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Fig. 6. (A) Complete sequence of kif12 as reported in the Dictyostelium genomic database (www.dictybase.org). Sequence analysis indicates the presence of a kinesin motor domain (orange) near the N terminus, with an N-terminal extension of ≈100 residues (black). The motor domain is followed by a central stalk (black) and then a C-terminal tail region (blue). Nuclear localization signals are indicated in green. (B) The construct used for knocking out kif12 contained a blasticidin gene as a marker in the center, flanked by two homologous stretches from the kif12 gene (≈500 bp each).

Fig. 7. Identification of the kif12 gene from the Dictyostelium genome. Left lane shows a 4.5-kb fragment of kif12 obtained by using RT-PCR from a total RNA preparation of Dictyostelium. Right lane is a molecular ladder.

Fig. 8. Cell volumes displayed for D kif12 cells (filled circles) compared with wild-type (wt) cells (open circles) grown on surfaces. Each point was determined by the average of four readings, each time counting at least 5 × 10⁴ cells with a model Z1 particle counter (Beckman Coulter).

Movie 1. Time series of phase-contrast images of a D kif12 cell attempting cytokinesis.

Movie 2. Time series of fluorescence images of a D kif12 cell rescued with GFP-Kif12 undergoing cytokinesis.

Movie 3. Time series of fluorescence images of a binucleate D kif12 cell rescued with GFP-Kif12 undergoing cytokinesis. Shown is a binucleate cell at upper left (even wild-type cells have a small fraction of binucleate cells in the population) that moves down to lower center and turns so that the two nuclei are on top of each other. As soon as the cell rounds up for the beginning of mitosis, the Kif12 exits the nuclei and the cytoplasm becomes obviously brighter with GFP-Kif12 fluorescence. Then the Kif12 appears along the spindle, and, finally, shows some concentration in the midbody. The cell divides into four cells.

Movie 4. Time series of fluorescence images of a wild-type (AX2) cell expressing GFP-myosin II undergoing cytokinesis.

Movie 5. Time series of fluorescence images of a D kif12 cell expressing GFP-myosin II undergoing attempted cytokinesis. The fluorescence intensity of GFP-myosin in the cleavage furrow is severely impaired. We examined 30 cells, and most showed no apparent increase in GFP-myosin fluorescence intensity in the furrow region at any stage of mitosis or cytokinesis. In the cell shown here, a few frames show a small amount of fluorescence increase that is transiently observed in the cortex near the unsuccessful furrow. However, it is seen only in certain frames, and the spatial and temporal intensity is significantly diminished compared with that consistently observed in movies of wild-type cells (Movie 2).