Interfering with Nup133-anchored dynein/dynactin impairs the tethering of centrosomes to the NE. (A) Time-lapse imaging of HeLa cells expressing EB3-GFP (green) and H2B-mCherry (red) and transfected with CENP-F (c and c′), NudE/EL siRNA duplexes (d and d′), or with a CFP-p50/dynamitin construct (e), or of cells stably expressing GFP-hNup133_CTD treated with hNup133 siRNAs and subsequently transfected with plasmids encoding EB3-GFP and H2B-mCherry (b). Time (in min:sec) was set at 0:00 when centrosome splitting just became detectable. Bars, 10 µm. See also Videos 1–6. (B) Tracks representing centrosome (CTR) movements in a control and a CENP-F–depleted cell (see Aa, Ac′, and Videos 1 and 4). Trajectories before centrosome separation (red) and tracks of the separated centrosomes (blue and green) were superimposed on a schematic representation of the cell border and nuclear position (gray shading) at the beginning of the video. White and black dots indicate the positions of centrosomes at the beginning and at the end of the videos, respectively. (C) Analysis of the centrosome–NE distance over time in HeLa cells expressing EB3-GFP and H2B-mCherry and treated with scramble or CENP-F siRNA duplexes. For each cell entering mitosis, the maximum distance between the centrosomes and the NE reached during the G2/M transition was plotted over the time centrosomes spent >3 µm away from the NE. Each dot represents a single cell. The number of cell quantified is indicated.