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First published online 8 May 2007
doi: 10.1242/jcs.005306

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RhoA-mediated apical actin enrichment is required for ciliogenesis and promoted by Foxj1

Pulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (70K): [in this window] [in a new window]   Fig. 1. The apical localization of actin in ciliated airway epithelial cells during differentiation. Differentiation was induced in primary culture mTECs under ALI conditions. (A) Photomicrographs of mTEC cultures stained for filamentous actin (phalloidin, red) and for the cilia marker -tubulin-IV (green) on the indicated day of ALI culture. Reconstructed confocal images show z-axis view below. An apical web-like structure is seen at ALI day 3 in ciliated cells. (B) Photomicrographs obtained as in A stained for actin (phalloidin, red) and basal body marker -tubulin (green). -tubulin is additionally present in paired nuclear centrioles within the microtubule organizing center as seen at ALI day 2. Bars, 10 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 2. The effect of pharmacological inhibition of actin assembly on ciliogenesis. (A) First nine panels from left: photomicrographs showing actin (phalloidin), basal body (-tubulin) and cilia (-tubulin-IV) localization in mTEC cultures at ALI day 3, obtained 1 day after treatment at ALI day 2 with vehicle, cytochalasin D or latrunculin B. Bar, 10 µm. Three panels on right: representative scanning electron micrographs (SEMs) of mTEC cultures on ALI day 4 obtained 1 day after treatment at ALI day 3. Bar, 1 µm. (B) Percentage of cells containing basal bodies localized at the apical membrane as a proportion of total cells containing basal bodies at the apical membrane or within the cytoplasm (identified by immunostaining for -tubulin). Data are the mean ± s.d.; *P<0.05, significance compared with vehicle. (C) The percentage of cells with apical cilia (identified by immunostaining for -tubulin-IV) as a proportion of all cells (identified by nuclear staining with DAPI). Data are the mean ± s.d.; *P<0.05, significance compared with vehicle.

View larger version (96K): [in this window] [in a new window]   Fig. 3. The effect of Rho GTPase inhibition on epithelial cell differentiation. (A) mTEC cultures treated with vehicle, mevasatin, or toxin B for 24 hours on ALI day 2 were harvested one day later and stained for actin (phalloidin, red) and basal bodies (-tubulin, green). Different fields are shown for each marker assayed. Bar, 10 µm. (B) Scanning electron micrograph of mTEC on ALI day 4 after treatment with mevastatin on ALI day 3 showing microvilli but absent cilia. (C) Transmission electron photomicrograph of mTEC treated with toxin B as in B. Vehicle treated cells show normal basal bodies docked at the apical membrane with ciliary axonemes (arrowheads). In toxin B treated cells, apical basal bodies are absent, but centrioles are present within the cytoplasm (magnification shown in the box). Microvilli are intact at the apical membrane (arrows) Bar, 1 µm in B,C.

View larger version (109K): [in this window] [in a new window]   Fig. 4. The effect of C3 exotoxin on ciliogenesis. (A) Confocal photomicrographs of actin stained with phalloidin (red) and of -tubulin (green) in mTEC cultures on ALI day 3 and ALI day 5 following treatment with vehicle or C3 exotoxin initiated on ALI day 2 and day 4, respectively. Reconstructed z-axis images are below. (B) The percentage of apical -tubulin in all cells with basal bodies (apical or cytoplasmic) and apical -tubulin in total cells treated as in A. Data are the mean ± s.d. (n=3 experiments); *P<0.05, significance compared with vehicle. (C) Basolateral -catenin (red) and DAPI-stained cell nuclei (blue) in ALI day 3 cultures following treatment with vehicle or C3 exotoxin at ALI day 2. Bars, 10 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 5. The effect of Foxj1 on actin assembly and RhoA activation. (A) Confocal photomicrographs of actin (phalloidin, red) and basal body (-tubulin, green) localization in mTEC cultures (ALI longer than day 7) from wild-type (WT) or Foxj1–/– mice. Reconstructed confocal z-axis images are below. (B) 3D reconstruction of confocal microscopy images from A showing the loss of apical actin in Foxj1–/– compared to WT mTECs. (C) Western blot showing RhoA and RhoB GTPase activation in non-differentiated mTECs infected with AdGFP or AdFoxj1. GTP-bound RhoA and RhoB were isolated from cell lysates by binding to rhotektin-agarose, and subjected to immunoblot analysis. RhoA and RhoB were detected in the same membrane re-probed with specific antibodies. (D) Scanning densitometry of immunoblots from C showing active relative to total Rho. (E) Western blot showing Rac1 GTPase activation in mTEC cultured as in C, treated with hepatocyte growth factor (HGF, 50 ng/ml, 4 hours) or transfected with adenovirus vector expressing GFP or Foxj1. Rac1-GTP was isolated from cell lysates of undifferentiated mTECs by binding to P21-binding-domain-bound agarose and immunoblots analyzed. (F) Scanning densitometry of blots shown in E.

View larger version (90K): [in this window] [in a new window]   Fig. 6. The effect of dominant-negative RhoA on ciliogenesis and ezrin localization. (A) Adenovirus-mediated overexpression of dominant-negative RhoA (AdRhoAN19) in cells co-expressing cilia marker Sp17 (arrows). At ALI day 0, mTEC cultures were infected with a tetracycline regulated (tet-off) adenovirus vector system (AdtTA) to control the expression of AdRhoAN19 by continuous incubation without or with doxycyline (Dox), then immunostained at ALI day 5. Bar, 10 µm. (B) -tubulin-IV, -tubulin, ezrin and Foxj1 in mTEC cultures infected with RhoAN19 and AdtTA as in A treated without or with doxycyline. (C) The percentage of cells transfected with RhoAN19 with apical localization of ciliated cell markers (-tubulin, -tubulin, ezrin) and Foxj1 (nuclear) in the absence or presence of doxycyline. Data are the mean values ± s.d.; *P<0.05, significance in the absence of doxycyline. (D) Western blots showing expression of ezrin and moesin in mTEC cultured on plastic dishes treated with siRNA containing scrambled (Scr) or three pooled ezrin-specific (Ezr) sequences. (E) -tubulin-IV and ezrin in mTEC cultures treated with medium, Scr or pool ezrin siRNA sequences at ALI day 2 through ALI day 5, then immunostained for cilia (-tubulin-IV, green) and ezrin (red) at ALI day 5. (F) The percentage of cells co-expressing -tubulin-IV and ezrin (mean ± s.d.) following treatment with media, scrambled sequence (scram) siRNA or ezrin-specific siRNA. Shown are the mean values ± s.d. (from three independent experiments); *P<0.05, significance compared with control conditions.