Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules. Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules

Tubulin, one of the most abundant cytoskeletal building blocks, exhibits extensive isotype diversity in humans. Displaying high similarity, whether these distinct isotypes form cell-type and context specific microtubule structures is poorly understood. Studying a cohort of 11 patients with the motile ciliopathy primary ciliary dyskinesia as well as mouse mutants, we report mutations in the TUBB4B isotype specifically perturb centriole and cilium biogenesis. We demonstrate that distinct TUBB4B mutations differentially affect microtubule dynamics and cilia formation in a dominant negative manner. Finally, structure-function studies reveal that different TUBB4B mutations disrupt distinct tubulin interfaces allowing clear stratification of patients into three classes of ciliopathic disease. These findings illustrate that specific tubulin isotypes have unique and non-redundant subcellular functions and establishes the missing link between human tubulinopathies and ciliopathies. Overall design: To investigate whether the loss of TUBB4B protein was due to an effect at the RNA level and to see what effect this loss had on the overall trancriptional landscape of multi-ciliated cells, especially on other beat tubulin isotypes which could theoretically compensate for the loss we sequenced the whole transcriptomes of mouse tracheas from 5 WT, 4 HET and 3 Hom mice.

微管蛋白（tubulin）是丰度最高的细胞骨架构建模块之一，在人类中展现出广泛的同工型（isotype）多样性。尽管各类同工型序列相似性较高，但这些不同的同工型是否能够形成细胞类型特异性及环境特异性的微管结构，目前仍不甚明晰。我们以11名活动性纤毛病患者——原发性纤毛运动障碍（primary ciliary dyskinesia, PCD）患者——以及小鼠突变体为研究队列，首次报道TUBB4B同工型的突变会特异性干扰中心粒与纤毛的生物发生过程。我们证实，不同的TUBB4B突变会以显性负性效应的方式，对微管动力学及纤毛形成产生差异化影响。后续的结构-功能研究表明，不同的TUBB4B突变会通过破坏不同的微管蛋白互作界面，可将患者明确划分为三类纤毛病亚型。上述研究结果证明，特定的微管蛋白同工型拥有独特且不可替代的亚细胞功能，同时填补了人类微管蛋白病与纤毛病之间长期缺失的关联环节。整体实验设计：为探究TUBB4B蛋白的缺失是否由RNA水平的调控异常所导致，并明确该缺失对多纤毛细胞整体转录谱的影响——尤其是对理论上可代偿该缺失的其他β微管蛋白同工型的影响——我们对5只野生型（wild type, WT）、4只杂合型（heterozygous, HET）以及3只纯合型（homozygous, Hom）小鼠的气管组织进行了全转录组测序。