Cilium-autonomous regulation of tubulin transport by intraflagellar transport in Chlamydomonas reinhardtii
Craft, Julie Melissa
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The microtubule-based axoneme is the defining characteristic of all eukaryotic flagella and cilia. Assembly of this structure during ciliogenesis requires a vast translocation of the microtubule subunit, the alpha/beta tubulin heterodimer, from the cytoplasm to the site of growth, the distal tip of cilia. Herein this work, we investigate tubulin transport in cilia, inlcuding the means of transport, its regulation, and timing. In Chlamydomonas reinhardtii, we express fluorescently tagged alpha- and beta-tubulins and observe via in vivo total internal reflection fluorescence (TIRF) microscopy. Both subunits are bona fide cargoes of intraflagellar transport (IFT), the devoted ciliary active transport system; tubulin also diffuses into the cilium. The protruding C-terminal E-hooks of alpha- and beta-tubulin are not essential for transport by IFT, but removal of beta-tubulin E-hook significantly reduced transport frequency suggesting that is does provide enhanced stability of the heterodimer-IFT particle interaction. The transport frequency of GFP-tubulin is strongly upregulated during ciliary growth and IFT trains carry more tubulin as cargo than during non-growth. Thus cells regulate the amount of tubulin cargo on IFT trains; the differential loading of IFT could explain this phenomenon. Further, cells possessing both growing and non-growing cilia selectively elevate IFT particles carrying tagged tubulin in only the actively growing cilia. We posit that cells regulate the amount of cargo loading; upon receipt of an incomplete growth status signal, the cells respond with an increase of cargo loading in a cilium-autonomous manner. Likewise, cells approximately double the intraciliary pool of soluble tubulin. We propose that IFT functions to increase the local soluble concentration of tubulin at the growing tip to facilitate timely axonemal elongation. Thus, IFT functions as a tubulin pump to enrich tubulin in cilia; high tubulin concentrations will promote microtubule extension, and inasmuch, ciliary growth.