Microridge-like structures anchor motile cilia
Takayuki Yasunaga, Johannes Wiegel, Max D. Bergen, Martin Helmstädter, Daniel Epting, Andrea Paolini, Özgün Çiçek, Gerald Radziwill, Christina Engel, Thomas Brox, Olaf Ronneberger, Peter Walentek, Maximilian H. Ulbrich & Gerd Walz
Nat Commun. 2022 Apr 19;13(1):2056. doi: 10.1038/s41467-022-29741-3.
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Several tissues contain cells with multiple motile cilia that generate a fluid or particle flow to support development and organ functions; defective motility causes human disease. Developmental cues orient motile cilia, but how cilia are locked into their final position to maintain a directional flow is not understood. Here we find that the actin cytoskeleton is highly dynamic during early development of multiciliated cells (MCCs). While apical actin bundles become increasingly more static, subapical actin filaments are nucleated from the distal tip of ciliary rootlets. Anchorage of these subapical actin filaments requires the presence of microridge-like structures formed during MCC development, and the activity of Nonmuscle Myosin II. Optogenetic manipulation of Ezrin, a core component of the microridge actin-anchoring complex, or inhibition of Myosin Light Chain Kinase interfere with rootlet anchorage and orientation. These observations identify microridge-like structures as an essential component of basal body rootlet anchoring in MCCs.
Fig. 1: Development of the subapical actin in multiciliated cells (MCCs).
a While the apical actin cytoskeleton reaches its final density by stage 25, the subapical actin continues to develop until stage 29 (scale bar, 2 µm). b Basal body (ciliary) rootlets, labeled with GFP-Clamp (green), are orthogonally aligned to the plasma membrane at stage 21-22. The right panels represent magnified insets marked by white squares. The dashed line depicts the apical plasma membrane, the solid line separates the apical and subapical actin layer (scale bar, 2 µm). c Actin filaments, labeled with phalloidin (red, white arrow heads) start originating from ciliary rootlets (GFP-Clamp) at stage 22–25. The right panels represent magnified insets marked by white squares (scale bar, 2 µm). d After nucleation of actin filaments (phalloidin, red), rootlets are tilted towards the apical plasma membrane at stage 26-27. The right panels represent magnified insets marked by white squares. The white arrowheads indicate subapical actin filaments, projecting toward the apical plasma membrane. The dashed white line depicts the apical plasma membrane, the solid line separates the apical and subapical actin layer (scale bar, 5 µm). e While rootlets align orthogonally to the plasma membrane (dashed line) at stage 21-22, subapical actin filaments, extending from the tip of the rootlet, connect the rootlets with the plasma membrane at stage 29 (scale bar, 2 µm). f Actin nucleation at the tip of the anterior rootlet coincides with rootlet polarization and orientation along the longitudinal body axis (white arrows). Subapical actin filaments (phalloidin, red) are nucleated from the tip of the rootlet (white arrowheads) (right upper insert; Clamp-GFP, green). The pseudo-colored actin heat map depicts phalloidin signals (white arrowheads), originating at the distal end of rootlets (magenta) (right lower insert) (scale bar, 1 µm). g Rootlets are laterally elongated at stage 29 as compared to stage 20. Representative data of two independent experiments, depicting the results of 12 cells at stage 20, and 5 cells at stage 29. Each circle represents the average length of rootlets in a multiciliated cell (mean ± SD; Mann–Whitney-U test). Source data are provided as a Source Data file. h The diagram explains the apparent elongation of apically imaged (x-y) rootlets after nucleation of subapical actin.
Fig. 2: Basal body rootlet tilting between stage 20-21 and stage 30.
A Serial sections of multiciliated cells were performed from 7 different cells (one embryo) fixed at stage 20-21 and from 5 different cells (one embryo) fixed at stage 30. Ten cilia were analyzed at each stage by transmission electron microscopy (scale bars, 500 nm). B 3D reconstructions were performed, using Reconstruct (Movie 1). C The minimal angle between rootlets and basal bodies (upper panel), and the minimal distance from the tip of the basal body rootlet to the plasma membrane (lower panel) significantly decreased between stage 20-21 and stage 30 (see Movie 2 for the applied workflow) (mean ± SD; Mann–Whitney-U test). Source data are provided as a Source Data file.
Fig. 8: Microridge-like structures anchor basal body rootlets.
a Multiciliated cells of the Xenopus epidermis form microridge-like structures, requiring Nonmuscle Myosin II (NMII). b Actin filaments, connecting the Fhod3-labeled tip of the anterior rootlet to Ezrin-containing microridges, anchor motile cilia into their final position. c Fhod3 localizes adjacent to FAK to the tip of the anterior rootlet. Ezrin interacts with Fhod3 and represents part of the actin-anchoring complex at the apical plasma membrane.
Adapted with permission from National Library of Medicine on behalf of Nature Communications: Yasunaga et al. (2022). Microridge-like structures anchor motile cilia. Nat Commun. 2022 Apr 19;13(1):2056. doi: 10.1038/s41467-022-29741-3.
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