Dgt2–6 proteins are important for MT generation within the spindle and bipolar spindle formation. (A) MT density specifically inside the spindle decreased in the absence of Dgt2–6 or Dgrip71. Signal intensities at the centrosome and within the spindle (red boxes) were measured. Ratio of spindle and centrosome signal intensity after Dgt2–6 and Dgrip71 knockdowns (0.43–0.55; n = 5 each) were significantly (P < 0.0001) lower than in control (1.81 ± 0.15 SEM; n = 10). (B) A high-resolution still image of MT (green) and kinetochore marker CENP-ACid (red) after double Cnn/Dgt5 RNAi. Spindle morphology and chromosome alignment were severely impaired. (C–F) FRAP analysis of GFP-tubulin for control (n = 13), Dgt5 (n = 14), and Cnn (n = 17) RNAi spindles. Cells were arrested in metaphase by Cdc16 RNAi, entire half spindles (excluding the centrosome) were photobleached (time 0), and relative GFP intensities were plotted (with SEM) for the half spindles (D, yellow), spindle equators (E, yellow), and pole regions (F, yellow). Only partial fluorescence recovery, mostly from the kinetochores (C, arrows), was observed in the absence of Dgt5, whereas recovery was seen in the whole bleached area in control or Cnn spindles (t1/2 = 30 s). The initial steep recovery (0–5 s) is likely because of diffusion of GFP-tubulin in the cytoplasm because it was also detected for the cells in which MTs were depolymerized by colcemid (not depicted). See also Video 4 . Bars, 5 μm.
