title: Observing planar cell polarity in multiciliated mouse airway epithelial cells.
abstract: The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type.
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Eszter K
authors.initials: EK
authors.lastname: Vladar
authors.affiliation: Department of Biology, Stanford University, Stanford, CA, USA.
authors.forename: Yin Loon
authors.initials: YL
authors.lastname: Lee
authors.affiliation: Department of Biology, Stanford University, Stanford, CA, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: Stearns
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Axelrod
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000032:analysis; D016764:Cell Polarity / Q000502:physiology*; D002923:Cilia / Q000502:physiology*; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D005455:Fluorescent Antibody Technique / Q000379:methods; D051157:Frizzled Receptors / Q000032:analysis; D008168:Lung / Q000166:cytology / Q000502:physiology; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000379:methods; D061251:Primary Cell Culture / Q000379:methods; D020545:Respiratory Mucosa / Q000166:cytology / Q000502:physiology*; D015398:Signal Transduction; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D014132:Trachea / Q000166:cytology* / Q000601:surgery
chemical_list: D002352:Carrier Proteins; D051157:Frizzled Receptors; C111048:Fzd6 protein, mouse; D008565:Membrane Proteins; C528120:Vangl1 protein, mouse
keywords: Ciliogenesis; De novo basal body formation; Inton retaining transcripts; Marsilea; Post-transcriptional regulation; Rapid development; Spermatogenesis
references.citation: J Microsc. 2006 Dec;224(Pt 3):213-32
references.pmid: 17210054
references.citation: Curr Biol. 2009 Jun 9;19(11):924-9
references.pmid: 19427216
references.citation: Biol Cell. 1985;55(1-2):147-50
references.pmid: 2937490
references.citation: Biophys J. 2012 Jun 20;102(12):2926-35
references.pmid: 22735543
references.citation: Mol Biol Cell. 2014 Oct 1;25(19):2919-33
references.pmid: 25103236
references.citation: J Cell Biol. 2011 Oct 3;195(1):19-26
references.pmid: 21949415
references.citation: Histochem J. 2000 Nov;32(11):669-71
references.pmid: 11272806
references.citation: Biomed Opt Express. 2012 Sep 1;3(9):1978-92
references.pmid: 23024894
references.citation: J Biophys Biochem Cytol. 1961 Oct;11:179-205
references.pmid: 13898346
references.citation: J Cell Biol. 2007 Oct 22;179(2):321-30
references.pmid: 17954613
references.citation: Am J Physiol Lung Cell Mol Physiol. 2002 Dec;283(6):L1315-21
references.pmid: 12388377
references.citation: Nat Methods. 2012 Jul;9(7):671-5
references.pmid: 22930834
references.citation: J Vis Exp. 2013;(78). doi: 10.3791/50343
references.pmid: 23963287
references.citation: Curr Biol. 2012 Dec 4;22(23):2203-12
references.pmid: 23122850
references.citation: Nat Cell Biol. 2013 Dec;15(12):1434-44
references.pmid: 24240477
references.citation: J Cell Biol. 2007 Jul 2;178(1):31-42
references.pmid: 17606865
references.citation: Dev Dyn. 2012 Aug;241(8):1301-9
references.pmid: 22689329
references.citation: Nat Methods. 2012 Jul;9(7):676-82
references.pmid: 22743772
references.citation: Cell. 2010 Sep 3;142(5):773-86
references.pmid: 20813263
references.citation: Nature. 2007 May 3;447(7140):97-101
references.pmid: 17450123
references.citation: Methods Enzymol. 2013;525:285-309
references.pmid: 23522475
references.citation: J Cell Biol. 2010 Jul 26;190(2):165-75
references.pmid: 20643879
references.citation: Nat Cell Biol. 2012 Nov;14(11):1159-68
references.pmid: 23086239
references.citation: Cold Spring Harb Perspect Biol. 2009 Sep;1(3):a002964
references.pmid: 20066108
references.citation: Methods Mol Biol. 2013;945:123-43
references.pmid: 23097105
references.citation: Proc Natl Acad Sci U S A. 2014 Jul 29;111(30):E3129-38
references.pmid: 25024228
references.citation: Nat Cell Biol. 2010 Apr;12(4):341-50
references.pmid: 20305650