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Chemically inducible diffusion trap at cilia reveals molecular sieve–like barrier

Abstract

Primary cilia function as specialized compartments for signal transduction. The stereotyped structure and signaling function of cilia inextricably depend on the selective segregation of molecules in cilia. However, the fundamental principles governing the access of soluble proteins to primary cilia remain unresolved. We developed a methodology termed 'chemically inducible diffusion trap at cilia' to visualize the diffusion process of a series of fluorescent proteins ranging in size from 3.2 nm to 7.9 nm into primary cilia. We found that the interior of the cilium was accessible to proteins as large as 7.9 nm. The kinetics of ciliary accumulation of this panel of proteins was exponentially limited by their Stokes radii. Quantitative modeling suggests that the diffusion barrier operates as a molecular sieve at the base of cilia. Our study presents a set of powerful, generally applicable tools for the quantitative monitoring of ciliary protein diffusion under both physiological and pathological conditions.

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Figure 1: Chemically inducible diffusion trap of soluble proteins inside primary cilia.
Figure 2: Exponential dependence of influx rate on size.
Figure 3: Localization of ciliary diffusion barriers.
Figure 4: Analysis of the diffusion barrier at primary cilia.

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Acknowledgements

We thank A. Seki (Stanford University) for the 5HT6 construct, S. Takeda (University of Yamanashi) for the GFP-IFT88 construct and M. Wolfgang (Johns Hopkins University) for β-Gal and luciferase constructs. We also thank D.N.R., T.K., H.I. and S.T. for helpful discussions. This study was supported in part by the US National Institutes of Health (the Baltimore Polycystic Kidney Disease Research and Clinical Core Center provided pilot funds GM092930 and P30 DK090868 to Takanari Inoue and R00CA129174 and R21NS074091 to R.R.) and other grants (Grant-in-Aid for Challenging Exploratory Research 23650197 to H.N. and Pew Foundation to R.R.).

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Contributions

Y.-C.L., S.C.P. and Takanari Inoue generated DNA constructs, and Y.-C.L., S.C.P. and J.J. performed cell biology experiments. B.L. analyzed data with R.R., P.N., A.L. and Takanari Inoue, and P.N. performed biochemical experiments with R.R. H.N. performed FRAP experiments with Takafumi Inoue, Y.-C.L., B.L. and R.R., and Takanari Inoue wrote the paper.

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Correspondence to Takanari Inoue.

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Lin, YC., Niewiadomski, P., Lin, B. et al. Chemically inducible diffusion trap at cilia reveals molecular sieve–like barrier. Nat Chem Biol 9, 437–443 (2013). https://doi.org/10.1038/nchembio.1252

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