Establishment of BB alignment during the MCC differentiation of MTECs revealed by long-term, high-resolution live imaging. (A) Linear alignment of BBs (double arrows) in GFP-centrin2 MTECs at air–liquid interface (ALI) 21 d. (B) Illustration of mature polarized ciliated cells with linearly aligned BBs, depicted as green circles at the base of each cilium. (C) Immunofluorescence (IF) image of the GFP-centrin2 signal in BBs (top left, green), and electron micrograph (EM) image showing BBs (top right, black rings). Arrows, 200 nm. Timeline of live imaging and ALI culture (bottom). (D) GFP-centrin2 transgenic MTECs during live imaging. (E) Live imaging of GFP-centrin2 MTECs (see Video 2), which exhibited four BB patterns during BB-alignment development. The degree of alignment analyzed using the index of alignment (Ia) is shown as numeric values (E, bottom). Bar shows the range of local Ias seen in the patterns. (F) Schematic drawing of representative live images showing each pattern. Bars, 5 µm.