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.