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 Chlamydomonas, 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. In vivo 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运动的鞭毛内运输 (IFT) 进入纤毛。然而，每种途径在为轴索组装提供微管蛋白方面的各自贡献仍然未知。使用衣藻，我们通过改变 IFT74N-IFT81N 微管蛋白结合模块和微管蛋白的 C 端 E 钩来减弱 GFP-β-微管蛋白的基于 IFT 的微管蛋白转运。E-hook 缺陷的 GFP-β-微管蛋白被整合到轴突微管中，但其通过 IFT 的转运频率在对照细胞中降低了约 90%，并且当 IFT81 的微管蛋白结合位点丧失能力时基本上被消除。尽管 IFT 大幅减少，但轴丝中 E-hook 缺陷的 GFP-β-微管蛋白的比例仅适度减少。体内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 多。外推到内源性微管蛋白，数据表明扩散提供了轴突组装所需的大部分微管蛋白。我们建议纤毛发生仍然需要微管蛋白的 IFT，因为它通过扩散增加了提供给纤毛尖端的微管蛋白池，从而确保在快速纤毛生长期间游离微管蛋白保持在正端微管组装的临界浓度。