Microridge-like structures anchor motile cilia

Abstract

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. 3. Localization of Fhod3 in multiciliated cells (MCCs).

a Delphilin, labeled with GFP, co-localizes with BFP-labeled Centrin to the basal bodies of MCCs. Actin filaments were visualized by co-expression of RFP-Utrophin. b Fhod1 (GFP-Fhod1) assumes a position in between surrounding actin filaments (phalloidin, red) and basal bodies (BFP-Centrin). c GFP-Fhod3 localizes to the tip of the RFP-Clamp-labeled anterior rootlet. The lower panel shows magnified Clamp- and Fhod3-labeled images. d Fhod3 associates with anterior and posterior rootlets. The posterior rootlets (arrowheads) are oriented orthogonally to the plasma membrane. Depicted are diagonal views after 3D reconstruction of confocal images (IMARIS). Images were obtained at stage 29-30; scale bars, 5 µm).

Fig. 4. Fhod3 localizes to the tip of the anterior rootlet.

a Fhod3 (RFP, red) localizes to the distal end of the anterior rootlets (labeled with BFP-Clamp, blue) in between phalloidin (white)-labeled actin filaments and GFP-labeled FAK (scale bar, 5 µm). Magnified images depict the pair-wise comparison of fluorescent proteins. b The x-z projection reveals the presence of Fhod3 at the distal tip of the anterior basal body rootlet within the subapical actin layer. The left upper image depicts the maximal intensity projection of the confocal section used for 3D reconstruction (scale bar, 1 µm). The right upper picture represents a magnification of the phalloidin (white)-labeled image, depicting the border (dashed line) between apical and subapical (arrowhead) actin layer. While Fhod3 (RFP, red) labeled the tip of the anterior rootlet (BFP-Clamp, blue), FAK (GFP, green) localized to the more proximal part of the rootlet (arrowhead). Magnified images depict the pair-wise comparison of fluorescent proteins. Images were obtained at stage 29-30.

Fig. 5. Microridge formation coincides with actin and Ezrin organization.

a Scanning electron microscopy (SEM) revealed that microridges form at stage 26, and continued to increase in complexity towards stage 40, when cilia start to retract (scale bars, 1 µm). b Ezrin, labeled with GFP, follows the organization of actin bundles that surround the basal bodies, labeled by BFP-Centrin in the merged image (scale bars, 5 µm). c Correlation between SEM and phalloidin (red)-label actin filaments revealed that the actin bundles observed in the apical actin layer follow the microridges detected by SEM (scale bars, 1 µm). d High-resolution confocal imaging reveals that actin bundles (phalloidin, red) arising from the anterior rootlet, connect to FERM-labeled microridges. Images were obtained at stage 29-30.

Fig. 6. Ezrin and Nonmuscle Myosin II are essential for microridge formation.

a While the actin cytoskeleton, labeled by phalloidin (red) co-localizes with microridges visualized by scanning electron microscopy (SEM), both actin cytoskeleton and microridge architecture are completely disrupted after knockdown of ezrin (scale bar ctl MO, 4.5 µm; scale bar ezrin MO, 9 µm). b Inhibition of Myosin Light Chain Kinase (MLCK) by ML7 disrupted microridge development (scale bars, 9 µm). c Twenty cells obtained from 2 DMSO- or ML7 (40 µM) treated Xenopus embryos were visualized by SEM and analyzed by U-net. Principal component analysis (PC scores) separated the DMSO- and ML7-treated MCCs based on seven different parameters (microridge length, MCC cell area, microridge area, microridge number, branches, thickness, and microridge to cell ratio). While the surface area of MCCs treated with ML7 was increased, the number of microridges, the number of branch points and the microridge thickness was significantly reduced, decreasing the ratio between cell surface area and the area occupied by microridges (mean; Mann–Whitney U test). Images were obtained at stage 29-30. Source data are provided as a Source Data file.

Fig. 7. Optogenetic manipulation of Ezrin function in multiciliated cells of the Xenopus epidermis.

a To test whether the Arabidopsis thaliana phytochrome system mediates protein interactions in MCCs in response to red light (660 nm), the phytochrome interaction factor (PIF) fused to GFP (GFP-PIF) was co-injected with phytochrome B (PHYB) fused to mCherry, localized to the plasma membrane by the C-terminal CAAX motif of Kras (PHYB-mCherry-CAAX). In response to red light, PHYB-mCherry-CAAX recruited GFP-PIF to the plasma membrane, while GFP-PIF remained in the cytoplasm after exposure to far-red light (740 nm) (scale bars, 10 µm). b The N-terminal FERM domain of Ezrin (SMART, http://smart.embl-heidelberg.de/) was fused to PIF, while the C-terminal part, containing the actin-binding domain, was fused to PHYB. While red light (660 nm) maintains the Ezrin fusion, far-red right light (740 nm) creates two Ezrin fragments, generating a dominant-negative effect. mRNAs were injected at the 4-cell stage and phycocyanobilin (PCB) (15 µM) was added at stage 6. To maintain the Ezrin in an assembled state, Xenopus embryos were exposed to red light until stage 18. At stage 20, embryos were split into two groups. One group was exposed to far-red light for 3 h to create Ezrin fragments and then returned to dark light, maintaining the dissociation. The other group was first kept in the dark, then returned to red light, keeping the Ezrin fragments assembled. c Far-red light (740 nm) disrupted rootlet tilting, revealed by shortening of the apparent rootlet length, and increased the rootlet circular standard deviation (CSD), demonstrating more randomly aligned rootlets. Depicted are the results of two independent experiments, using 5 embryos per condition. The symbols display the results for individual multiciliated cells (660 nm: 16 cells; 740 nm: 13 cells). Three cells, all derived from one embryo and depicted as black circles, were identified as outliers (ROUT, Q = 1%). The significance level was p = 0.0754 including the three outliers, and p < 0.0001 excluding the outliers (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.