Figure 6. HsSAS-6 Levels Must Be Limited to Restrict Procentriole Formation.

(A and B) U2OS cells transfected with HsSAS-6 or HsSAS-6-ΔKEN, fixed after 72 hr and stained with antibodies against centrin (green) and HsSAS-6 (red). Representative images of cells without (A) or with (B) excess centrin foci are shown.

(C) Cells transfected with GFP, HsSAS-6, or HsSAS-6-ΔKEN fixed after 72 hr and stained with antibodies against centrin and GFP or centrin and HsSAS-6. The number of centrin foci in GFP-positive cells (for GFP) or in cells with elevated HsSAS-6 levels (for HsSAS-6 and HsSAS-6-ΔKEN) was quantified (n ≥ 100 for each of three independent experiments). Error bars, 95% confidence intervals.

(D) HeLa cells were cotransfected with GFP and HsSAS-6 or GFP and HsSAS-6-ΔKEN, and HsSAS-6 levels were analyzed after 48 hr by western blot. GFP was used as a transfection and loading control. The images are from one membrane and a single exposure. Note that there is significantly more HsSAS-6 accumulating in cells expressing HsSAS-6-ΔKEN, as compared with HsSAS-6.

(E and F) U2OS cells transfected with HsSAS-6-ΔKEN, fixed after 24 hr and stained with antibodies against centrin (green, shown in E and F) and HsSAS-6 (red, shown in E) or Odf2 (red, shown in F). Arrows point to a centriole that seeds formation of three (E) or two (F) centrin foci. Scale bar, 500 nm.

(G) U2OS cells expressing centrin-GFP were transfected with HsSAS-6-ΔKEN. Seventy-two hours later, cells were screened using fluorescence microscopy (top panel; arrow points to the area shown on the EM images below; note also the other centrin-GFP focus), fixed, and analyzed by serial section EM (1–4: selected sections spanning the entire centriole and associated procentrioles; scale bar, 500 nm). Arrows point to the centriole; arrowheads, to procentrioles, three of which are present in this particular case.