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The IN/OUT assay: a new tool to study ciliogenesis
Ira Kukic1, Felix Rivera-Molina1, Derek Toomre1
1Department of Cell Biology, Yale School of Medicine, New Haven, CT 06510 USA.
Cilia
|August 6, 2016
Summary
A new assay reveals distinct ciliary states, showing most primary cilia are partially or fully intracellular. This tool enables better study of cilia assembly and function.
Area of Science:
- Cell Biology
- Molecular Biology
Background:
- Primary cilia function as cellular antennae, crucial for cell signaling.
- Cilia assembly involves intracellular initiation and extracellular emergence, but current assays lack stage monitoring.
Purpose of the Study:
- To develop a novel assay for visualizing and quantifying distinct primary cilia assembly stages.
- To characterize intermediate ciliary states and their molecular components.
Main Methods:
- Developed a new assay detecting antibody access to a fluorescently tagged ciliary transmembrane protein.
- Classified cilia into three states: 'inside,' 'outside,' or 'partial' based on antibody accessibility.
Main Results:
- Most primary cilia in RPE cells were found to be partially or fully intracellular, undetectable by conventional methods.
- Ciliary states dynamically change during starvation-induced ciliogenesis.
- Identified colocalization of EHD1, Septin 9, and glutamylated tubulin within the ciliary pocket.
Conclusions:
- The IN/OUT assay provides a quantifiable method to visualize diverse ciliary stages.
- This assay facilitates molecular and functional studies of the ciliary pocket and intermediate ciliary states.
