Loss of Bardet-Biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia - PubMed (original) (raw)

. 2008 Mar 4;105(9):3380-5.

doi: 10.1073/pnas.0712327105. Epub 2008 Feb 25.

Sara L Farmen, Thomas O Moninger, Thomas R Businga, Michael P Andrews, Kevin Bugge, Charles C Searby, Darryl Nishimura, Kim A Brogden, Joel N Kline, Val C Sheffield, Michael J Welsh

Affiliations

Loss of Bardet-Biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia

Alok S Shah et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Mutations in a group of genes that contribute to ciliary function cause Bardet-Biedl syndrome (BBS). Most studies of BBS have focused on primary, sensory cilia. Here, we asked whether loss of BBS proteins would also affect motile cilia lining the respiratory tract. We found that BBS genes were expressed in human airway epithelia, and BBS2 and BBS4 localized to cellular structures associated with motile cilia. Although BBS proteins were not required for ciliogenesis, their loss caused structural defects in a fraction of cilia covering mouse airway epithelia. The most common abnormality was bulges filled with vesicles near the tips of cilia. We discovered this same misshapen appearance in airway cilia from Bbs1, Bbs2, Bbs4, and Bbs6 mutant mice. The structural abnormalities were accompanied by functional defects; ciliary beat frequency was reduced in Bbs mutant mice. Previous reports suggested BBS might increase the incidence of asthma. However, compared with wild-type controls, neither airway hyperresponsiveness nor inflammation increased in Bbs2(-/-) or Bbs4(-/-) mice immunized with ovalbumin. Instead, these animals were partially protected from airway hyperresponsiveness. These results emphasize the role of BBS proteins in both the structure and function of motile cilia. They also invite additional scrutiny of motile cilia dysfunction in patients with this disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

BBS4 and BBS2 localized to cilia and related structures in primary cultures of differentiated human airway epithelia. Airway epithelia were infected with Ad-3xFlag-tagged-BBS4 (A and B) or BBS2 (C) and immunostained 28 h later. Each image shows a stack of X-Y confocal images on the left, a single X-Z image in the upper right (the dashed line indicates the top of the filter on which epithelia grew), and a 3D surface projection in the lower right. (A) BBS4 (red) localized in multiple puncta beneath the cilia in the area of the basal body. Acetylated α-tubulin (green) is a marker of cilia (45). Nuclei were stained with DAPI (blue). (B) BBS4 (red) localized adjacent to or with pericentrin (green). Pericentrin staining occurred at the basal bodies in the apical portion of ciliated cells and near the nucleus of basal cells in centrioles (24). (C) BBS2 (red) localized in the region of cilia (acetylated α-tubulin, green) and the basal body. Control epithelia treated with secondary antibodies showed no staining (data not shown). (Scale bars: 10 μm.)

Fig. 2.

Fig. 2.

Loss of BBS2 changed the distribution of cilia length. Cilia length was measured by using light microscopy of H&E sections. _Bbs2_−/− and wild-type cilia showed the same average length: 4.1 μm. However, _Bbs2_−/− cilia exhibited a greater variation in length; variance for wild-type cilia (0.20) differed from _Bbs2_−/− cilia (1.22); P < 0.001, Levene test.

Fig. 3.

Fig. 3.

Loss of BBS protein produced abnormal morphology of motile cilia. Images are SEMs of airway epithelial cultures from wild-type (A), _Bbs2_−/− (B), and _Bbs4_−/− (C) mice and of the in vivo tracheal surface from wild-type (D), _Bbs2_−/− (E), and _Bbs4_−/− (F) mice. B shows cilia with bulges at their distal end and along the shaft. Some of the cilia show sprouts at their end, giving a glove-with-fingers-like appearance; also see Inset. B and C also show cup-shaped cilia. SEMs from in vivo tissue showed similar abnormalities but less frequently. We have never observed these abnormalities in wild-type epithelia. In addition to cilia, the apical surface contains microvilli of variable number and length. (Scale bars: 1 μm.)

Fig. 4.

Fig. 4.

Loss of BBS proteins caused a build up of vesicles in motile cilia. Data are TEM images from wild-type (A), Bbs2 null (B), and Bbs1 mutant (M390R) (C and D) primary cultures of differentiated airway epithelia. B shows bulges at the distal end of two cilia; the one on the right has a cup-like shape, and both contain vesicles. (Inset) Vesicles adjacent to an intact microtubule structure. D shows that the vesicles have a bilayer membrane. (Scale bars: A–C, 0.5 μm; D, 100 nm.)

Fig. 5.

Fig. 5.

_Bbs2_−/− and _Bbs4_−/− mice did not have increased airway hyperresponsiveness. Mice were immunized with ovalbumin and challenged with methacholine. We studied _Bbs2_−/− and _Bbs4_−/− mice and their wild-type littermates. Airway hyperresponsiveness was measured noninvasively by using whole-body plethysmography and recording enhanced pause (Penh) (A) or by using an invasive small-animal ventilator (Flexivent) (B). The Bbs mice showed reduced airway hyperresponsiveness. n = 8 for _Bbs2_−/− and wild type; n = 6 for _Bbs4_−/− and wild type. Asterisks indicate P < 0.05.

Fig. 6.

Fig. 6.

Loss of BBS1 and BBS4 reduced ciliary beat frequency but did not alter mucociliary transport. (A) Data are ciliary beat frequency in differentiated airway epithelial cultures. n = 8 mice. Asterisks indicate P < 0.001. (B) Mucociliary transport rates did not significantly differ between wild-type and Bbs null mice. n = 6 mice. Data are mean ± SEM.

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References

    1. Mykytyn K, Sheffield VC. Establishing a connection between cilia and Bardet–Biedl syndrome. Trends Mol Med. 2004;10:106–109. - PubMed
    1. Beales PL, Elcioglu N, Woolf AS, Parker D, Flinter FA. New criteria for improved diagnosis of Bardet–Biedl syndrome: Results of a population survey. J Med Genet. 1999;36:437–446. - PMC - PubMed
    1. Moore SJ, et al. Clinical and genetic epidemiology of Bardet–Biedl syndrome in Newfoundland: A 22-year prospective, population-based, cohort study. Am J Med Genet A. 2005;132:352–360. - PMC - PubMed
    1. Nachury MV, et al. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 2007;129:1201–1213. - PubMed
    1. Tobin JL, Beales PL. Bardet–Biedl syndrome: Beyond the cilium. Pediatr Nephrol. 2007;22:926–936. - PMC - PubMed

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