Screening the composition of tubulin isotypes reveals the most abundant TUBB4B for ciliary polarity in ependymal cells

Cilia are microtubule-based organelles that protrude from the surface of various eukaryotic cell types. Microtubules are assembled by α/β-tubulin heterodimers, all of which comprise multiple isotypes encoded by distinct genes. However, the composition and function of tubulin isotypes in cilia are largely unclear. Here, we successfully labeled the endogenous α-tubulin and β-tubulin isotypes with HA or GFP tag in cultured mouse ependymal cells (EPCs) via the CRISPR/Cas9 system. TUBA1A, TUBA1B, TUBA1C, TUBB2A, TUBB2B, TUBB4B, and TUBB5 were identified to be incorporated in ependymal cilia, with TUBB4B showing the highest expression. Overexpression assay revealed that the ependymal cilia did not display a preference for the entrance of specific tubulin isotypes. Furthermore, luciferase reporter assay showed that the expression of TUBB4B in EPCs was specifically regulated by the ciliogenesis factor FOXJ1. TUBB4B deficiency disrupted planar polarity of EPCs and impaired cerebrospinal fluid flow, resulting in hydrocephalus. This study reveals the composition of tubulin isotypes in ependymal cilia and the specific role of FOXJ1-promoted TUBB4B in ciliary motility.