Ciliogenesis is a multi-step process, including centriole/basal body assembly, maturation and docking to the cell surface where it templates the growth of the cilium. The structural junction between the basal body and the axoneme (transition zone) acts as a filter between the cellular and the ciliary compartment and houses many proteins involved in human ciliopathies. Two transition zone modules (MKS and NPHP modules) are required for cilium formation/function that act together with the Cep290 as a ciliary gate. The transmembrane protein MKS2/TMEM216 protein is one of the MKS module. In order to get a better understanding of the function of MKS2/TMEM216 in motile cilia formation, we turned to Paramecium, which is covered with thousands of motile cilia.
Three Paramecium genes encoding three expressed MKS2 proteins were identified in the Paramecium genome as a result of whole genome duplication. These three proteins are highly similar. Using GFP-tagged MKS2 expression we demonstrated, using confocal microscopy and electron microscopy, that MKS2 is localized specifically at the transition zone of ciliated basal bodies on Triton-X100 membrane remnants at the level of the axosomal plate, most probably corresponding to the ciliary necklace. To get an understanding of the MKS2 function, we performed two kinds of experiments: knock-down of the three MKS2 genes by bacterial feeding and expression of one Paramecium gene carrying one of the two human mutations (R 73L and L114R) causing respectively Joubert and Meckel syndromes. RNAi knocked-down cells showed the presence of cilia and, unlike control cells, all basal bodies were ciliated. This result suggests a deregulation of the ciliogenesis after knocking-down MKS2. Ultrastructural analyses of MKS2-deficient cells revealed an excess of membrane protrusions and an accumulation of vesicles, suggesting defective membrane trafficking in these cells. We demonstrate that introduction of the human mutations in one Paramecium gene leads to mislocalization of the expressed protein.
Altogether, the Paramecium model allowed us to localize precisely MKS2 in the transition zone. We demonstrate its involvement in the regulation of ciliogenesis. The mislocalization of the human mutated protein opens the way to understand the mechanisms leading to ciliogenesis defects in the human pathology.