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First published online 2 March 2004
doi: 10.1242/jcs.00978

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Foxj1 regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells

Developmental Biology Research Unit, The Edward R. Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (54K): [in a new window]   Fig. 1. Calpastatin is decreased in the absence of Foxj1. (A) RNase protection of E15.5 lung RNA with a [32P]cRNA probe for calpastatin. Relative transcript abundance is shown in comparison with wild-type lung tissue (± s.e.m.). Samples were corrected for loading by comparison with GAPDH transcript abundance. (B) Western blot of E15.5 lung tissue with calpastatin primary antibody. Full-length tissue calpastatin is a 110 kDa protein and a truncated erythrocyte calpastatin isoform is a 70 kDa protein. GAPDH (36 kDa) abundance was used as a protein loading control. (C,D) Immunohistochemistry of foxj1+/+ (C) and foxj1-/- (D) lung tissue with primary antibody to calpastatin and peroxidase detection. Scale bar: 5 µm.

View larger version (58K): [in a new window]   Fig. 2. No change in calpain 2 in the absence of Foxj1. (A) Western blot of E15.5 lung tissue with primary antibody to calpain 2. Calpain 2 is an 80 kDa protein. Comparison was made to GAPDH (36 kDa) to correct for protein loading. (B,C) Immunohistochemistry of foxj1+/+ (B) and foxj1-/- (C) lung tissue with primary antibody to calpain 2 and peroxidase detection. Scale bar: 5 µm.

View larger version (72K): [in a new window]   Fig. 3. Ezrin is decreased in the absence of Foxj1. (A) Western blot of E15.5 lung tissue with primary antibody to ezrin. Ezrin is an 80/81 kDa protein. Comparison was made to GAPDH (36 kDa) to correct for protein loading. (B) RT-PCR for ezrin expression in E15.5 lung tissue. A 170 bp ezrin product is detected in both foxj1+/+ and foxj1-/- lung tissue. (C,D) Immunofluorescence of foxj1+/+ (C) and foxj1-/- (D) in mouse trachea with primary antibody to ezrin or acetylated -tubulin and detection of fluorescence. Scale bar: 10 µm.

View larger version (65K): [in a new window]   Fig. 4. EBP-50 is decreased in the absence of Foxj1. (A,B) Immunofluorescence of foxj1+/+ (A) and foxj1-/- (B) in mouse trachea with primary antibody to EBP-50 or acetylated -tubulin and fluorescence detection. Scale bar: 20 µm.

View larger version (146K): [in a new window]   Fig. 5. Ezrin is associated with the basal bodies of ciliated epithelial cells. (A) Immunoelectron microscopy of tracheal epithelium with primary antibody for ezrin demonstrates gold particles associated with basal bodies (arrows) at the cell apex. Gold particles are also associated with the apical microvilli (arrowheads). No significant gold particles are seen in association with axonemal structures (*). Scale bar: 250 nm. (B) Transmission electron microscopy of trachea from a foxj1+/+ mouse incubated with secondary gold-labeled antibody only. Microvilli (arrowheads) extend from the cell apex between axonemes. Basal bodies are localized at the cell apex (arrows). (C) Transmission electron microscopy of trachea from a foxj1-/- mouse incubated with primary antibody for ezrin and secondary gold-labeled antibody. No gold particles are detected in association with basal bodies that are distributed throughout the cytoplasm (arrows). No gold particles are associated with the microvilli that are severely flattened. No axonemal structures are present. Scale bar: 200 nm (B,C).

View larger version (173K): [in a new window]   Fig. 6. Partial reversal of the foxj1-/- phenotype of no cilia with calpain inhibitor II treatment. Images D, E, F show higher magnification of A, B, C, respectively. (A,D) By scanning electron microscopy, multiple ciliated cells are observed in explanted E17.5 foxj1+/+ trachea cultured for 96 hours (arrows). Cilia cover most of the apical cell surface. (B,E) By scanning electron microscopy, no 9+2 cilia are detected in explanted E17.5 foxj1-/- trachea cultured for 96 hours. 9+0 monocilia are observed (arrowheads). (C,F) Explanted foxj1-/- trachea cultured in the presence of 10 mM calpain inhibitor II for 96 hours demonstrates formation of cilia (arrows). The number of cilia per cell is less than in foxj1+/+ trachea. 9+0 monocilia are also present (arrowheads). Scale bars: 5 µm (A-C); 500 nm (D-F).

View larger version (62K): [in a new window]   Fig. 7. Calpain inhibition results in apical relocalization of basal bodies in foxj1-/- trachea. (A,B) By transmission electron microscopy, basal bodies (arrowheads) are found to be apically localized in an E17.5 foxj1+/+ tracheal explant (A), but distributed throughout the cytoplasm of foxj1-/- tracheal explant (B); both cultured for 96 hours. (C) Treatment of cultured E17.5 foxj1-/- tracheal explant with 10 mM calpain II inhibitor results in localization of basal bodies (arrowheads) to cell apex. Scale bars: 500 nm (A,B); 250 nm (C).

View larger version (81K): [in a new window]   Fig. 8. Calpain inhibition results in apical localization of ezrin and EBP-50 in foxj1-/- trachea. (A) Immunofluorescence of untreated, E17.5 foxj1-/- tracheal explant cultured for 96 hours with primary antibody to ezrin and acetylated -tubulin. (B) Immunofluorescence of E17.5 foxj1-/- tracheal explant cultured for 96 hours in the presence of 10 mM calpain inhibitor II with primary antibody to ezrin and acetylated -tubulin. (C) Immunofluorescence of E17.5 foxj1-/- tracheal explant cultured for 96 hours in the presence of 10 mM calpain inhibitor II with primary antibody to EBP-50 and acetylated -tubulin. Scale bars: 20 µm (A); 2.5 µm (B); 5 µm (C).