Requirement for MTs in establishing and maintaining BB alignment. (A and B) Effects of nocodazole on the BB patterns in GFP-centrin2 MTECs. (A) Comparison of three different conditions of nocodazole treatment. Time in hours:minutes. (B) Measurement of Ia from cells shown in A. No treatment (black circles), short-term treatment (green triangles) followed by washout (black triangles), and long-term treatment (green squares). A, alignment; P, partial alignment; S, scatter; F, floret. (C and D) Different responses to nocodazole (Noc) treatment in early- versus late-stage cells. (C) More prominent clustering (arrows) appeared in the early-stage cells (top row) in the presence of nocodazole compared with late-stage cells (bottom row). See Video 8. Time stamps shown in hours:minutes. (D) Temporal changes in Ia in early- and late-stage cells. Black lines represent late-stage cells (those belong to scatter/partial alignment pattern), and red lines show early-stage cells (those belong to floret pattern). Green region indicates the period of nocodazole treatment. (E) Apical MTs in MTECs at ALI 6 d treated with 6.6 µM nocodazole for 2 h showed a lower intensity of α-tubulin staining compared with nontreated MTECs. (F) Immunostained images with an anti–keratin 8 mAb after nocodazole treatment revealed prominent keratin bundling. (G) Nocodazole treatment altered the normal IF network visualized by EM. Coincident with the depolymerization of MTs (white arrows), IFs (white arrowheads) formed thick bundles surrounding individual BB clusters. (H) The impaired BB pattern induced by nocodazole treatment was a floret-like pattern with random BB directions. Insets are magnified regions. Bars: (A, C, E, F, and H) 10 µm; (G) 1 µm.