Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Aug 29;7(9):124.
doi: 10.3390/cells7090124.

CFAP70 Is a Novel Axoneme-Binding Protein That Localizes at the Base of the Outer Dynein Arm and Regulates Ciliary Motility

Affiliations
Free PMC article

CFAP70 Is a Novel Axoneme-Binding Protein That Localizes at the Base of the Outer Dynein Arm and Regulates Ciliary Motility

Noritoshi Shamoto et al. Cells. .
Free PMC article

Abstract

In the present study, we characterized CFAP70, a candidate of cilia-related protein in mice. As this protein has a cluster of tetratricopeptide repeat (TPR) domains like many components of the intraflagellar transport (IFT) complex, we investigated the domain functions of particular interest in ciliary targeting and/or localization. RT-PCR and immunohistochemistry of various mouse tissues demonstrated the association of CFAP70 with motile cilia and flagella. A stepwise extraction of proteins from swine tracheal cilia showed that CFAP70 bound tightly to the ciliary axoneme. Fluorescence microscopy of the cultured ependyma expressing fragments of CFAP70 demonstrated that the N-terminus rather than the C-terminus with the TPR domains was more important for the ciliary localization. When CFAP70 was knocked down in cultured mouse ependyma, reductions in cilia beating frequency were observed. Consistent with these observations, a Chlamydomonas mutant lacking the CFAP70 homolog, FAP70, showed defects in outer dynein arm (ODA) activity and a reduction in flagellar motility. Cryo-electron tomography revealed that the N-terminus of FAP70 resided stably at the base of the ODA. These results demonstrated that CFAP70 is a novel regulatory component of the ODA in motile cilia and flagella, and that the N-terminus is important for its ciliary localization.

Keywords: cryo-electron tomography; outer dynein arm; tetratricopeptide repeat containing protein.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Detection of Cfap70 mRNA and protein in mouse. (A) Semi-quantitative RT-PCR analysis of various cells and tissues. To the left of the gel, the positions and sizes (bp) of DNA ladders are indicated. (B) Confocal microscopy of tissue sections immunostained for CFAP70 (green) and acetylated α-tubulin (AcTub, red) along with DAPI (blue). As a negative control (N.C.), a tracheal section was immunostained using mouse anti-AcTub and rabbit normal IgG. All of the CFAP70-positive epithelia and spermatocytes have motile 9 + 2 cilia and flagella. Bars, 10 µm.
Figure 2
Figure 2
Comparison of the intra-ciliary distribution between CFAP70 and IFT88. (A) Confocal microscopy of mouse tracheal tissue immunostained for CFAP70 (top panels) and IFT88 (bottom panels). Bar, 5 µm. (B) Isolated swine tracheal cilia were extracted sequentially with 0.1% Triton X-100, 0.6 M KCl, and 0.6 M KI. The resulting supernatants (TX, KCl, and KI, respectively) and final pellet were analyzed by Western blot using antibodies against the indicated ciliary proteins. To the left of the gel, the positions and sizes (kDa) of molecular weight standards are indicated.
Figure 3
Figure 3
Changes in the ciliary localization of CFAP70 fragments in primary culture of mouse ependyma. (A) Schematic diagrams of mouse CFAP70 protein and its deletion constructs made in this study. The 1141-amino-acid protein has clusters of tetratricopeptide repeat (TPR) domains (indicated by filled boxes) at the C-terminal half. (B) Western blot analysis for validation of the expression of recombinant CFAP70 proteins fused with a GFP tag at the C-terminus. 293T cells were transfected with the lentiviral expression plasmids and the whole cell lysates were analyzed. The calculated molecular weights are as follows: CFAP70-FL-GFP, 155 kDa; CFAP70-N-GFP, 95 kDa; CFAP70-C-GFP, 87 kDa. To the left of the gel, the positions and sizes (kDa) of molecular weight standards are indicated. (C) Confocal microscopy of cultured ependyma transduced with lentiviral particles carrying the above constructs. The cells were immunostained for AcTub (red) and GFP (green). Bars, 10 µm.
Figure 4
Figure 4
Knockdown of Cfap70 in cultured mouse ependyma. (A) Western blot analysis to assess the knockdown efficiency of lentiviral shRNA in cultured mouse ependyma. Left, whole cell lysates of ependyma treated with viral particles carrying mock or Cfap70 shRNA were analyzed. The lysate of untreated ependyma was also loaded as a control. To the left of the gel, the positions and sizes (kDa) of molecular weight standards are indicated. The band of endogenous CFAP70 with a calculated size of 128 kDa, but a non-specific band of ~250 kDa (asterisk), was diminished in the sample treated with Cfap70 shRNA. The immunoblot for pan actin shows equal loading. Right, quantification of CFAP70 protein levels. Data are presented as the mean ± SD (n = 3). (B) Cilia beat frequency was measured based on high-speed video microscopy data. * p < 0.05 versus mock-treated control. Data are presented as the mean ± SD (n = 18). (C) Ciliary length was measured based on the z-stack images of immunostaining for AcTub. * p < 0.05 versus mock-treated control. Data are presented as the mean ± SD (n = 20 for mock sh, n = 24 for Cfap70 sh).
Figure 5
Figure 5
Characterization of a Chlamydomonas reinhardtii mutant lacking FAP70. (A) Analysis of the sequence similarity between mouse CFAP70 and Chlamydomonas FAP70 proteins. The dot plot was generated by a BLASTP program [46,47], where CFAP70 (1141 amino acids) is represented on the X-axis and FAP70 (1104 amino acids) is represented on the Y-axis. Matched sequence alignments are shown as lines. Shown on the top of the plot is the CFAP70 schematic diagram shown in Figure 3A. (B,C) The swimming speed (B) and flagellar beat frequency (C) of Chlamydomonas. Wild-type, three single mutants (fap70, oda2, ida4, and ida1), two double mutants (fap70 oda2 and fap70 ida4), and the fap70 mutant expressing FAP70-N-BCCP (FAP70-N-BCCP) were analyzed. Data are presented as the mean ± SD (n = 20). (D) Representative tracings of the flagellar beating form. (E) Western blot analysis of FAP70, IC140 (inner dynein arm), and DRC3C (dynein regulatory complex) protein levels in wild-type and three different mutants (fap70, oda1, and oda2). Twenty micrograms of axonemal protein lysate were loaded on each lane. To the left of the gel, the positions and sizes (kDa) of molecular weight standards are indicated. (F) Top, a 0.6 M KCl extract from fap70::FAP70-N-BCCP-HA axonemes was separated by MonoQ anion-exchange chromatography column. The protein peaks of known dynein arm complexes are labeled. Bottom, the MonoQ fractions were separated by SDS-PAGE and FAP70 and outer dynein arms (ODAs) were detected using anti-HA and anti-IC2 antibodies, respectively.
Figure 6
Figure 6
Analysis of the localization of FAP70 protein by cryo-electron tomography. (A) A cross-sectional view of one out of nine doublet microtubules of the axoneme. The flagella of the fap70 mutant rescued by FAP70-N-BCCP were purified, and the axoneme was labeled with streptavidin and biotinylated cytochrome c to increase the density of the BCCP tag. The resulting three-dimensional (3D) structure of the subject by cryo-electron tomography was compared with the wild-type reference data, and the tag densities found only in the subject are labeled in red. The arrow indicates the direction of the views in B. (B) Longitudinal view. The tag densities were located at the base of the ODAs.

Similar articles

See all similar articles

Cited by 5 articles

References

    1. Ibañez-Tallon I., Heintz N., Omran H. To beat or not to beat: Roles of cilia in development and disease. Hum. Mol. Genet. 2003;12:27R–35R. doi: 10.1093/hmg/ddg061. - DOI - PubMed
    1. Leigh M.W., Pittman J.E., Carson J.L., Ferkol T.W., Dell S.D., Davis S.D., Knowles M.R., Zariwala M.A. Clinical and genetic aspects of primary ciliary dyskinesia/artagener syndrome. Genet. Mdicine Off. J. Am. Coll. Med. Genet. 2009;11:473–487. doi: 10.1086/597422.Tumor. - DOI - PMC - PubMed
    1. Satir P., Christensen S.T. Overview of structure and function of mammalian cilia. Annu. Rev. Physiol. 2007;69:377–400. doi: 10.1146/annurev.physiol.69.040705.141236. - DOI - PubMed
    1. Ishikawa T. Axoneme structure from motile cilia. Cold Spring Harb. Perspect. Biol. 2017;9 doi: 10.1101/cshperspect.a028076. - DOI - PMC - PubMed
    1. Summers K.E., Gibbons I.R. Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin sperm. Proc. Natl. Acad. Sci. USA. 1971;68:3092–3096. doi: 10.1073/pnas.68.12.3092. - DOI - PMC - PubMed
Feedback