Fig 2: nNOS is essential for cilia spacing pattern and rotational polarity.(A, B) Cby1/ODF2 pair demonstrates high degree of coordination of rotational polarity within single ciliated cells and between neighboring cells in the trachea and alignment with the direction of the flow. Scale bar is 5 µm in (A, B, C, D, E, F). (C, D) ODF2 staining reveals the spacing pattern of basal bodies as parallel rows, oriented perpendicularly to the direction of the flow. (E) nNOS KO mutant cells with mild phenotype have preserved rotational polarity and spacing pattern of the basal bodies within single cells, which is poorly coordinated with the neighboring cells. (F, G) nNOS KO mutant ciliated cells with more severe phenotype lack rotational polarity both within the cell and between neighboring ciliated cells, and the spacing pattern of the basal bodies is scrambled. (H, I, J) Microtubules are anchored to the most basal aspects of the basal feet (labeled by ODF2) in the ciliated cells in the trachea (H, scale bar is 1 µm). Microtubules connect all basal bodies into a regular joint network in the ciliated cells of the trachea in wild type (I); (J) – higher magnification. (K, L) In the nNOS KO cells basal feet are aberrantly connected with microtubules, with some basal feet having excessive connections with the microtubules and others left out of the connections. (H, I, J, K, L) Scale bars are 1 µm in (H, I, K) and in (J, L). Pearson correlation coefficient for the overlap between fluorescent signals of ODF2 (Alexa-568) and microtubules (Alexa-488) is indicated. (M) Fraction of the basal feet connected to the microtubules in wild-type and nNOS KO ciliated cells (20 and 25 cells of each genotype analyzed, correspondingly). (N, O) Transmission electron microscopy shows a sparse network of microtubules connecting basal feet in nNOS KO, leaving some basal feet out of the network, as compared with wild type.

Fig 3: Localization of mGFP–WDR34. (A) mGFP–WDR34 (green) localizes to a cytosolic pool and to primary cilia, colocalizing with acetylated tubulin (red). Labeling of (B) ?-tubulin, (C) ODF2 and (D) OFD1 (all red) demonstrate the accumulation of mGFP–WDR34 at the base of the cilium with some labeling evident along its length. Insets show enlargements of the areas indicated in the main images. (E) Cells expressing mGFP–WDR34 were fixed with paraformaldehyde and labeled to detect acetylated tubulin and WDR34, as indicated. Two examples are shown. (F) Cells expressing GFP were processed as in E to demonstrate an absence of GFP from primary cilia. Scale bars: 5 µm.

Fig 4: 3D molecular architecture of DAPs and sDAPs.(a) Relative localization of DAP and sDAP proteins in radial and lateral directions revealing the slanted arrangement of a DAP (dotted line) and the triangular structure of an sDAP (dashed line). (b) A 3D model of a mother centriole, illustrating the localization of various sDAP and DAP proteins for one of the possible arrangements when all nine sDAPs are present. (c) An axial view of the 3D model in panel (b) viewed from the distal end of the centriole, illustrating the radial positions of CEP89 and various sDAP proteins. (d) Lateral view of the model in panel (c). (e) Close view of the sDAP and the DAP in panel (d). ODF2 localizes at both ends of the sDAP and close to the centriole wall; CEP89 localizes on the DAP as well as in the sDAP region.

Fig 5: Localization of nNOS in the ciliated cells of trachea.(A) Immunostaining for nNOS shows its expression in the ciliated cells of the mouse trachea and absence in the nNOS-null mutant trachea (inset): nNOS: red; acetylated a-tubulin: blue (here and below the fluorophores for multiple labels are indicated on the figures by the corresponding colors). Scale bars are 5 µm in (A, B, C, D). (B) Detection of nNOS expression in the tracheal ciliated cells of nNOS-CreER/Ai9 mice. dTomato expression, driven by nNOS promoter, is observed in the ciliated cells, with the sample counterstained with phalloidin and acetylated a-tubulin. (C) nNOS is detected in the ciliary axonemes in the trachea, counterstained with phalloidin. (D) nNOS is detected in the apical cortex of the ciliated cell of the trachea and in the ciliary axonemes, stained with antibodies to nNOS and acetylated a-tubulin. (E) In cultured mouse tracheal ciliated cells nNOS is associated with basal bodies along their entire height. Z section from SIM in the inset. Scale bar is 2 µm. (F, G, H, I, J, K) In the ciliated cells of the mouse trachea, nNOS is associated with the cortical apical actin grid and cortical microtubules. Scale bar is 2 µm. (F) The image is focused on the apical-most area of the ciliated cells where nNOS is associated with the cortical actin grid; inset shows the entire cell (top view). (G) The same cell region as in (F), but flipped over to show the interaction of nNOS with the cortical network of microtubules located below the actin grid; inset shows the entire cell as in (F). (H, I, J, K) Z sections across the apical area of the ciliated cell of the tracheal explant, revealing the positions of the apical cortical actin grid and the microtubules (H); nNOS association with the cortical microtubules (I); nNOS association with the cortical actin grid (J); and nNOS association with both actin and microtubule components of the cortical cytoskeleton (K). (L) In the most basal aspects of the basal bodies nNOS is associated with microtubules and the basal feet (cultured mouse ciliated cells). Scale bars are 1 µm for (L, M, N). (M) nNOS is associated with basal bodies, labeled for ?–tubulin, and with basal feet, labeled for ODF2 (cultured mouse ciliated cells); higher magnification in the inset. (N) nNOS is not detected in the cilia rootlets in cultured ciliated cells labeled for rootletin. Z section in the inset.