Fig. 5

Impaired NE recruitment and Nesprin-2 interaction by BicD2 K775X mutation. a Subcellular distributions of BicD2 and dynein in WT and mutant BicD2-transfected cells. U2OS cells were transfected with constructs expressing WT or mutant (K775X, T703M, and R501P) HA-BicD2 proteins and synchronized at G0 phase by serum starvation for 24 h. BicD2 and dynein showed prominent NE distribution revealed by HA and DIC antibodies. BicD2 and dynein were more dispersed in the cytoplasm in cells expressing the K775X mutant, but not the T703M or R501P mutant. Cells were stained with DAPI (blue) to show the cell nuclei. Bar = 5 μm. b Subcellular distribution of BicD2 WT or K775X (red) in cultured cortical neurons. E17 neurons were isolated from mouse brains electroporated with BicD2 WT or K775X along with GFP (green) by in utero electroporation at E14. While BicD2 WT mainly localized on the NE, K775X was dispersed in the cytoplasm. Cells were stained with DAPI (blue) to show the cell nuclei. Bar = 5 μm. c Western blots of dynein and BicD2 in the nuclear and cytoplasmic fractions from cells expressing BicD2 WT or K775X. K775X mutation leads to decreases in the amount of dynein (judged by both DHC and DIC antibodies) and BicD2 (judged by HA antibody) in the nuclear fraction and increases in the cytosolic fraction. Lamin A/C and α-tubulin were used as the nuclear and cytosolic marker, respectively. d Quantification of DHC, DIC and BicD2 in nuclear and cytosolic fractions (n > 3 replicated experiments in each groups). Error bars represent SEM. **P < 0.01, ****P < 0.0001. Student’s t test. e GST pull-down assay to determine the interaction between BicD2 and Nesprin-2. Cell lysate expressing BicD2 WT or K775X were incubated with glutathione-agarose beads preloaded with GST or GST-tagged Nesprin-2 C-terminus (left panel). 2% of the input (right panel) was the positive control. Western blot using HA antibody showed that BicD2 WT but not K775X was pulled down by Nesprin-2 C-terminus. f These results suggested a dynein-BicD2-Nesprin-2 link to the NE