Ectopic Root localizes to mother centrioles in testes and distributes asymmetrically to NB centrosomes. (A) Ectopic expression of Root in Kc167 cells forms rootlet-like fibers (arrows) that associate with the centrioles marked by γ-Tub (arrowheads) but not the MTs marked by α-Tub. (B) During spermatogenesis, GFP-Root associates with centrioles and has localization patterns that vary with cell type. In polar spermatocytes, GFP-Root forms fibrous structures both inside and between the centriole pairs. In mature spermatocytes, which have long, engaged centriole pairs, GFP-Root localizes at the base of the mother centrioles (arrow). 3D-structured illumination microscopy images distinguish the mother centriole from the daughter (the daughter grows from the side of the mother) and show that GFP-Root localizes at the entrance to the mother centriole lumen. In spermatids, GFP-Root localizes to the proximal end of the centriole (arrows). γ-Tub and Bld10 mark the centrioles. (C) GFP-Root localizes asymmetrically to NB centrosomes: higher at the mother than the daughter. The mother and daughter centrosomes are distinguished by higher level of the pericentriolar material protein Cnn at the daughter. Phospho-Histone H3 (pH3) marks the mitotic cells. (D) Representative images showing distributions of GFP-Root, Cnn, γ-Tub, Plp, and Bld10 in NB centrosomes. Cnn or γ-Tub that distributes significantly more to daughter centrosomes is used to distinguish the mother and daughter centrosomes. (E) Quantification of asymmetric distribution of proteins between the mother and the daughter centrosomes in NBs. Total signal intensity of the mother plus the daughter centrosome is 100%. The distribution of protein in the daughter or the mother centrosome was calculated as 100% × D/(D + M) or 100% × M/(D + M), where D was the signal intensity in the daughter centrosome and M was the intensity in the mother. Numbers of NBs measured are indicated in the bars. Bars: (A–C) 10 µm; (B and C, zoom) 500 nm; (D) 500 nm. See also Fig. S4.