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Proc Natl Acad Sci U S A
2020 Jun 16;11724:13571-13579. doi: 10.1073/pnas.1907335117.
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CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia.
Robinson AM
,
Takahashi S
,
Brotslaw EJ
,
Ahmad A
,
Ferrer E
,
Procissi D
,
Richter CP
,
Cheatham MA
,
Mitchell BJ
,
Zheng J
.
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Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a "9 + 2" microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3
tm1a/tm1a
, exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3
tm1a/tm1a
mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3
tm1a/tm1a
, MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.
Fig 1. Phenotypes of the Camsap3tm1a mouse model. (A) Structure of the Camsap3tm1a allele with an RNA-processing signal/lacZ-trapping element inserted between exons 6 and 7 (indicated as yellow boxes) of the Camsap3 gene. (B) CAMSAP3 expression is significantly decreased in Camsap3tm1a/tm1a. Relative intensities in arbitrary units of CAMSAP3 bands from three independent Western blots using brain lysates from P10 to 11 littermates. Intensities are normalized to tubulin-loading control bands (detected by anti–α-tubulin). (C and D) Body weight is significantly decreased in Camsap3tm1a/tm1a. Weight of neonatal mice ages P3 to 4 (C) and older adult mice ages P184 to 248 (D) versus genotype. (E and F) Fertility and litter size are significantly decreased in Camsap3tm1a/tm1a. Fertility rate (E) and mean litter sizes (F) for breeding pairs of the indicated genotypes. M, male; F, female. (G) Food finding is significantly decreased in older adult Camsap3tm1a/tm1a. Mean number of times mice were found in the center of a cage with hidden food versus genotype. Het, Camsap3tm1a/+; Homo, Camsap3tm1a/tm1a. n, number of animals; n.s., not statistically significant. Error bars are standard deviations from the means.
Fig 2. Camsap3tm1a/tm1a mice have obstructed nasal cavities and abnormal nasal cavity structure. (A–D) Mucin blocks the nasal airway and obstructs the olfactory epithelium in older Camsap3tm1a/tm1a mice. PAS staining for glycoproteins in coronal nasal sections in WT (A) showing a patent anterior nasal airway (A) with a central bony septum at the level of the vomeronasal organ compared with mucin buildup (B) (purple) in Camsap3tm1a/tm1a with a deviated septum (arrows). WT (C) patent nasal airway with a central septum at a level where the ethmoid turbinates (T) are covered primarily with olfactory sensory epithelium compared with Camsap3tm1a/tm1a (D) with almost total airway (a) blockage with mucin (purple). (Scale bars, 500 µm.) (E–H) Mucin blocks the nasal airway of young adult Camsap3tm1a/tm1a mice. PAS staining shows a normal opening to the Eustachian tube (Et) in WT mouse (E), while its Camsap3tm1a/tm1a littermate exhibited mucin accumulation (m) and folding of epithelial cell layers (white arrows) in the Eustachian tube (F). (Scale bars, 50 µm.) Micro-CT images showing dark and radiopaque regions at the back of the nasal cavity in WT (G) and Camsap3tm1a/tm1a (H), respectively. White arrows indicate blockage. A calibration bar is as indicated. Hu, Hounsfield units. (I–M) Representative rendered 3D images obtained by thresholding of CT data in WT and Camsap3tm1a/tm1a are shown. Snout details from the region outlined in red in I are shown for different orientations for WT (J and L) and Camsap3tm1a/tm1a (K and M) mice. An orientation axis for each figure is shown in the corresponding inset. The white arrows in (L) and (M) point at the snout region with most structural differences between the WT and Camsap3tm1a/tm1amouse.
Fig. 3. MCCs from Camsap3tm1a/tm1a have abnormal ciliary morphology. SEM images of nasal MCCs from WT (A and C) and its Camsap3tm1a/tm1a littermate (B, D, and E) at P3 and 20. Arrows in B point to long and curved cilia in Camsap3tm1a/tm1a mice. Nearby microvilli are indicated by asterisks. (E) The surface of MCCs from a Camsap3tm1a/tm1a at P20. Yellow arrows indicate the layer of fibrous substances. (Scale bars, 2 µm.)
Fig. 4.
Reduction of CAMSAP3 causes impaired mucociliary transport. (A) Nasal ciliary beat frequency in hertz (beats per second) for motile cilia of nasal tissues at age P3 versus genotype. Replicates (n) are as indicated. (B) Velocity of fluorescent beads in micrometers per second of control (Ctrl) Xenopus embryos injected with morpholinos versus Camsap3 morpholino-injected embryos. n, number of fluorescent beads.
Fig. 5. CAMSAP3 is located at the base of axonemes and at the basal bodies. (A–F) Immunofluorescent images of nasal MCCs from WT (A, C, and E) and Camsap3tm1a/tm1a (B, D, and F) at P3 (C and D) and 30 (A, B, and E–G) are shown. Antibodies include anti–CAMSAP3-M (green), anti–γ-tubulin (red), anti–acetylated-α-tubulin (red in A and B and violet in C–G), and Hoechst 33342 (blue, nuclei). Respiratory epithelium (RE). (G) SIM image showing the upper CAMSAP3 line (green dots) and lower line of basal bodies (γ-Tub, red dots). (Scale bars, 100 µm [A and B], 10 µm [C–F], 5 µm [C′, D′, and E′], and 1 µm [G].) C′, D′, and E′ are enlarged images of the boxed regions in C–E showing anti–γ-tubulin (red) and anti–CAMSAP3-M (green) channels.
Fig. 6. Reduction of CAMSAP3 expression in mice disrupts the polarity of basal bodies in MCCs. (A) Motile cilium structure. (B) A representative image showing disorientated basal feet found in two MCCs (indicated with numbers 1 and 2) in the nasal cavity of Camsap3tm1a/tm1a (P30). (Scale bar, 2 µm.) (C) High-magnification image of the boxed region in B. Yellow arrows indicate basal feet with various orientations. (Scale bar, 500 nm.)
Fig. 7. Reduction of CAMSAP3 expression disrupts the polarity of basal bodies in MCCs of Xenopus embryos. Confocal images of Xenopus embryos injected with RNAs for RFP-centrin and CLAMP-GFP together with either control- (A) or Camsap3-MO (B). Boxed regions from A and B are shown in A′ and B′, respectively. (Scale bars, 5 µm.) (C) Quantification of MCC polarity. Each arrow represents the mean cilia orientation for a given cell, while arrow length represents 1/r, namely the variation in orientation within the cell (long arrows indicate low variation and short arrows indicate high variation).
Fig. 8.
CAMSAP3 is needed for the formation of central MT pairs in axonemes. TEM images showing MT arrays in axonemes from an adult P30 WT (A) compared with a P30 Camsap3tm1a/tm1a (B). Five different axoneme MT configurations (a–e) are listed in C. Red arrows point to inner and outer dynein arms in outer MT doublets. (Scale bars, 500 nm [A and B] and 100 nm [C]
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