1. Cell Biology
  2. Developmental Biology

Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling

  1. Maia Kinnebrew
  2. Ellen Jean Iverson
  3. Bhaven B Patel
  4. Ganesh V Pusapati
  5. Jennifer H Kong
  6. Kristen A Johnson
  7. Giovanni Luchetti
  8. Kaitlyn M Eckert
  9. Jeffrey G McDonald
  10. Douglas F Covey
  11. Christian Siebold
  12. Arun Radhakrishnan  Is a corresponding author
  13. Rajat Rohatgi  Is a corresponding author
  1. Stanford University School of Medicine, United States
  2. Stanford University School of medicine, United States
  3. University of Texas Southwestern Medical Center, United States
  4. Washington University School of Medicine, United States
  5. University of Oxford, United Kingdom
https://doi.org/10.7554/eLife.50051
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  1. Maia Kinnebrew
  2. Ellen Jean Iverson
  3. Bhaven B Patel
  4. Ganesh V Pusapati
  5. Jennifer H Kong
  6. Kristen A Johnson
  7. Giovanni Luchetti
  8. Kaitlyn M Eckert
  9. Jeffrey G McDonald
  10. Douglas F Covey
  11. Christian Siebold
  12. Arun Radhakrishnan
  13. Rajat Rohatgi
(2019)
Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling
eLife 8:e50051.
https://doi.org/10.7554/eLife.50051
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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

  1. Maia Kinnebrew

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7344-8231

  2. Ellen Jean Iverson

    Department of Biochemistry, Stanford University School of medicine, Stanford, United States
    Competing interests
    No competing interests declared.

  3. Bhaven B Patel

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.

  4. Ganesh V Pusapati

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.

  5. Jennifer H Kong

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.

  6. Kristen A Johnson

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.

  7. Giovanni Luchetti

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.

  8. Kaitlyn M Eckert

    Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.

  9. Jeffrey G McDonald

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.

  10. Douglas F Covey

    Department of Developmental Biology, Washington University School of Medicine, St Louis, United States
    Competing interests
    No competing interests declared.

  11. Christian Siebold

    Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6635-3621

  12. Arun Radhakrishnan

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    arun.radhakrishnan@utsouthwestern.edu
    Competing interests
    Arun Radhakrishnan, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7266-7336

  13. Rajat Rohatgi

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    For correspondence
    rrohatgi@stanford.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7609-8858

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

  1. Duojia Pan, UT Southwestern Medical Center and HHMI, United States

Publication history

  1. Received: July 12, 2019
  2. Accepted: October 25, 2019
  3. Accepted Manuscript published: October 28, 2019 (version 1)
  4. Accepted Manuscript updated: October 30, 2019 (version 2)
  5. Version of Record published: November 12, 2019 (version 3)
  6. 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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  1. Maia Kinnebrew
  2. Ellen Jean Iverson
  3. Bhaven B Patel
  4. Ganesh V Pusapati
  5. Jennifer H Kong
  6. Kristen A Johnson
  7. Giovanni Luchetti
  8. Kaitlyn M Eckert
  9. Jeffrey G McDonald
  10. Douglas F Covey
  11. Christian Siebold
  12. Arun Radhakrishnan
  13. Rajat Rohatgi
(2019)
Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling
eLife 8:e50051.
https://doi.org/10.7554/eLife.50051

Categories and tags

Further reading

    1. Cell Biology
    2. Developmental Biology

    Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes

    Maia Kinnebrew et al.
    Research Advance Updated

    A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.

    1. Biochemistry and Chemical Biology
    2. Developmental Biology

    Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

    Giovanni Luchetti et al.
    Research Article Updated

    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.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in Caenorhabditis elegans

    Haikel Dridi et al.
    Research Article Updated

    Age-dependent loss of body wall muscle function and impaired locomotion occur within 2 weeks in Caenorhabditis elegans (C. elegans); however, the underlying mechanism has not been fully elucidated. In humans, age-dependent loss of muscle function occurs at about 80 years of age and has been linked to dysfunction of ryanodine receptor (RyR)/intracellular calcium (Ca2+) release channels on the sarcoplasmic reticulum (SR). Mammalian skeletal muscle RyR1 channels undergo age-related remodeling due to oxidative overload, leading to loss of the stabilizing subunit calstabin1 (FKBP12) from the channel macromolecular complex. This destabilizes the closed state of the channel resulting in intracellular Ca2+ leak, reduced muscle function, and impaired exercise capacity. We now show that the C. elegans RyR homolog, UNC-68, exhibits a remarkable degree of evolutionary conservation with mammalian RyR channels and similar age-dependent dysfunction. Like RyR1 in mammals, UNC-68 encodes a protein that comprises a macromolecular complex which includes the calstabin1 homolog FKB-2 and is immunoreactive with antibodies raised against the RyR1 complex. Furthermore, as in aged mammals, UNC-68 is oxidized and depleted of FKB-2 in an age-dependent manner, resulting in ‘leaky’ channels, depleted SR Ca2+ stores, reduced body wall muscle Ca2+ transients, and age-dependent muscle weakness. FKB-2 (ok3007)-deficient worms exhibit reduced exercise capacity. Pharmacologically induced oxidization of UNC-68 and depletion of FKB-2 from the channel independently caused reduced body wall muscle Ca2+ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca2+ transients and function. Taken together, these data suggest that UNC-68 oxidation plays a role in age-dependent loss of muscle function. Remarkably, this age-dependent loss of muscle function induced by oxidative overload, which takes ~2 years in mice and ~80 years in humans, occurs in less than 2–3 weeks in C. elegans, suggesting that reduced antioxidant capacity may contribute to the differences in lifespan among species.