Fig. 9.

Nuclear polarization in migrating torsinA^+/+ and torsinA^−/−MEFs. To evaluate nuclear polarization, confluent monolayers of MEFs were serum-starved for 24 hours, then a wound was made through the monolayer and cells were stimulated with 2 μM lysophosphatidic acid (LPA; Sigma-Aldrich) (Gomes and Gundersen, 2006). Three hours later, the cells were fixed and immunostained for pericentrin to label the centrosome (green) and for β-tubulin to label microtubules (red), with DAPI staining (blue) for nuclei. The nucleus was scored as polarized (+) when the centrosome was localized between the nucleus and the leading wound edge and as non-polarized (-) in any other location. Representative images are shown for torsinA^+/+ (left) and torsinA^−/− (right) cells along the wound edge. One hundred cells of each type were evaluated for orientation of the centrosome between the nucleus and the leading edge in each of three experiments using two different MEF preparations of each type. TorsinA^+/+ cells showed 85±5% nuclei in the polarized location, whereas torsinA^−/− cells showed only 38±7% in the polarized position at this time point (values are mean percentage ± s.e.m., n=3 experiments, P<0.001). Magnification: 20×.