Simultaneous Measurement of Smoothened Entry Into and Exit From the Primary Cilium

Ciliary accumulation of signaling proteins must result from a rate of ciliary entry that exceeds ciliary exit, but approaches for distinguishing ciliary entry vs. exit are lacking. Using a photoconvertible fluorescent protein tag, we establish an assay that allows a separate but simultaneous examination of ciliary entry and exit of the Hedgehog signaling protein Smoothened in individual cells. We show that KAAD-cyclopamine selectively blocks entry, whereas ciliobrevin interferes initially with exit and eventually with both entry and exit of ciliary Smoothened. Our study provides an approach to understanding regulation of ciliary entry vs. exit of Hedgehog signaling components as well as other ciliary proteins.

信号蛋白在纤毛内的聚集，必然源于其纤毛内流入速率高于流出速率，但目前尚缺乏可区分纤毛蛋白流入与流出的研究方法。本研究借助光转换荧光蛋白标签，构建了一种可在单个细胞中分别同时检测刺猬（Hedgehog）信号通路关键蛋白平滑蛋白（Smoothened）的纤毛内流入与流出过程的实验方法。实验结果表明，KAAD-环杷明（KAAD-cyclopamine）可选择性阻断纤毛内平滑蛋白的流入过程，而西利布雷文（ciliobrevin）最初仅干扰其流出过程，最终则同时阻断其流入与流出过程。本研究为解析刺猬信号通路组分乃至其他纤毛蛋白的纤毛内流入与流出调控机制提供了可行的研究策略。