Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA.
Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
J Cell Sci. 2020 Sep 11;133(17):jcs249805. doi: 10.1242/jcs.249805.
Tubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using , we attenuated IFT-based tubulin transport of GFP-β-tubulin by altering the IFT74N-IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP-β-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP-β-tubulin in the axoneme was only moderately reduced. imaging showed more GFP-β-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.
微管蛋白通过扩散和基于动力的鞭毛内运输（IFT）进入纤毛。然而，每种途径在为轴丝组装提供微管蛋白方面的各自贡献仍然未知。使用 ，我们通过改变 IFT74N-IFT81N 微管蛋白结合模块和微管蛋白 C 末端 E 钩，削弱了 GFP-β-微管蛋白的基于 IFT 的运输。E 钩缺陷的 GFP-β-微管蛋白被纳入轴丝微管，但在对照细胞中，其 IFT 的运输频率降低了约 90%，而当 IFT81 的微管蛋白结合位点失活时，运输则基本被消除。尽管 IFT 强烈减少，但 E 钩缺陷的 GFP-β-微管蛋白在轴丝中的比例仅适度降低。 成像显示，通过扩散进入纤毛的 GFP-β-微管蛋白颗粒比通过 IFT 的多。外推到内源性微管蛋白，数据表明扩散提供了用于轴丝组装的大部分微管蛋白。我们提出，IFT 的微管蛋白仍然是纤毛发生所必需的，因为它通过扩散增加了供应给纤毛尖端的微管蛋白池，从而确保在快速纤毛生长过程中，那里的游离微管蛋白保持在正端微管组装的临界浓度。
