Figure 1.
Cnn Initially Incorporates into the Center of the PCM and Then Spreads Outward
(A) Confocal images show the behavior of GFP-Cnn before and after photobleaching (t = 0 s) at centrosomes in embryos with intact MTs.
(B–E) Confocal images show the behavior of Dendra2-Cnn (pseudo-colored red; B and D) and photoconverted Dendra2-Cnn (pseudo-colored green; B–E) at centrosomes in embryos. Dendra2-Cnn was converted at the center of the PCM at t = 0 and its distribution followed over time in the presence (B and C) or absence (D and E) of MTs. Red arrowheads (B and C; t = 1:30) indicate flares of Dendra2-Cnn at the periphery of the PCM that do not contain any photoconverted protein; white arrowheads (B and C; t = 8:30 and 10:30) indicate flares of Dendra2-Cnn at the periphery of the PCM that now contain photoconverted molecules that were originally generated in the middle of the PCM. The centrosome in (B) and (C) duplicated during the movie and the second centrosome is indicated with an arrow as it moves away (t = 8:30). Note how MT depolymerization (D and E) largely blocks the outward movement of photoconverted Dendra2-Cnn, although a dark hollow develops in the center of the PCM (arrow, E; t = 19:30), indicating that some outward movement has occurred.
(F) Confocal images from a FRAP experiment reveal that MT depolymerization does not block the incorporation of GFP-Cnn into the center of the PCM (compare F to A).
(G) Graph displays the total levels of unbleached centrosomal GFP-Cnn during M-phase in Drosophila embryos where MTs have been depolymerized (red line). The dotted black line indicates the maximal levels of centrosomal GFP-Cnn during a normal mitosis (that normally only lasts 3–4 min and during which time GFP-Cnn levels remain constant; Conduit et al., 2010); MT depolymerization leads to M-phase arrest, allowing measurements to be taken for a longer period, and Cnn continues to steadily accumulate at centrosomes over this time.
(H) A super-resolution 3D-SIM image of centrosomes in a living cnn null mutant embryo injected with mRNA encoding GFP-Cnn. Red arrows indicate hollows that likely contain a centriole. Error bars = SE.