Patterns of dynein movements are shown by plotting distance from the MTOC over time. Typical examples of bidirectional movements are shown, with each single run represented by a different color. Time intervals between adjacent frames are 302 mseconds. Retrograde movements appear as descending distance from the MTOC with time, whereas anterograde movements appear as the opposite.

View larger version (36K): [in a new window]   Fig. 7. Dynein traffic at the microtubule ends. (a) Dynein traffic near the MTOC (see Movie 4 and Movie 5 at jcs.biologists.org ). Arrows along the microtubule tracks indicate movements into and out of the MTOC. Back-to-back arrows represent bidirectional movement. In this example, three in, three out and two bidirectional movements were observed during an 8.785 second period. (b) Dynein traffic at the microtubule plus end (Movie 6 at jcs.biologists.org ). The microtubule track is partially highlighted, with the plus end resembling a comet-tail. Arrows indicate movements into and out of the microtubule plus end. An example of this structure can also be observed in Fig. 3c (arrowhead). Scale bars, 5 µm.

View larger version (32K): [in a new window]   Fig. 8. A schematic representation of dynein traffic in the cell. The gray linear network represents interphase microtubules, and the black box in the center is the centrosome. Dynein signals are shown as colored dots, with red representing dynein probably being the active motor responsible for the movement and the blue representing dynein being passively transported by an undefined plus-end-directed microtubule-based motor, most likely a kinesin. N, nucleus; V vesicle. The + and - symbols mark the ends of microtubules. For simplicity, cargo is not shown except in the inset. See the discussion for details.