Decision letter: Cholesterol accessibility at the ciliary membrane controls hedgehog signaling

The primary cilium has been found to be associated with a number of cellular signaling pathways, such as vertebrate hedgehog signaling, and implicated in the pathogenesis of diseases affecting multiple organs, including the neural tube, kidney, and brain. The primary cilium is the site where a subset of the cell's membrane proteins is enriched. However, pathways that target and concentrate membrane proteins in cilia are not well understood. Processes determining the level of proteins in the ciliary membrane include entry into the compartment, removal, and retention by diffusion barriers such as the transition zone. Proteins that are concentrated in the ciliary membrane are also localized to other cellular sites. Thus it is critical to determine the particular role for ciliary compartmentalization in sensory reception and signaling pathways. Here we provide a brief overview of our current understanding of compartmentalization of proteins in the ciliary membrane and the dynamics of trafficking into and out of the cilium. We also discuss major unanswered questions regarding the role that defects in ciliary compartmentalization might play in disease pathogenesis. Understanding the trafficking mechanisms that underlie the role of ciliary compartmentalization in signaling might provide unique approaches for intervention in progressive ciliopathies.

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Faculty Opinions recommendation of Cholesterol accessibility at the ciliary membrane controls hedgehog signaling.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736821530.793567431 ◽

2019 ◽

Author(s):

Robert Parton

Keyword(s):

Hedgehog Signaling ◽

Ciliary Membrane

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CCRK/CDK20 regulates ciliary retrograde protein trafficking via interacting with BROMI/TBC1D32

PLoS ONE ◽

10.1371/journal.pone.0258497 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258497

Author(s):

Tatsuro Noguchi ◽

Kentaro Nakamura ◽

Yuuki Satoda ◽

Yohei Katoh ◽

Kazuhisa Nakayama

Keyword(s):

Extracellular Vesicles ◽

Protein Trafficking ◽

Hedgehog Signaling ◽

Intraflagellar Transport ◽

Wild Type ◽

Ciliary Membrane ◽

Ciliary Protein ◽

Exogenous Expression ◽

Turnaround Process

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.

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Cholesterol accessibility at the ciliary membrane controls hedgehog signaling

eLife ◽

10.7554/elife.50051 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 16

Author(s):

Maia Kinnebrew ◽

Ellen J Iverson ◽

Bhaven B Patel ◽

Ganesh V Pusapati ◽

Jennifer H Kong ◽

...

Keyword(s):

Plasma Membrane ◽

Birth Defects ◽

Hedgehog Signaling ◽

Signal Transmission ◽

Membrane Cholesterol ◽

Signaling Proteins ◽

Signaling System ◽

Ciliary Membrane ◽

Patched 1 ◽

Hedgehog Signal

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.

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Author response: Cholesterol accessibility at the ciliary membrane controls hedgehog signaling

10.7554/elife.50051.025 ◽

2019 ◽

Cited By ~ 3

Author(s):

Maia Kinnebrew ◽

Ellen J Iverson ◽

Bhaven B Patel ◽

Ganesh V Pusapati ◽

Jennifer H Kong ◽

...

Keyword(s):

Hedgehog Signaling ◽

Author Response ◽

Ciliary Membrane

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Sphingomyelin suppresses Hedgehog signaling by restricting cholesterol accessibility at the ciliary membrane

10.1101/699819 ◽

2019 ◽

Author(s):

Maia Kinnebrew ◽

Ellen J. Iverson ◽

Bhaven B. Patel ◽

Ganesh V. Pusapati ◽

Jennifer H. Kong ◽

...

Keyword(s):

Hedgehog Signaling ◽

Direct Interaction ◽

Subcellular Location ◽

Signaling System ◽

Ciliary Membrane ◽

Cellular Processes ◽

Cholesterol Levels ◽

Collateral Effects ◽

Patched 1 ◽

Hedgehog Signal

AbstractTransmission of the Hedgehog signal across the plasma membrane by Smoothened is proposed to be triggered by its direct interaction with cholesterol. 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 here that Smoothened activation and Hedgehog signaling are driven by a biochemically defined fraction of membrane cholesterol, termed accessible cholesterol. Increasing accessible cholesterol levels by depletion of sphingomyelin, which sequesters cholesterol in complexes, potentiates Hedgehog signaling. By inactivating the transporter-like protein Patched 1, Hedgehog ligands trigger an increase in cholesterol accessibility in the ciliary membrane, the subcellular location for Smoothened signaling. Thus, compartmentalization of Hedgehog signaling in the primary cilium may allow cholesterol accessibility to be used as a second messenger to mediate the communication between Patched 1 and Smoothened, without causing collateral effects on other cellular processes.

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Role of the Cilia Membrane in Control of Microtubule Sliding

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002683 ◽

1980 ◽

Vol 38 ◽

pp. 612-615

Author(s):

Edna S. Kaneshiro

Keyword(s):

Control Mechanism ◽

Beat Frequency ◽

Surface Membrane ◽

Ciliary Membrane ◽

Ciliary Beating ◽

Ciliary Reversal ◽

Voltage Dependent ◽

Reversal Mechanism ◽

And Function

It is currently believed that ciliary beating results from microtubule sliding which is restricted in regions to cause bending. Cilia beat can be modified to bring about changes in beat frequency, cessation of beat and reversal in beat direction. In ciliated protozoans these modifications which determine swimming behavior have been shown to be related to intracellular (intraciliary) Ca2+ concentrations. The Ca2+ levels are in turn governed by the surface ciliary membrane which exhibits increased Ca2+ conductance (permeability) in response to depolarization. Mutants with altered behaviors have been isolated. Pawn mutants fail to exhibit reversal of the effective stroke of ciliary beat and therefore cannot swim backward. They lack the increased inward Ca2+ current in response to depolarizing stimuli. Both normal and pawn Paramecium made leaky to Ca2+ by Triton extrac¬tion of the surface membrane exhibit backward swimming only in reactivating solutions containing greater than IO-6 M Ca2+ Thus in pawns the ciliary reversal mechanism itself is left operational and only the control mechanism at the membrane is affected. The topographic location of voltage-dependent Ca2+ channels has been identified as a component of the ciliary mem¬brane since the inward Ca2+ conductance response is eliminated by deciliation and the return of the response occurs during cilia regeneration. Since the ciliary membrane has been impli¬cated in the control of Ca2+ levels in the cilium and therefore is the site of at least one kind of control of microtubule sliding, we have focused our attention on understanding the structure and function of the membrane.

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