An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier

• DOI: 10.1083/jcb.201212024
• Publication Date: 2013-10-08T02:57:42Z
• Authors: David K. Breslow; Elena F. Koslover; Federica Seydel; Andrew J. Spakowitz; Maxence V. Nachury
• Keywords: 0301 basic medicine; Kidney Disease; Biomedical and clinical sciences; Cell Membrane Permeability; Time Factors; 1.1 Normal biological development and functioning; Recombinant Fusion Proteins; Transfection; Medical and Health Sciences; Models, Biological; Time-Lapse Imaging; Fluorescence; Cell Line; Diffusion; Mice; 03 medical and health sciences; Underpinning research; Models; Animals; Cilia; Research Articles; Microscopy; Microscopy, Video; Cell Membrane; Proteins; Reproducibility of Results; Video; Biological Sciences; Biological; Molecular Weight; Biological sciences; Actin Cytoskeleton; Protein Transport; Microscopy, Fluorescence; Nuclear Pore; Biochemistry and Cell Biology; Generic health relevance; Developmental Biology

Specific proteins are concentrated within primary cilia, whereas others remain excluded. To understand the mechanistic basis of entry into cilia, we developed an in vitro assay using cells in which the plasma membrane was permeabilized, but the ciliary membrane was left intact. Using a diffusion-to-capture system and quantitative analysis, we find that proteins >9 nm in diameter (∼100 kD) are restricted from entering cilia, and we confirm these findings in vivo. Interference with the nuclear pore complex (NPC) or the actin cytoskeleton in permeabilized cells demonstrated that the ciliary diffusion barrier is mechanistically distinct from those of the NPC or the axon initial segment. Moreover, applying a mass transport model to this system revealed diffusion coefficients for soluble and membrane proteins within cilia that are compatible with rapid exploration of the ciliary space in the absence of active transport. Our results indicate that large proteins require active transport for entry into cilia but not necessarily for movement inside cilia.