Figure 2.

The Initial Incorporation of Asl-GFP into Newly Assembled Centrioles Can Be Inhibited with Anti-DSas-4 or Anti-Asl Antibodies

(A and B) Fluorescence images show two regions (“control” and “DSas-4-blocked”) of an embryo expressing Asl-GFP (green) that has been injected with Texas-red-labeled anti-DSas-4 antibodies (red) at the start of mitosis (columns I and II) and several minutes later after the centrioles have separated at the start of S phase (columns III and IV). The control region (columns I and III) is far from the injection site (see Figures S3B and S3C), so no antibodies are detectable; the DSas-4-blocked region (columns II and IV) is close to the injection site, and the antibodies bind to the centrioles. The schematic at the top of each panel illustrates how the DSas-4 antibodies bind to centrioles in mitosis (II) and block the subsequent incorporation of Asl-GFP into the new centriole at the start of S phase, but they do not interfere with Asl-GFP localization at the old centriole (IV).

(C) The graph quantifies Asl-GFP levels in new centrioles in early S phase in the control region and in the DSas-4-blocked region (n = 16 centrioles from three injected embryos), as shown schematically above the graph. Error bars indicate the SEM.

(D and E) Fluorescence images show two regions of an embryo expressing DSas-4-GFP (green) injected with Texas-red-labeled anti-Asl antibodies (red), presented as in (A) and (B) above. The schematics illustrate how the anti-Asl antibodies do not perturb the localization of DSas-4-GFP that is already incorporated into the centrioles at the time of antibody injection but bind the endogenous (nonfluorescent) Asl molecules (gray) in the cytoplasm and so block their incorporation into the new centriole.

(F) The graph quantifies DSas-4-GFP levels in centriole pairs in early S phase in either control or Asl-blocked regions (n = 40 centriole pairs from four embryos), as illustrated schematically above the graph. Note that we compare centriole pairs instead of individual centrioles in this experiment because we cannot distinguish old and young control centrioles based on DSas-4-GFP levels alone. Scale bars, 2 μm. Error bars indicate the SEM.

See also Figure S3 and the Supplemental Experimental Procedures.