Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling
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
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.
Reviewing Editor
- Duojia Pan, UT Southwestern Medical Center and HHMI, United States
Version history
- Received: July 12, 2019
- Accepted: October 25, 2019
- Accepted Manuscript published: October 28, 2019 (version 1)
- Accepted Manuscript updated: October 30, 2019 (version 2)
- Version of Record published: November 12, 2019 (version 3)
- Version of Record updated: November 15, 2019 (version 4)
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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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
- Neuroscience
Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.