In vivo Imaging of IFT in Chlamydomonas Flagella

Lechtreck, Karl‐Ferdinand

doi:10.1016/b978-0-12-397945-2.00015-9

Cited by 33 publications

(40 citation statements)

References 44 publications

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“…Flagella attached to the coverslip were partially photobleached by using a 491-nm epifluorescence beam of ∼3.3 nm diameter. Images were recorded at 20 images/s for 25 s ( photobleach occurred 5 s after the onset of the recording) and analyzed by ImageJ as described in Lechtreck (2013). To increase the contrast by ImageJ, 'Brightness/Contrast' of all movies was adjusted identically; the value of 'Maximum' was decreased from 255 to 185.…”

Section: Tirf Microscopy Of Fluorescently Tagged α-Tubulinmentioning

confidence: 99%

Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin

Kubo

Brown

Bellvé

et al. 2016

Journal of Cell Science

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102

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The assembly and maintenance of most cilia and flagella rely on intraflagellar transport (IFT). Recent in vitro studies have suggested that, together, the calponin-homology domain within the IFT81 N-terminus and the highly basic N-terminus of IFT74 form a module for IFT of tubulin. By using Chlamydomonas mutants for IFT81 and IFT74, we tested this hypothesis in vivo. Modification of the predicted tubulin-binding residues in IFT81 did not significantly affect basic anterograde IFT and length of steady-state flagella but slowed down flagellar regeneration, a phenotype similar to that seen in a strain that lacks the IFT74 N-terminus. In both mutants, the frequency of tubulin transport by IFT was greatly reduced. A double mutant that combined the modifications to IFT81 and IFT74 was able to form only very short flagella. These results indicate that, together, the IFT81 and IFT74 N-termini are crucial for flagellar assembly, and are likely to function as the main module for IFT of tubulin.

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Section: Tirf Microscopy Of Fluorescently Tagged α-Tubulinmentioning

confidence: 99%

Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin

Kubo

Brown

Bellvé

et al. 2016

Journal of Cell Science

Self Cite

102

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“…Visualization and recording of IFT were performed according to published protocols Lechtreck, 2013Lechtreck, , 2016. Videos were recorded using a Nikon Eclipse Ti total internal reflection fluorescence microscope.…”

Section: Ift Measurementsmentioning

confidence: 99%

Intraflagellar transport protein IFT52 recruits IFT46 to the basal body and flagella

Wan

Täschner

et al. 2017

Journal of Cell Science

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Cilia are microtubule-based organelles and perform motile, sensing and signaling functions. The assembly and maintenance of cilia depend on intraflagellar transport (IFT). Besides ciliary localization, most IFT proteins accumulate at basal bodies. However, little is known about the molecular mechanism of basal body targeting of IFT proteins. We first identified the possible basal body-targeting sequence in IFT46 by expressing IFT46 truncation constructs in an ift46-1 mutant. The C-terminal sequence between residues 246-321, termed BBTS3, was sufficient to target YFP to basal bodies in the ift46-1 strain. Interestingly, BBTS3 is also responsible for the ciliary targeting of IFT46. BBTS3::YFP moves bidirectionally in flagella and interacts with other IFT complex B (IFT-B) proteins. Using IFT and motor mutants, we show that the basal body localization of IFT46 depends on IFT52, but not on IFT81, IFT88, IFT122, FLA10 or DHC1b. IFT52 interacts with IFT46 through residues L285 and L286 of IFT46 and recruits it to basal bodies. Ectopic expression of the C-terminal domain of IFT52 in the nucleus resulted in accumulation of IFT46 in nuclei. These data suggest that IFT52 and IFT46 can preassemble as a complex in the cytoplasm, which is then targeted to basal bodies.

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“…TIRF illumination typically excites fluorophores in a ~30–300 nm region which is well below the optical section thickness of most confocal fluorescence microscopes; signal intensity decreases exponentially with distance from the cover glass effectively eliminating fluorescence from the cell body. We use a home-build through-the-objective TIRF system, which has been previously described in detail [3]; commercially available TIRF systems will work just as well.

Place the chamber onto the microscope with the large 24×60 mm coverslip facing the objective; center the spot of adhered cells into the middle of the objective.

Using the microscope’s standard light source focus onto the plane in which the flagella are attached to the cover glass.

…”

Section: Methodsmentioning

confidence: 99%

“…TIRF imaging has been applied to study protein transport in cilia of various systems including Tetrahymena thermophila and mammalian cells [1,2]. The flagella of Chlamydomonas reinhardtii are excellently suited to study protein transport by TIRFM because the cells naturally adhere via their two 12-µm long flagella to smooth surfaces such as cover glass [3]. This which will immobilize the cells and the flagella will be positioned within the TIRF excitation range while the cell bodies remain out of reach.…”

Section: Introductionmentioning

confidence: 99%

Methods for Studying Movement of Molecules Within Cilia

Lechtreck

2016

Methods in Molecular Biology

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Summary The assembly of cilia and eukaryotic flagella (interchangeable terms) requires the import of numerous proteins from the cell body into the growing organelle. Proteins move into and inside cilia by diffusion and by motor-based intraflagellar transport (IFT). Many aspects of ciliary protein transport such as the distribution of unloading sites and the frequency of transport can be analyzed using direct in vivo imaging of fluorescently tagged proteins. Here, we will describe how to use total internal reflection fluorescence microcopy (TIRFM) to analyze protein transport in flagella using in the unicellular alga Chlamydomonas reinhardtii, a widely used model for cilia and cilia-related disease.

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In vivo Imaging of IFT in Chlamydomonas Flagella

Cited by 33 publications

References 44 publications

Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin

Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin

Intraflagellar transport protein IFT52 recruits IFT46 to the basal body and flagella

Methods for Studying Movement of Molecules Within Cilia

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