Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier.

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 (C-IDTc) to visualize the diffusion process of a series of fluorescent proteins ranging in size from 3.2 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.

初级纤毛作为信号转导的特殊区域发挥作用。纤毛的定型结构和信号传导功能不可分割地依赖于纤毛中分子的选择性分离。然而，控制可溶性蛋白质进入初级纤毛的基本原理仍未解决。我们开发了一种称为“纤毛化学诱导扩散陷阱”（C - IDTc）的方法，以观察一系列大小从3.2纳米到7.9纳米的荧光蛋白扩散进入初级纤毛的过程。我们发现，纤毛内部可允许大至7.9纳米的蛋白质进入。这组蛋白质在纤毛中积累的动力学受到其斯托克斯半径呈指数限制。定量建模表明，扩散屏障在纤毛基部作为分子筛起作用。我们的研究提供了一套强大的、普遍适用的工具，用于在生理和病理条件下对纤毛蛋白扩散进行定量监测。