Dgt is not involved in chromatin-mediated MT nucleation but is needed for chromosome alignment and K-fiber formation. (A) MT regrowth assay after control Cnn (top) or double Cnn/Dgt5 (bottom) RNAi. MTs (green) initially appeared around chromatin (blue) in both samples, whereas γ-tubulin (red) failed to localize to the MTs in the absence of Dgt5. The double RNAi cells failed to form robust bipolar spindles. (B) Delayed chromosome alignment and anaphase onset in the bipolar spindle in the absence of Dgt6 (mCherry-tubulin [red] and Mis12-GFP [green]). See also Video 5 . Mean duration (and SEM) from NEBD to metaphase and from metaphase to anaphase onset is plotted (control; RNAi against pBluescript sequences). (C) kMTs visualized after 10 min of colcemid treatment. Some kinetochores (Mis12-mCherry) do not associate with K-fibers (GFP-tubulin) in the absence of Cnn/Dgt5. See text for quantitation. Boxes on the left indicate the enlarged areas shown on the right. Bars, 5 μm.