title: Recording and analyzing IFT in Chlamydomonas flagella.
abstract: The transport of materials to and from the cell body and tips of eukaryotic flagella and cilia is carried out by a process called intraflagellar transport, or IFT. This process is essential for the assembly and maintenance of cilia and flagella: in the absence of IFT, cilia cannot assemble and, if IFT is arrested in ciliated cells, the cilia disassemble. The major IFT complex proteins and the major motor proteins, kinesin-2 and osm-3 (which transport particles from the cell body to ciliary tips) and cytoplasmic dynein 1b (which transports particles from ciliary tips to the cell body) have been identified. However, we have little understanding of the structure of the IFT particles, the cargo that these particles carry, how cargo is loaded and unloaded from the particles, or how the motor proteins are regulated. The focus of this chapter is to provide methods to observe and quantify the movements of IFT particles in Chlamydomonas flagella. IFT movements can be visualized in paralyzed or partially arrested flagella using either differential interference contrast (IFT) microscopy or, in cells with fluorescently tagged IFT components, with fluorescence microscopy. Methods for recording IFT movements and analyzing movements using kymograms are described.
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
authors.forename: William
authors.initials: W
authors.lastname: Dentler
authors.forename: Kristyn
authors.initials: K
authors.lastname: Vanderwaal
authors.forename: Mary E
authors.initials: ME
authors.lastname: Porter
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002696:Chlamydomonas / Q000166:cytology / Q000378:metabolism; D005407:Flagella / Q000378:metabolism / Q000648:ultrastructure; D008853:Microscopy / Q000295:instrumentation / Q000379:methods; D020409:Molecular Motor Proteins / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism*
chemical_list: D020409:Molecular Motor Proteins; D015800:Protozoan Proteins
references.citation: Annu Rev Genomics Hum Genet. 2006;7:125-48
references.pmid: 16722803
references.citation: Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5519-23
references.pmid: 8516294
references.citation: Mol Biol Cell. 2005 Mar;16(3):1341-54
references.pmid: 15616187
references.citation: Curr Biol. 2007 Feb 6;17(3):193-202
references.pmid: 17276912
references.citation: Methods Cell Biol. 1995;47:263-71
references.pmid: 7476498
references.citation: J Cell Biol. 2008 Jan 14;180(1):17-21
references.pmid: 18180369
references.citation: Eukaryot Cell. 2007 Aug;6(8):1411-20
references.pmid: 17573545
references.citation: Nat Cell Biol. 2004 Nov;6(11):1109-13
references.pmid: 15489852
references.citation: J Cell Biol. 2005 Aug 15;170(4):649-59
references.pmid: 16103230
references.citation: J Cell Biol. 2001 Apr 2;153(1):13-24
references.pmid: 11285270
references.citation: J Cell Biol. 1998 Dec 14;143(6):1591-601
references.pmid: 9852153
references.citation: J Cell Biol. 1995 Dec;131(6 Pt 1):1517-27
references.pmid: 8522608