First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00830
Foxj1 is required for apical localization of ezrin in airway epithelial cells
Tao Huang1,
Yingjian You1,
Melanie S. Spoor1,
Edward J. Richer1,
Vrinda. V. Kudva1,
Renee C. Paige2,
Michael P. Seiler3,
Janice M. Liebler4,
Joseph Zabner3,
Charles G. Plopper2 and
Steven L. Brody1,*
1 Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA
2 Department of Cell Biology and Anatomy, University of California, Davis, CA 95616 USA
3 Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242, USA
4 Department of Medicine, University of Southern California, Los Angeles, CA 90033, USA
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Fig. 1. Ezrin is expressed in the apical domain of ciliated airway epithelial cells in vivo. (A) Mouse lungs were immunostained with anti-ezrin (clone 3C12, green) and ß-tubulin-IV (red) antibodies to identify cilia. (B) Ezrin localization detected as in A highlighted by confocal microscopy with diffusion interference contrast. (C) Mouse lungs were immunostained with anti-ezrin (clone 3C12, green) and anti-Clara cell secretory protein (CCSP) antibody (red). Tissue sections (6 µm) were incubated with primary antibodies and detected using fluorescent-labeled secondary antibodies. Representative sections are shown. Bars 10 µm (A,C); 7 µm (B).
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Fig. 2. Ezrin localization at the apical membrane immediately precedes maturation in a population of highly differentiated epithelial cells. (A) Primary culture MTE wild-type cells differentiated for 7 days on semipermeable, supported membranes under ALI conditions were immunostained for ezrin (red) and ß-tubulin-IV (green) expressed in cilia then imaged by immunofluorescent microscopy and merged. Nuclei were detected in the same field by DAPI staining. Bar, 10 µm. (B) Primary culture MTE cells grown and immunostained as in A were harvested on the indicated days (d) and imaged by confocal microscopy at the level of the apical membrane (x,y). Images were reconstructed to generate z-axis images. Bar, 10 µm. (C) MTE cells cultured as in A were partitioned into detergent-soluble and -insoluble fractions as described (Algrain et al., 1993 ) at indicated days and 10 µg of protein was subjected to immunoblot analysis for detection of ezrin (clone 3C12), ß-catenin (ß-cat) and actin. Representative data from four independent preparations are shown.
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Fig. 3. Apical ezrin is developmentally regulated and dependent on Foxj1 expression. (A) Lung sections from indicated mouse embryonic day (E) were immunostained for Foxj1 (red) and ezrin (clone 3C12, green). At E14.5, auto-fluorescense is present in red blood cells at the bottom edge. Bar, 10 µm. (B) Tissue sections from lung or sinus of wild-type (+/+) or Foxj1-null (–/–) mouse were immunostained for Foxj1 (red) and ezrin (green) as in A. Representative sections are shown. Bar, 30 µm.
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Fig. 5. Decreased ezrin threonine phosphorylation in Foxj1-null airway epithelial cells. (A) Wild-type (+/+) and Foxj1-null (–/–) primary MTE cells cultured as in Fig. 2 and analyzed at ALI day 14 were immediately fixed in TCA, calycA or control buffer then 5 µg of protein was subjected to immunoblot analysis with phosphothreonine (P-T) ERM-specific and other indicated antibodies. Phosphothreonine ezrin (P-T-ezrin) and moesin (P-T-moe) are indicated by arrows. Ezrin (clone 3C12) and moesin (Upstate Biotech) were detected in whole cell lysates. (B) P-T-ezrin and P-T-moesin expressed as a ratio of ß-catenin (ß-cat) determined by densitometry from immunoblots as in A. Data represents the mean and standard deviation of expression from four independent preparations. The asterisk indicates a difference in means (P<0.05). (C) Expression of moesin in the detergent-insoluble fraction of primary culture MTE cells from wild-type or Foxj1-null cells from ALI day 14. Fractions were prepared and 5 µg of protein per lane were analyzed as in Fig. 2C.
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Fig. 6. Apical expression of moesin is independent of Foxj1 expression in airway epithelial cells. (A) Wild-type (+/+) and Foxj1-null (–/–) MTE cells were cultured and immunostained for expression of ezrin (clone 3C12; green) and moesin (red) at indicated day (d) as in Fig. 2B. Confocal microscopy images were reconstructed to generate z-axis images. Bar, 10 µm. (B) Mouse lung cells were immunostained for moesin (Franck et al., 1993) (red) and ezrin (green). Arrow indicates a rare cell expressing only apical moesin. Bar, 10 µm. (C) Wild-type MTE cells from ALI day 10 (left) or mouse lung cells from wild-type and Foxj1-null mice (right) were immunostained for expression of moesin (red) and CCSP (green). Bar, 10 µm.
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Fig. 7. Apical membrane moesin cannot compensate for ezrin in Foxj1-null cells. (A) MTE cells were grown to ALI day 10 as in Fig. 2, then immunostained for expression of either EBP50 (red) or ß2AR (red) together with ezrin (green). Immunofluorescent microscopy images focused on the apical membrane of cells in identical fields analyzed for expression of ezrin, EBP50, or ß2AR then merged. Bar, 10 µm. (B) MTE cells from wild-type (+/+) and Foxj1-null (–/–) mice cultured as in A, immunostained for expression of either EBP50 or ß2AR (both green) together with lateral cell junction membrane protein E-cadherin (E-cad, red) and imaged by confocal microscopy at the level of the apical membrane (x,y). Images were reconstructed to generate z-axis images. Bar, 10 µm. (C) Tissue sections from wild-type and Foxj1-null mouse lungs were immunostained for expression of indicated apical ezrin-dependent and basolateral protein as in Fig. 1. Arrows mark apical expression of ß2AR. Bar, 10 µm.
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© The Company of Biologists Ltd 2003
