Imaging of the Ciliary Cholesterol Underlying the Sonic Hedgehog Signal Transduction.

Abstract

Primary cilia are antenna-like structures that develop on the surface of quiescent G0-phase cells and receive extracellular signals including sonic hedgehog (Shh) for embryogenesis and adult tissue homeostasis. In mammalian cells, cholesterol activates the seven-transmembrane protein Smoothened to transduce the Shh signal. Germline mutations of the DHCR7 gene encoding the cholesterol biogenesis enzyme 7-dehydrocholesterol reductase cause Smith-Lemli-Opitz syndrome with ciliopathy-related symptoms such as polycystic kidney and polydactyly, implying that cholesterol is indeed involved in ciliary functions. Notably, it has been reported that the cholesterol in ciliary membranes is significantly more abundant than that in the rest of the plasma membrane. However, several studies have failed to image the enriched ciliary cholesterol. Here, we propose a set of protocols for the sensitive imaging of ciliary cholesterol using the fluorescent small compound Filipin III and the green fluorescent protein tagged Domain 4 of the exotoxin Perfringolysin O derived from the anaerobic bacterium Clostridium perfringens. These cholesterol probes should be powerful tools for understanding the physiological and pathological roles of ciliary cholesterol in the context of Shh signaling in mammalian cells.