Characterization of membrane structures regulating primary ciliogenesis by quantitative isotropic ultrastructure imaging

The trafficking, docking, and fusion of membrane vesicles at the mother centriole (MC) are important for primary cilium construction. Here, we determined the three-dimensional (3D) membrane ultrastructures, and associated proteins, involved in this primary cilium assembly mechanism upstream of axoneme growth. Our work suggests that the enlargement of small vesicles docked to the MC is a key trigger for ciliogenesis progression, a process requiring the MC distal appendage protein CEP164. These vesicles appear to fuse to form tubular C-shaped intermediates and an unprecedented toroidal membrane intermediate. The formation of these previously uncharacterized tubular membrane ciliogenesis intermediates is orchestrated by the membrane trafficking regulators EHD1 and RAB8, and is associated with the IFT-B complex protein IFT88. Remarkably, we show that EHD1, through its membrane tubulation function, regulates ciliogenesis progression by directly promoting CP110/CEP97 removal from the MC cap. The establishment of these tubular membrane structures is also associated with the recruitment of the ciliary gate transition zone proteins. Together, these findings redefine the architectural framework of early ciliogenesis and underscore the utility of isotropic ultrastructural imaging combined with quantitative 3D analysis for elucidating mechanisms of membrane trafficking and organelle biogenesis.