Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry (original) (raw)
Figure 4
BICD2 is required for targeting dynein/dynactin to the NE and AL.
(A–D) Control HeLa cells or HeLa cells treated with 10 µM nocodazole for 1 h were fixed with paraformaldehyde (A–C) or cold methanol (D) and stained for the indicated endogenous proteins. Dynactin is visualized with an antibody to p150Glued and dynein with an antibody to dynein IC. Colors used for the overlays are indicated above the corresponding images. Note that BICD2 shows two types of localization: it colocalizes either with Rab6 or with RanBP2. In cells where BICD2 associates with RanBP2-positive NE and AL, dynein and dynactin are also targeted to these structures. (E) HeLa cells were transfected with a control siRNA (upper panels) or BICD2#1 siRNA (bottom panels). Three days later, cells were treated with 10 µM nocodazole for 5 h, fixed with cold methanol, and stained for endogenous RanBP2, phosphorylated histone H3, and dynein IC. NE staining by dynein antibodies is indicated by an arrow. Colors used for the overlays are indicated above the corresponding images. Note that dynein is enriched at the NE and annulate lamellae in control phospho-histone H3 positive cell, but not in BICD2-knockdown cell. (F) Percentage of HeLa cells positive for phosphorylated histone H3 that show strong accumulation of dynein IC at the RanBP2-positive NE and AL in control or BICD2-depleted cells 3 d after siRNA transfection. Only the cells with clearly visible AL were included in the quantification. Error bars represent SD; ∼25–30 cells were counted in two experiments.