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In Vivo Imaging of IFT in Chlamydomonas Flagella

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In Vivo Imaging of IFT in Chlamydomonas Flagella

Karl F Lechtreck. Methods Enzymol.

Abstract

Intraflagellar transport (IFT) is a specialized intracellular transport which is required for the assembly and maintenance of cilia and eukaryotic flagella. IFT protein particles move bidirectionally along the flagella in the space between the flagellar membrane and the axonemal doublets. The particles consist of more than 20 different polypeptides and are transported by kinesin-2 from the cell body to the flagellar tip and by cytoplasmic dynein back to the cell body. Chlamydomonas reinhardtii is unique in that IFT can be visualized by two distinct microscopic approaches: differential interference contrast (DIC) and tracking of fluorescently tagged IFT proteins. In vivo imaging of IFT is critical to determine, for example, the role of individual proteins in the IFT pathway and how flagellar proteins are transported by IFT. Here, the microscopic requirements and the procedures for the imaging of IFT by DIC and by total internal reflection fluorescence microscopy will be described. Kymograms, graphical representations of spatial position over time, provide a convenient way to analyze in vivo recordings of IFT. In the future, multicolor in vivo imaging of IFT and its cargoes will be used to understand how flagella are assembled, maintained, and repaired.

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