Tppp3 determines basal body positioning and identity of respiratory cilia via microtubule assembly and sphingolipid homeostasis

Abstract

Cilia are hair-like organelles that protrude from the cell surface. In mammals, tracheal multiciliated cells (MCCs) play an important role in elimination of hazardous microorganisms by driving a unidirectional mucus flow. Although uniform orientation of ciliary beating is critical for the unidirectional flow, it remains unknown how MCCs establish uniform orientations and maintain identities of hundreds of ciliary membranes. This study focuses on investigating the roles of Tubulin Polymerization Promoting Family Member 3 (Tppp3) in MCC function. We generated a Tppp3-deficient mouse (Tppp3^{∆ex2-4/∆ex2-4}; Tppp3 knockout (KO)) and found that the Tppp3 KO mouse exhibited cough and hyposmia phenotype. The loss of Tppp3 disrupted the apical microtubules (MTs) meshwork in the tracheal MCCs, leading to random orientation and alignment of basal bodies (BBs) of the motile cilia. Unexpectedly, aberrant ciliary membrane fusions occurred in the trachea of the Tppp3 KO mice. We examined the underlying molecular mechanism of the ciliary membrane fusion by isolating the tracheal cilium. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis as well as pharmacological analysis revealed that hyperaccumulation of a long chain ceramide at the ciliary membrane caused the membrane fusion. In addition, sensory cilia formation was impaired in the olfactory sensory neuron of the Tppp3 KO mice. Due to the lack of Tppp3, dendritic MT assembly that underlies long-range migration of BBs toward the cell surface was impaired. These findings demonstrate that Tppp3, as well as the defined intracellular MT architecture, regulate proper orientation/subcellular positioning of BBs and the independency of individual motile cilium membranes.

Keywords: basal body; microtubule; motile cilia; olfactory sensory neuron; trachea.

Fig. 1.

Characterization of Tppp3 KO mice. (A) RT-PCR analysis of Tppp3 in the mouse organs. Tppp3 was mainly expressed in the brain, the olfactory epithelium, the lung, and the heart. (B) Strategy for generating Tppp3 KO mice. Two guide RNAs were designed to delete exon3 and exon 4. Unexpectedly, however, the two guide RNAs had deleted exon2, exon3, and exon4 by chance. (C) Protein expression of Tppp3 in the lung of the WT and the Tppp3 KO mice. β-actin was used for loading control. (D) Survival curve of the WT mice (+/+), Tppp3 heterozygotic mutant mice (+/−), and the Tppp3 homozygotic mutant mice (−/−). The number of mice used for the survival curve analysis: N = 15 for WT (+/+) mice, N = 10 for the heterozygotic mutant (+/−) mice, N = 13 for the homozygotic mutant (−/−) mice. P = 0.9601(Tppp3 +/+ vs. Tppp3 +/−) and 0.2362(Tppp3 +/+ vs. Tppp3 −/−) analyzed by Log-rank (Mantel-Cox) test, respectively. (E) Litter sizes for breeding pairs of the indicated genotype mice. (F) Body weights for the WT and the Tppp3 KO mice. (G) Recording of sound of breath for the WT and the Tppp3 KO mouse. The recording time was 40 s. (H) Hidden buried food assay. The time data spent for finding a buried food were plotted for the WT and the Tppp3 KO mice.

Fig. 2.

Tppp3 regulated the orientation and the subcellular regular positioning of BBs of the motile cilia in the mouse trachea via MT assembly. (A–C) DIC images of multiple motile cilia in the trachea of the WT and the Tppp3 KO mice. (Scale bars: 5 µm.) Arrows indicated orientation of the cilia in (B). Arrowhead denoted cilia that showed complete synchronized motion in (C). (D) CBF of trachea cilia in the WT and the Tppp3 KO mice. The number of cells was 17 for WT and 18 for Tppp3 KO mice, respectively. The data were obtained from in total N = 3 mice (three WT and three KO mice). (E) Transmission electron micrographs (TEM) of the BBs in the WT and the Tppp3 KO mice. Arrows indicated orientation of the BF. (Scale bars: 500 nm.) (F) Analysis of orientation of BBs by IF of BBs (ODF2) and BFs (Centriolin). The number of cells was 13 for WT and 10 for Tppp3 KO mice, respectively. The data were obtained from in total N = 3 mice (Scale bars: 5 µm in the Left column and 1 µm in the Right column.) (G) Analysis of subcellular positioning of BBs by g-tubulin immunostaining. The number of cells was 20 for WT and 26 for Tppp3 KO mice, respectively. The data were obtained from in total N = 3 mice (Scale bars: 5 µm.) (H) TEM micrographs of MTs in the apical region of the trachea MCCs. (Scale bars: 500 nm.) (I) Observation of the apical MTs of the trachea MCCs by IF. The apical MTs were visualized by α-tubulin immunostaining. (Scale bars: 5 µm in the Left column and 1 µm in the Right column.) (J) Subcellular localization of Tppp3 in the trachea MCCs. (Scale bars: 5 µm in the Left column and 1 µm in the Right three columns.)

Fig. 3.

Upregulation of long chain ceramide caused the aberrant ciliary membrane fusion in the trachea of the Tppp3 KO mice. (A) LC–MS analysis of isolated trachea cilium revealed that a long chain ceramide (Cer 18:1;O2/24:0) was increased in the trachea ciliary membrane of the Tppp3 KO mice compared with that in the WT mice. Gray: not significant. Red: upregulation. Blue: Down regulation. White: Triacyl glycerol. The data were obtained from in total N = 7 mice (seven WT and seven Tppp3 KO mice). (B) SEM revealed that aberrant trachea ciliary fusion occurred in the Tppp3 KO mice. Values of the vertical axis in the graph indicated the percentage of total area of cells that showed ciliary membrane-fusion. The data were obtained from in total N = 4 mice (Scale bars: 10 µm.) (C) TEM micrographs of cross-section of the trachea cilia. Ciliary membrane fusions occurred in the Tppp3 KO mice. (Scale bars: 500 nm.) (D) Subcellular localization of ceramide. IF analysis revealed that ceramide was localized in the trachea cilium of the WT mice. In the Tppp3 KO mice, expression level of ciliary localized ceramide was significantly enhanced compared with that in the WT mice. Number of cells was 651 for WT mice and 294 cells for the Tppp3 KO mice. The data were obtained from in total N = 3 mice (Scale bars: 10 µm.) (E) SEM observations revealed exogenous treatment of the long chain ceramide (Cer 18:1;O2/24:0) caused the trachea ciliary membrane fusion but the abnormal fusion was rescued by endogenous ceramide synthesis inhibition. Values of the vertical axis in the graph indicated the percentage of total area of cells that showed ciliary membrane-fusion. The data were obtained from in total N = 4 mice per individual assay. (Scale bars: 10 µm.) The experimental procedures of ex vivo culture of the whole trachea were described in Materials and Methods.

Fig. 4.

Tppp3 regulated subcellular centrioles/BBs positioning via assembly of the dendritic MTs architecture in the mouse OSN. (A) Observation of olfactory sensory cilia and BBs. Olfactory sensory cilia and BBs were visualized by α-tubulin and ODF2 immunostaining, respectively. The total number of cells was 2066 cells for WT mice and 1,341 cells for the Tppp3 KO mice. The data were obtained from in total N = 3 mice. (Scale bars: 5 µm in the Left column and 1 µm in the Right column.) Further, a proportion of OSNs that lacks the apical BBs was measured. The total number of cells was 3018 cells for WT mice and 3445 cells for the Tppp3 KO mice. The data were obtained from in total N = 3 mice. (B) TEM micrographs of the knob of mouse OSN. Arrowheads indicated BBs. (Scale bars: 1 µm.) (C) En face view of the mouse OSNs. OMP (shown in green) was a mature OSN marker protein. Actin/Phalloidin visualized the apical outline of the OSNs. The value of the vertical axis in the graph denoted percentage of OMP-positive mature OSNs in 127 µm ×127 µm area (D) Sagittal cryosection IF analysis of the olfactory epithelium and visualization of centrioles. Centrioles were visualized by immunostaining of centrin. The value of the vertical axis in the graph denotes the number of the centriole cluster in 127 µm ×127 µm area. The data were obtained from N = 3 mice. (E) Sagittal cryosection IF analysis of the olfactory epithelium for visualization of the dendritic MTs. The dendritic MTs architectures were visualized by immunostaining of TUBB3/β3-tubulin. The total number of cells was 57 cells for WT mice and 57 cells for the Tppp3 KO mice. The data were obtained from in total N = 3 mice [Scale bars: 20 µm and 5 µm (magnified image).] (F) Subcellular localization of Tppp3 in the mouse OSN. Tppp3 was localized along the dendritic MT. (Scale bars: 5 µm.)