Figure 4.

γ-Tubulin RNAi in Dividing Cells of Arabidopsis Roots Affect Cytokinesis and the Organization of Cell Files.

(A) Whole-mount α-tubulin (green) and γ-tubulin (red) immunolabeling and DAPI staining of DNA (blue) of control uninduced seedlings having regularly arranged cell files. γ-Tubulin in the root cells of the control exhibits bipolar localization from prophase (arrowheads) to telophase when it accumulates in the phragmoplast and the forming cell plate area.

(B) Whole-mount α-tubulin (green) and γ-tubulin (red) immunolabeling and DAPI staining of DNA (blue) of RNAi-expressing seedlings with mild phenotypes and reduced γ-tubulin levels 10 d after dexamethasone induction. Shown are a merged image (left panel), α-tubulin, γ-tubulin, DAPI staining, and DIC image (four small right panels). MTs are still present in mitotic cells, with spindles focused to acentrosomal poles, but cytokinesis became defective. The phragmoplasts are misaligned (arrows), and the organization of cell files is disrupted. The yellow line indicates the shape of a binuclear cell in the DIC image. Dividing cells are surrounded by enlarged cells with callose deposits.

(C) Collapsed spindle with MTs randomly arranged in the vicinity of chromosomes in a cell with severely depleted γ-tubulin 15 d after induction with dexamethasone.

(D) Misorientation of cell division planes in roots expressing ethanol-induced RNAi for 10 d.

(E) Phragmoplast with bundled and disorganized MTs in roots expressing dexamethasone-induced RNAi for 10 d.

(F) Early solid phragmoplast persists between separated nuclei with already decondensed chromatin, as revealed by DAPI staining. The expanded ring-like phragmoplast is present in this late stage of cytokinesis in the control cells (A).

(G) Cells are often binuclear in seedlings 20 d after induction, with severe depletion of γ-tubulin.

Bars = 10 μm.