Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry (original) (raw)
Figure 3
BICD2 to localizes to the nuclear pores at the NE.
(A) HeLa cells were fixed with cold methanol and stained with antibodies against BICD2 and nucleoporins (MAB414). These panels show focal planes in the middle part of the nuclei. (B) Enlargements of the flattened NE surface from the same cells as in (A) (the area from which the enlarged image is taken is indicated in (A) by a white rectangle). The insets show further enlargements, in which individual NPC can be distinguished. Colors used for the overlays are indicated above the corresponding images. Note that most NPC dots display signal in both channels, indicating that BICD2 and MAB414 antigens (NPCs) co-localize. (C) Representative fluorescence intensity profile of BICD2 (green) and MAB414 staining (red) at the NE surface. The profile was obtained using Linescan function of MetaMorph at the position indicated by a blue line in (B). Vertical axes are in arbitrary units. Note that most peaks are present in both BICD2 and MAB414 channels, although their intensities often differ, in agreement with the fact that BICD2 is not a nucleoporin. (D) Correlation between BICD2 and MAB414 signals in Figure 3B. The intensity of each pixel in the green and red channel is represented by a dot. (E,F) The same images and analysis as in (B) and (D), but with the MAB414 panel rotated by 180°. Note that there is no co-localization between the green and red signals and no correlation is visible in the plot. (G) Average coefficient of linear correlation between BICD2 and MAB414 signals, determined from plots such as in (B) and (D) obtained for 19 cells. Note that the coefficient is much higher for properly aligned images compared to rotated ones, indicating that co-localization is not spurious. Error bars represent SD.