Imaging intraflagellar transport in mammalian primary cilia.
The primary cilium is a specialized organelle that projects from the surface of many cell types. Unlike its motile counterpart it cannot beat but does transduce extracellular stimuli into intracellular signals and acts as a specialized subcellular compartment. The cilium is built and maintained by the transport of proteins and other biomolecules into and out of this compartment. The trafficking machinery for the cilium is referred to as IFT or intraflagellar transport. It was originally identified in the green algae Chlamydomonas and has been discovered throughout the evolutionary tree. The IFT machinery is widely conserved and acts to establish, maintain, and disassemble cilia and flagella. Understanding the role of IFT in cilium signaling and regulation requires a methodology for observing it directly. Here we describe current methods for observing the IFT process in mammalian primary cilia through the generation of fluorescent protein fusions and their expression in ciliated cell lines. The observation protocol uses high-resolution time-lapse microscopy to provide detailed quantitative measurements of IFT particle velocities in wild-type cells or in the context of genetic or other perturbations. Direct observation of IFT trafficking will provide a unique tool to dissect the processes that govern cilium regulation and signaling.
Besschetnova
Tatiana Y
TY
Renal Division, Department of Medicine, Brigham and Women's Hospital and Department of SystemsBiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Roy
Barnali
B
Shah
Jagesh V
JV
D000818:Animals; D001692:Biological Transport / Q000502:physiology*; D002460:Cell Line; D002923:Cilia / Q000378:metabolism / Q000648:ultrastructure; D005407:Flagella / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D008322:Mammals; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D011993:Recombinant Fusion Proteins
Mol Biol Cell. 2006 Sep;17(9):3781-92
16775004
Nat Genet. 2008 Apr;40(4):403-410
18327258
Mol Biol Cell. 2005 Jan;16(1):270-8
15496456
Cell. 2009 Apr 3;137(1):32-45
19345185
J Cell Biol. 2005 Aug 15;170(4):649-59
16103230
Mol Biol Cell. 2008 Apr;19(4):1450-61
18199688
J Cell Biol. 2001 Oct 29;155(3):405-14
11684707
J Cell Biol. 2000 Oct 30;151(3):709-18
11062270
J Cell Biol. 1995 Dec;131(6 Pt 1):1517-27
8522608
J Cell Biol. 1969 May;41(2):600-19
5783876
Cell. 2004 May 14;117(4):527-39
15137945
Nature. 1999 Apr 22;398(6729):674
10227290
Semin Cell Dev Biol. 2008 Dec;19(6):494-501
18692148
Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5519-23
8516294
Mol Biol Cell. 2008 Mar;19(3):929-44
18094047
Nat Methods. 2005 Dec;2(12):905-9
16299475
Cell. 2007 Jun 15;129(6):1201-13
17574030
Curr Top Dev Biol. 2008;85:23-61
19147001
Mol Biol Cell. 2005 Mar;16(3):1341-54
15616187
J Cell Biol. 1998 May 18;141(4):993-1008
9585417
Science. 1994 Feb 11;263(5148):802-5
8303295
J Cell Sci. 2006 Oct 1;119(Pt 19):3935-43
16954145
Dev Biol. 2004 Oct 1;274(1):56-69
15355788
Nucleic Acids Res. 1990 Jun 25;18(12):3587-96
2194165