The Io values taken from all of the samples exhibit a bimodal distribution, in which a group of cells with low (0.7) Io values within 2 d. During the progressive refinement of the BB orientation over time, the Io exhibited a rapid increase around air–liquid interface (ALI) days 6–7.
S2 B and Materials and methods for details).
3, B and C), and then estimated the degree of uniformity of BB orientation by introducing the index of orientation ( Io) (see Fig. S2 A), we determined the orientation of each BB ( Fig. By immunostaining Odf2 ( Nakagawa et al., 2001) and centriolin ( Gromley et al., 2003), which mark the positions of BBs and BF, respectively ( Fig. We examined the development of tracheal BB orientation in the course of cell maturation and its correlation with BB alignment.
#Fastcam mc2 1 skin#
The establishment of BB alignment is correlated with the refinement of BB orientationĪlthough earlier studies ( Guirao et al., 2010 Werner et al., 2011) showed that the interplay between BB position and orientation underlies proper ciliary beating in Xenopus embryonic skin and mouse brain, little is known about this process in tracheal MCCs. Collectively, our live imaging system of MCCs with quantitative analyses revealed that the BB array adopted four stereotypical patterns, from a clustering floret pattern to a linear alignment during MCC differentiation. We also observed occasional intermittent bursts of BB velocity caused by the large cellular deformation caused by the cell’s reconnection with a neighboring cell ( Fig. After BB alignment was established, only minor changes occurred. We found that the BBs moved most rapidly when they transitioned from the floret to the scatter pattern and then gradually slowed as they approached the alignment configuration ( Fig. 2 B), with a nearly constant number of BBs ( Fig. Over the course of MCC culture, the Ia value revealed a long-term ascending trend ( Fig. BBs in the floret pattern moved in and out of the area of the originally assigned floret cluster, and as a result, sets of BBs that later formed a line did not originate from a single floret ( Fig. We next tracked the spatiotemporal dynamics of individual BBs by imaging GFP-centrin2. This model showed the ability of cytoskeletal systems to self-organize BBs into specific patterns. Finally, we constructed a mathematical model for BB alignment based on an active hydrodynamic theory. We found that the development of the BB alignment pattern is highly dynamic and is achieved through a characteristic process, which we classified into four stereotypical patterns of BB array: “floret,” “scatter,” “partial alignment,” and “alignment.” Perturbing apical MTs caused the linear BB alignment pattern to revert to a floret-like pattern, and a genetic loss of BF that decreased MT density led to a failure to achieve BB alignment. To follow BB pattern formation during MCC maturation, we developed a novel long-term and high-resolution live-imaging system in which cultured tracheal MCCs could be maintained as constantly moving specimens with beating cilia. This striking pattern prompted us to address the mechanism by which these BBs become regularly aligned beneath the apical membrane. In the present study, we examined mouse tracheal MCCs in which the BBs are linearly aligned, a pattern also found in oviduct MCCs. We constructed a theoretical model, which indicated that the apical cytoskeleton, acting like a viscoelastic fluid, provides a self-organizing mechanism in tracheal MCCs to align BBs linearly for mucociliary transport. The BB alignment was disrupted by disturbing apical microtubules with nocodazole and by a BF-depleting Odf2 mutation. During MCC differentiation, the BB array adopted four stereotypical patterns, from a clustering “floret” pattern to the linear “alignment.” This alignment process was correlated with BB orientations, revealed by double immunostaining for BBs and their asymmetrically associated basal feet (BF).
To study this mechanism, we developed a long-term and high-resolution live-imaging system and used it to observe green fluorescent protein–centrin2–labeled BBs in cultured mouse tracheal MCCs. The mechanism for BB alignment is unexplored. Airway MCCs have large numbers of BBs, which are uniformly oriented and, as we show here, align linearly. Multiciliated cells (MCCs) promote fluid flow through coordinated ciliary beating, which requires properly organized basal bodies (BBs).
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