Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry (original) (raw)
Figure 7
Dynein, kinesin-1, and BICD2 control the localization of AL in G2 phase.
(A) HeLa cells were transfected with control, DHC#1, p150Glued, or KIF5B#1 siRNAs, fixed with paraformaldehyde 3 d later, and stained for endogenous BICD2, RanBP2, and cyclin B1. In the overlays, BICD2 is shown in green and RanBP2 in red. The outline of the cyclin B1-positive cell is indicated. Note AL displacement to the cell periphery in cyclin B1-positive cells depleted of dynein or dynactin, and the centripetal relocalization of AL in kinesin-1 (KIF5B) depleted cyclin B1-stained cell. (B) GFP-RanGAP1 stable HeLa cell line was imaged with a 2 or 3 min time interval 2 d after transfection with the control, p150Glued, or KIF5B#1 siRNAs. 0 min indicates the first frame after NEB (defined as the time when GFP-RanGAP1 enters the nucleus). Contrast is inverted. Arrows show the accumulation of AL at the cell periphery or the cell center. Peripheral displacement of AL after dynein or dynactin knockdown occurred at 1 h±30 min before mitotic onset (mean ± SD, measured in 6 and 10 cells, respectively); strong accumulation of AL near the nucleus in KIF5B depleted cells started at 3 h±30 min before NEB (mean ± SD, measured in 18 cells). (C) AL localization after different siRNA co-transfections. Black bars represent the percentage of HeLa cells showing a strong AL accumulation near the nucleus (like in the bottom panel in Figure 7A) and gray bars illustrate the percentage of HeLa cells showing displacement of AL to the outmost cell periphery (like in the middle panels of Figure 7A). In each case, cells were transfected with a combination of two siRNAs: control, KIF5B or dynein HC siRNAs (as indicated on top of the panel) in combination with the control siRNA or the siRNAs against BICD1 and BICD2 (the cotransfected siRNA is indicated at the bottom), or the combination of KIF5B siRNAs and dynein HC siRNAs. Cells with AL accumulation in the cell center or the cell periphery were scored in KIF5B or dynein knockdown cells, respectively; in a double KIF5B/dynein HC knockdown, both types of cells were counted. Error bars represent SD. ∼30–100 cells were counted in three experiments. Cells with fully aggregated AL represent a minority of the population (∼20%) in control G2 cells but become predominant (∼75%) after depletion of kinesin-1. This effect can be suppressed by co-depletion of BICD2 or dynein HC, but not BICD1. Similarly, control cells never display peripheral AL localization, while ∼40% of dynein-depleted cyclin B1-positive cells show this phenotype, which can be suppressed by co-depletion of BICD2 or KIF5B, but not BICD1.