Fig. 6. APC localizes to the mother centriole. (A) APC co-localizes with DsRed-EB1 to the mother centriole. Sections of MDCK cells expressing DsRed-EB1 (red in a-n) showing the centrosome regions immunostained for centrosome markers pericentrin (blue in a-d), -tubulin (blue in e-h) or centrin (blue in j-n) and co-stained for the mother centriole marker p150^Glued/dynactin (green in b-d) or APC (green in f-h,k-n). DsRed-EB1 co-localizes with p150^Glued/dynactin at only one of the two centrosomes stained by pericentrin (a-d). DsRed-EB1 and APC preferentially localize to the same of two centrosomes marked by -tubulin (e-h) and to the same two centrioles of four centrioles marked by centrin (j-n). APC precedes EB1 at the second centrosome (e-h). Only two out of 25 analysed U-2 OS and MDCK cells in asynchronous cultures had no APC at the second centrosome, whereas seven out of 28 analysed cells had no EB1 at the second centrosome, indicating that APC localizes earlier to the second mother centriole than EB1. Bar, 1 µm. (B) U-2 OS centrosome region expressing GFP-centrin as a marker for centrioles (blue) and co-immunostained for APC (green) and for the mother centriole with p150^Glued/dynactin (red). Sections of this region have been deconvolved and recombined in a 3D rendering. The first column shows four centrioles in multiple angles of counterclockwise (ccw) rotation, with the two central centrioles too close to each other to be resolved as separate spots. The second and third columns show that APC tightly surrounds the mother centriole as marked by p150^Glued/dynactin and extends to a second centriole. (B') Schematic representation of 3D localization of proteins shown in B at a 205° angle. See Movie 4 (http://jcs.biologists.org/supplemental) for a 360° rotation and Materials and Methods for the resolution limits of the optical system. (C) U-2 OS centrosome region expressing GFP-centrin as a marker for centrioles (blue) and co-immunostained for EB1 (green) and for the mother centriole with p150^Glued/dynactin (red). Sections of this region have been deconvolved and recombined in a 3D rendering. The first column shows two centrioles in multiple angles of ccw rotation. The second and third columns show that EB1 and p150^Glued/dynactin cap one end of the mother centriole and continue in a MT-like filamentous extension from this end of the centriole. (C') Schematic representation of 3D localization of proteins shown in C at a 0° angle. See Movie 5 (http://jcs.biologists.org/supplemental) for a 360° rotation. (D) APC-EB1 interaction is not essential for localization of APC and EB1 to centrosomes. SW480 centrosome regions were co-immunostained for the centrosomal markers -tubulin (green in a,c) or pericentrin (green in d,f,h,j) and APC (red in b,c), EB1 (red in e,f) or p150^Glued (red in i,j). N-APC, EB1 and p150^Glued localize to centrosomes in SW480 cells that express a truncated form of APC without the EB1 binding site.