Primary cilia function as specialized compartments for signal transduction in many tissues. The stereotyped structure and signaling function of cilia are inextricably dependent on the selective segregation of molecules in cilia, demonstrated by the finding that damage to ciliary protein transport mechanisms lead to a family of human genetic diseases known as ciliopathies. Despite its importance, the fundamental principles governing the access of soluble proteins to primary cilia remain unresolved, primarily because it has not been possible to monitor the kinetics of protein movement into these organelles in unperturbed live cells. We have developed a methodology termed Chemically-Inducible Diffusion Trap at Cilia (C-IDTc) based on chemically-inducible protein dimerization and live-cell microscopy to visualize the diffusion process of a series of fluorescent proteins ranging in size from 32 to 79 � into primary cilia.  As a result, we found that the interior of the cilium was accessible to proteins as large as 79 � (~650 kDa). However, the kinetics of ciliary accumulation of this panel of proteins was exponentially limited by their Stokes radii. Quantitative modeling from our kinetic data suggests that the diffusion barrier operates as a molecular sieve at the base of cilia. In addition to elucidating key physical properties, our study presents a set of powerful, generally applicable tools for the quantitative monitoring of ciliary protein diffusion under both physiological and pathological conditions.

Chemically-inducible diffusion trap at cilia (C-IDTc) reveals molecular sieve-like barrier

246th National Meeting (2013)

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