Figure 5.

Stable Plk4 promotes excess daughter centriole formation, and slimb RNAi eliminates the S-phase centriole reduplication block by accumulating Plk4 on centrioles. (A) Slimb overlaps Plk4-SBM–EGFP localization on centrioles. Immunostaining of Slimb (red) and D-PLP centrioles (blue) in a transiently expressing coexpressing Nlp-EGFP (green nuclei) and SAS-6p Plk4-SBM–EGFP (green) interphase S2 cell. A representative centriole (arrowhead) is shown at a higher magnification (inset). (B) Anti-GFP immunoblots of lysates prepared from transiently expressing inducible Plk4-EGFP that were RNAi treated for the indicated proteins for 7 d. α-Tubulin was used as a loading control. Molecular mass is indicated in kilodaltons. (C and D) Transient coexpression of Nlp-EGFP (green nuclei) and Plk4-SBM–EGFP in day 5 cycling S2 cells. Plk4-SBM–EGFP labels one or more spots (green) on D-PLP–stained centrioles (red). Insets show select centrioles at a higher magnification. The cell in D shows an extreme example of centriole overduplication. White tracing marks cell borders. (E and F) Transmission electron micrographs of interphase cells expressing Plk4-SBM–EGFP for 5 d. Red arrows denote excess daughter centrioles emanating from mother centrioles shown in the cross section. The cell in E shows a normal mother–daughter centriole pair (orange arrow) adjacent to a mother with two daughters. Illustrations of these centrioles are shown in E′ and F′. (G) Transient expression of Plk4-EGFP/Nlp-EGFP (green) in day 3 RNAi-treated cells arrested in S phase for 2 d. D-PLP–labeled centrioles (red, arrowheads). Insets show centrioles at a higher magnification. (H) Stable expression of Plk4-EGFP (green) in a 24-h S phase–arrested cell treated with MG132 proteasome inhibitor. Insets show select D-PLP–labeled centrioles (red) at a higher magnification. Bars: (A–D) 5 µm; (E and F) 0.2 µm; (G and H) 2.5 µm.