Figure 1.

Motif 1 of Cnn is essential. (A) Schematic of Cnn protein showing the two conserved motifs at the amino and carboxyl termini. An expanded view of Motif 1 is shown with the 13 amino acids that are deleted in Cnn^Δ1 underlined in red. The alignments include the putative Cnn homologues in Aedes egypti (mosquito, A.e.), CDK5RAP2 from Gallus gallus (chicken, G.g.) and human (H.s.), Myomegalin/PDE4DIP from human, and Mto1p from S. pombe (S.p.). (B) Western blot showing the expression of GFP-Cnn^WT and GFP-Cnn^Δ1 in a cnn null mutant (cnn^hk21/cnn^25cn1) background (labeled “cnn-” above the lanes). The samples were loaded onto two gels and blotted, and one gel was probed with anti-Cnn, anti-γ-Tub, and anti-α-Tub (loading control) antibodies as indicated. The second blot, shown below the solid line, was probed with anti-GFP antibody. Immunostaining of cnn null embryos expressing GFP-Cnn^WT (C) and GFP-Cnn^Δ1 (D) shows localization to centrosomes and mutant phenotype. Anti-GFP signal is green, anti-α-Tub signal is red, and DNA is blue. The erratic spacing of mitotic figures, linked spindles, and aneuploid nuclei in D are all characteristic of cnn mutant embryos, showing that GFP-Cnn^Δ1 did not rescue. Note the abundance of centrosome pairs or clusters in the cnn^Δ1 mutant embryo.