Dataset Information

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

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.

SUBMITTER: Ishikawa H

PROVIDER: S-EPMC5028138 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Ishikawa Hiroaki H Marshall Wallace F WF

Methods in cell biology 20150307

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 ...[more]

PMID: 25837392

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Project description:Study Smoking and COPD are associated with decreased mucociliary clearance and healthy smokers have shorter cilia in the large airway than nonsmokers. Intraflagellar transport (IFT) is the process by which cilia are produced and maintained. We assessed expression of IFT-related genes in smokers and nonsmokers and evaluated cilia length in the large and small airway of nonsmokers, healthy smokers, and smokers with COPD. Methods Airway epithelium was obtained via bronchoscopic brushing. Affymetrix microarrays were used to evaluate IFT gene expression in 2 independent data sets from large and small airway. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject. Results All 40 IFT genes were expressed in the human large and small airway epithelium. In the large airway, 10 IFT genes were down-regulated and 1 up-regulated in smokers. In the small airway, 11 genes were down-regulated and 3 up-regulated in smokers. A set of 8 IFT genes was down-regulated in both data sets. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. Answer to the Question These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies. Gene expression was assessed for 40 intraflagellar transport related genes in the LAE of nonsmokers (n=21) and healthy smokers (n=31) and the SAE of an independent group of nonsmokers (n=28) and healthy smokers (n=69). Cilia length was assessed in a total of 228 airway epithelium samples, including 120 LAE samples and 108 SAE samples; a subset of the 228 samples is represented among the 149 samples in this Series.

Dataset Information

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

Publications

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

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