Sterols regulate ciliary membrane dynamics and hedgehog signaling in health and disease

The primary cilium is a specialized signaling hub whose function depends on a tightly regulated membrane composition. While its protein content is well-characterized, its lipid identity, particularly regarding sterols, remains poorly defined. Here, we used mass spectrometry-based lipidomics to map the sterol profile of isolated primary cilia from MDCK cells. We found that ciliary membranes are enriched in cholesterol and desmosterol while excluding precursors like 7-lathosterol and limiting others, suggesting a selective sterol barrier. Inhibiting cholesterol biosynthesis at distinct enzymatic steps led to sterol accumulation, altered ciliary membrane fluidity, and impaired Hedgehog signaling, including defective Smoothened (Smo) retention— even in the presence of a constitutively active form of Smo. These findings link sterol homeostasis to ciliary membrane properties and signaling fidelity. Our work provides a molecular framework for understanding Hedgehog-related phenotypes in disorders like Smith-Lemli-Opitz Syndrome, highlighting the importance of membrane lipid composition in developmental signaling.