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 required to construct the primary cilium. Here, we determined the three-dimensional (3D) membrane ultrastructures, and associated proteins, involved in primary cilium assembly upstream of axoneme growth. Our work reveals 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. We show these vesicles subsequently fuse to form tubular C-shaped and an unprecedented toroidal membrane intermediates, which ultimately organize into the ciliary vesicle covering the MC distal end. The formation of these previously uncharacterized tubular membrane ciliogenesis intermediates is orchestrated by the membrane trafficking regulators EHD1 and RAB8, and requires 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. This study changes the architectural framework for understanding ciliogenesis mechanisms and highlights the application of isotropic ultrastructure imaging and three-dimensional quantitative analysis in understanding membrane trafficking and organelle biogenesis mechanisms.