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

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.