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Methods Cell Biol. 2015;127:189-201. doi: 10.1016/bs.mcb.2015.01.002. Epub 2015 Mar 7.

Efficient live fluorescence imaging of intraflagellar transport in mammalian primary cilia.

Author information

1
Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.

Abstract

Intraflagellar transport (IFT) is a motile process critical for building most cilia, including those of mammalian cells. Defects in IFT lead to short or missing cilia, and in animals can cause defects in development, for example, in hedgehog-mediated signaling, as well as disease symptoms such as polycystic kidney disease or retinal degeneration. Understanding how IFT works is thus a high priority in ciliary biology. Imaging of living cells has played a key role in understanding the mechanism of IFT and this is particularly the case in mammalian cells where biochemical analysis of IFT is extremely difficult due to the difficulty of isolating cilia away from the rest of the cell. Imaging IFT in living mammalian cells requires solution to several problems: constructing cell lines that express fluorescent-protein-tagged IFT proteins, obtaining cell populations with a high degree of ciliation, confocal or TIRF imaging with sufficient time resolution and signal-to-noise ratio to observe the majority of IFT particles as they travel back and forth inside the cilium, and analyzing the image data to extract quantitative measurements of IFT. We describe optimized solutions to each of these technical challenges. Using the approaches described here, mammalian cultured cells become powerful platforms for quantitative analysis of IFT dynamics.

KEYWORDS:

IFT; Live imaging; Primary cilium; TIRF

PMID:
25837392
PMCID:
PMC5028138
DOI:
10.1016/bs.mcb.2015.01.002
[Indexed for MEDLINE]
Free PMC Article

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