Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling
- Stanford University School of Medicine, United States
- Stanford University School of medicine, United States
- University of Texas Southwestern Medical Center, United States
- Washington University School of Medicine, United States
- University of Oxford, United Kingdom
Abstract
Previously we proposed that transmission of the Hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.
Data availability
All data generated or analyzed are included in Supplementary Files 1-5 in this manuscript.
Article and author information
Author details
Arun Radhakrishnan
Funding
National Institutes of Health (GM118082)
- Rajat Rohatgi
American Heart Association (14POST20370057)
- Ganesh V Pusapati
American Heart Association (19POST34380734)
- Jennifer H Kong
National Institutes of Health (GM13251801)
- Jennifer H Kong
Ford Foundation (Pre-doctoral Fellowship)
- Giovanni Luchetti
National Institutes of Health (GM106078)
- Rajat Rohatgi
National Institutes of Health (HL20948)
- Kristen A Johnson
- Jeffrey G McDonald
- Arun Radhakrishnan
Welch Foundation (I-1793)
- Kristen A Johnson
- Arun Radhakrishnan
Cancer Research UK (C20724/A14414)
- Christian Siebold
Cancer Research UK (C20724/A26752)
- Christian Siebold
European Research Council (647278)
- Christian Siebold
National Science Foundation (Pre-doctoral Fellowship)
- Maia Kinnebrew
National Science Foundation (Pre-doctoral Fellowship)
- Ellen Jean Iverson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Kinnebrew et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling
eLife 8:e50051.
https://doi.org/10.7554/eLife.50051
Further reading
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- Biochemistry and Chemical Biology
- Developmental Biology
Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.
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- Cell Biology
The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.
