An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier
| Autor: | Maxence V. Nachury, David K. Breslow, Andrew J. Spakowitz, Elena F. Koslover, Federica Seydel |
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| Rok vydání: | 2013 |
| Předmět: |
Cell Membrane Permeability
Time Factors Recombinant Fusion Proteins Biology Transfection Models Biological Time-Lapse Imaging Article Cell Line Diffusion Mice 03 medical and health sciences 0302 clinical medicine Animals Cilia Nuclear pore Ciliary membrane Research Articles 030304 developmental biology 0303 health sciences Microscopy Video Cilium Cell Membrane Proteins Reproducibility of Results Cell Biology Actin cytoskeleton Axon initial segment In vitro Cell biology Molecular Weight Actin Cytoskeleton Protein Transport Membrane Microscopy Fluorescence Membrane protein Nuclear Pore 030217 neurology & neurosurgery |
| Zdroj: | Breslow, DK; Koslover, EF; Seydel, F; Spakowitz, AJ; & Nachury, MV. (2013). An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier. JOURNAL OF CELL BIOLOGY, 203(1), 129-147. doi: 10.1083/jcb.201212024. UC San Diego: Retrieved from: http://www.escholarship.org/uc/item/2nz052f1 The Journal of Cell Biology |
| ISSN: | 1540-8140 0021-9525 |
| DOI: | 10.1083/jcb.201212024 |
| Popis: | The ciliary permeability barrier is mechanistically distinct from other cellular diffusion barriers and allows soluble proteins under ∼100 kD in size to enter cilia in the absence of active transport. 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. |
| Databáze: | OpenAIRE |
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