Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45−/− mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Cilia are small microtubule-based structures found on the surface of most mammalian cells, which have key sensory and sometimes motile functions. Primary ciliary dyskinesia (PCD) is a type of ciliopathy caused by defects in motile cilia. The genetic basis of PCD is only partially understood. Studying a cohort of 11 human patients with PCD, we find thatde novomutations inTUBB4B, a beta tubulin isotype, cause three distinct classes of ciliopathic disease.In vivostudies in mice show thatTubb4bplays a specific role in cilia, building centrioles and axonemes in multiciliated cells. Examining the effects of specific TUBB4B variants in cells and in mice, we further demonstrate that distinctTUBB4Bmutations 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. Importantly, these molecular differences correlate with disease features. We show that tubulin heterodimer-impairing TUBB4B variants underlie nonsyndromic PCD, whilst additional renal and sensorineural ciliopathic features in a syndromic PCD subtype arise from microtubule lumenal interface-impaired TUBB4B variants. These findings suggest that specific tubulin isotypes have distinct and non-redundant subcellular functions, and demonstrate that human tubulinopathies can be drivers of ciliopathic syndromes.

Ciliopathies are clinically and genetically heterogeneous diseases. We studied three patients from two independent families presenting with features of Joubert syndrome: abnormal breathing pattern during infancy, developmental delay/intellectual disability, cerebellar ataxia, molar tooth sign on magnetic resonance imaging scans, and polydactyly. We identified biallelic loss-of-function (LOF)

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