Figure 2.

Microtubules Change from Centrosomal to Acentrosomal Nucleation/Anchoring during Early Invagination

(A–C) Surface projections show that at early stage 11, the ends of many apical MTs colocalize with the centrosomal protein asterless (A), but through midstage 11 this changes (B) so that by late stage 11, centrosomes labeled by asterless less frequently colocalize with MT foci (C). Asterless, green; tyrosinated α-tubulin, red; srcGFP, blue. Arrows point to centrosomes and the matching positions in the MT channel.

(D) Quantification of colocalization of MT bundle ends and centrosomes at early and late stage 11 (350 MT bundles from six different placodes for each stage; shown are mean ± SEM, p < 0.0001 using Student’s t test; see Table S1).

(E and F) Section views of late stage 11 placodes: Nod-LacZ, a marker of MT (−) ends (E, green), accumulates apically in a flat region of a placode, indicating that MT (−) ends are located apically, whereas an MT (+) end marker, Kin-LacZ, is found basally (F, green). Tyrosinated α-tubulin, red. The white dotted lines mark placodal cells.

(G and H) In surface projections of placodal cells, γ-tubulin becomes less tightly centrosome associated from early stage 11 to late stage 11. At early stage 11, the brightest γ-tubulin foci (green) colocalize with centrosomes (red) labeled by asterless (G). At late stage 11, in addition to centrosome foci, further noncentrosomal densities of γ-tubulin labeling have appeared within the placode (H, green arrows), not colocalizing with centrosomes marked by asterless. Panels are higher magnifications of boxes in Figures S1E and S1G.

(I) When asterless labeling (green) at late stage 11 is analyzed at higher laser power, it shows, in addition to strong labeling of centrosomes, many fainter acentrosomal foci that colocalize with apical MT foci (arrows). Acetylated α-tubulin, red; srcGFP, blue.

(J) Quantification of the mean noncentrosomal γ-tubulin fluorescence inside versus outside the placode (six placodes were analyzed for each stage; shown are mean ± SEM, p = 0.0069 using Student’s t test; see Table S1).

See also Figure S1.