Figure 3.

The Sas-4-Thr200-STP Motif Helps Recruit Both Polo and Asl to New Centrioles

(A and B) Asl-GFP (A, green) and Polo-GFP (B, green) localization in embryos injected with high levels of anti-Sas-4-pThr200 antibodies (red). Embryos are shown during mitosis and the following S phase; two regions from the same embryo are shown in which there is either a low concentration (unbound, left panels) or high concentration (bound, right panels) of the antibodies. Colored arrowheads indicate the same centrosomes in mitosis and then after centrosome separation; arrows highlight examples where binding of the antibody has perturbed the recruitment of Asl-GFP (2/2 embryos) or Polo-GFP (2/3 embryos) to new centrioles. Scale bars, 5 μm.

(C) Polo-GFP (green) localization at newly separated centrioles in embryos expressing WT Sas-4-mKate2 (mKate2 was used as a red fluorescent tag due to its relatively short fluorescence maturation time compared to other red fluorescent proteins; see Supplemental Experimental Procedures) or Sas-4-mKate2-Thr200-STP-motif mutant forms (red; as indicated). Note that Polo-GFP localizes symmetrically at newly separated centriole pairs in embryos expressing WT Sas-4-mKate (arrowheads), however, Polo-GFP localization is strongly disrupted or absent from new centrioles in embryos expressing T200A, P201G, or S199G mutant forms of Sas-4-mKate2. Arrows highlight examples where the mutant fusion protein has perturbed the recruitment of Polo-GFP to new centrioles, while arrowheads indicate the unperturbed older centrioles in these embryos. Scale bars, 5 μm (left panel) and 2 μm (right panel).

See also Figure S3.