The IN/OUT assay: a new tool to study ciliogenesis

Ira Kukic, Felix Rivera-Molina, Derek Toomre

Nearly all cells have a primary cilia on their surface, which functions as a cellular antennae. Primary cilia assembly begins intracellularly and eventually emerges extracellularly. However, current ciliogenesis assays, which detect cilia length and number, do not monitor ciliary stages. We developed a new assay that detects antibody access to a fluorescently tagged ciliary transmembrane protein, which revealed three ciliary states: classified as 'inside,' 'outside,' or 'partial' cilia. Strikingly, most cilia in RPE cells only partially emerged and many others were long and intracellular, which would be indistinguishable by conventional assays. Importantly, these states switch with starvation-induced ciliogenesis and the cilia can emerge both on the dorsal and ventral surface of the cell. Our assay further allows new molecular and functional studies of the 'ciliary pocket,' a deep plasma membrane invagination whose function is unclear. Molecularly, we show colocalization of EHD1, Septin 9 and glutamylated tubulin with the ciliary pocket. Together, the IN/OUT assay is not only a new tool for easy and quantifiable visualization of different ciliary stages, but also allows molecular characterization of intermediate ciliary states.