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


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2016-12

AUTHORS

Ira Kukic, Felix Rivera-Molina, Derek Toomre

ABSTRACT

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. More... »

PAGES

23

References to SciGraph publications

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/s13630-016-0044-2

DOI

http://dx.doi.org/10.1186/s13630-016-0044-2

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1018799707

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/27493724


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