Fig. 2: TIRF microscopy coupled with targeted cilia demembranation to reconstitute IFT train motility ex vivo. | Nature Communications

Fig. 2: TIRF microscopy coupled with targeted cilia demembranation to reconstitute IFT train motility ex vivo.

From: Tubulin tyrosination/detyrosination regulate the affinity and sorting of intraflagellar transport trains on axonemal microtubule doublets

Fig. 2

A Illustration of the reconstitution setup. Chlamydomonas cells are overlaid on synthetically polymerized microtubules adhered to a coverslip in a droplet of motility buffer on a TIRF microscope. A detergent back-filled capillary micropipette is immersed in the buffer from the top and centred using a three-axis micromanipulator. B Legend showing components of IFT trains (Top), and illustrative representations of an anterograde (Middle) and retrograde train (Bottom). C Illustration of relative positions of microtubules, cells, and capillary pipette. D Illustration of in vivo IFT train motility (Left) and ex vivo IFT train motility on polarity-marked microtubules (Right). â‘  Detergent shot from capillary pipette solubilizes ciliary membrane. â‘¡ IFT trains are released into the surrounding environment. Some motile trains continue to move (black arrow) on the demembranated cilia (parent axoneme), while â‘¢ others land and move (red arrows) on the polarity-marked microtubules (see also Supplementary Movie 3).

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