title: Analysis of ciliary import.
abstract: Cilia and flagella are microtubule-based organelles that play important roles in human health by contributing to cellular motility as well as sensing and responding to environmental cues. Defects in cilia formation and function cause a broad class of human genetic diseases called ciliopathies. To carry out their specialized functions, cilia contain a unique complement of proteins that must be imported into the ciliary compartment. In this chapter, we describe methods to measure the permeability barrier of the ciliary gate by microinjection of fluorescent proteins and dextrans of different sizes into ciliated cells. We also describe a fluorescence recovery after photobleaching assay to measure the entry of ciliary proteins into the ciliary compartment. These assays can be used to determine the molecular mechanisms that regulate the formation and function of cilia in mammalian cells.
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: John F
authors.initials: JF
authors.lastname: Dishinger
authors.forename: H Lynn
authors.initials: HL
authors.lastname: Kee
authors.forename: Kristen J
authors.initials: KJ
authors.lastname: Verhey
mesh_terms: D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002923:Cilia / Q000378:metabolism*; D003911:Dextrans / Q000032:analysis / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes; D006801:Humans; D051379:Mice; D008845:Microinjections; D008870:Microtubules / Q000378:metabolism*; D041681:NIH 3T3 Cells; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D003911:Dextrans; D005456:Fluorescent Dyes; D011994:Recombinant Proteins