Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways

Geneva, Ivayla; Tan, Han; Calvert, Peter D.

doi:10.1091/mbc.e16-07-0549

Cited by 15 publications

(27 citation statements)

References 35 publications

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“…A unique feature of mouse and rat SST 3 is selective targeting to primary neuronal cilia ( Händel et al, 1999 ) dependent on the presence of a conserved ciliary targeting motif within the third ICL ( 243 APSCQWVQAPACQ 255 ). This sequence is identical in mouse and rat and contains a dual (AX[A/S]XQ) ciliary targeting motif found in GPCRs efficiently localized to cilia ( Berbari et al, 2008a ; Jin et al, 2010 ; Geneva et al, 2017 ). In contrast, the third ICL of the human SST 3 contains only a single (AX[A/S]XQ) motif.…”

Section: Somatostatin Receptormentioning

confidence: 99%

International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature

et al. 2018

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Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein–coupled receptors (GPCRs) called somatostatin receptor (SST)1–5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST2 and SST5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature.

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Section: Somatostatin Receptormentioning

confidence: 99%

International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature

et al. 2018

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“…1B); this difference could be due to there being less RHO in the cilium or more RHO in the plasma membrane. It has been calculated that a cilium, representing a cylindrical shape lying on an apical membrane, should have 2.3 times as much signal from a given membrane protein as the adjacent membrane if the two regions contain the same concentration of the membrane protein (Geneva et al, 2017). However, this calculation assumes the apical membrane to be a planar surface, whereas, in polarized epithelial cells, the apical membrane consists of an array of microvilli.…”

Section: Rho In Cilia Of Imcd3 Cellsmentioning

confidence: 99%

The route of the visual receptor rhodopsin along the cilium

Chadha

Volland

Baliaouri

et al. 2019

Journal of Cell Science

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The photoreceptor outer segment is the most elaborate primary cilium, containing large amounts of rhodopsin (RHO) in disk membranes that grow from a connecting cilium. The movement of RHO along the connecting cilium precedes formation of the disk membranes. However, the route that RHO takes has not been clearly determined; some reports suggest that it follows an intracellular, vesicular route along the axoneme, possibly as an adaptation for the high load of delivery or the morphogenesis of the disk endomembranes. We addressed this question by studying RHO in cilia of IMCD3 cells and mouse rod photoreceptors. In IMCD3 cilia, fluorescence recovery after photobleaching (FRAP) experiments with fluorescently tagged RHO supported the idea of RHO motility in the ciliary plasma membrane and was inconsistent with the hypothesis of RHO motility within the lumen of the cilium. In rod photoreceptors, FRAP of RHO-EGFP was altered by externally applied lectin, supporting the idea of plasmalemmal RHO dynamics. Quantitative immunoelectron microscopy corroborated our live-cell conclusions, as RHO was found to be distributed along the plasma membrane of the connecting cilium, with negligible labeling within the axoneme. Taken together, the present findings demonstrate RHO trafficking entirely via the ciliary plasma membrane. This article has an associated First Person interview with the first author of the paper.

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“…For each cilium imaged, we calculated the ratio of mean ciliary fluorescence to mean plasma membrane fluorescence in a region surrounding the cilium (hereafter the "C/P ratio") (Geneva, Tan, and Calvert 2017) . We used this metric because myriocin treatment or cholesterol loading will lead to changes in probe staining at both the plasma membrane and the ciliary membrane.…”

Section: The Ciliary Membrane Is a Compartment With Low Cholesterol Amentioning

confidence: 99%

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

Kinnebrew

Iverson

Patel

et al. 2019

Preprint

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Transmission 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. We thank Kyle Travaglini and Onn Brandman for help with the MATLAB code for automated quantitation of probe fluorescence at primary cilia, Xiaohui Zha and Kevin Courtney for helpful discussions and protocols for sphingomyelin assays, and Suzanne Pfeffer for comments on the manuscript. Author contributionsRR and AR designed the project. BP analyzed the screen data, with the exception of the KEGG analysis which was performed by MK. BP and GP designed and cloned the CRISPR library focused on lipid-related genes. BP, GP and JK generated the mutant cell library and performed the HiSHH-Bottom10% screen. MK performed the LoSHH-Top5% screen. MK performed the experiments related to cholesterol genes and MK and EJI performed experiments related to sphingomyelin genes. MK, GL and KAJ performed the lipid probe staining experiments. MK and BP designed the computational pipeline for probe quantitation at primary cilia. CS and DFC contributed key conceptual and structural insights and experimental suggestions. RR and MK wrote the paper, with input from all authors.

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Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways

Abstract: Quantitative microscopy shows that protein-sorting signals have opposite effects on ciliary enrichment of G protein–coupled receptors in different cell types, revealing distinct ciliary trafficking mechanisms among ciliated cells.

Cited by 15 publications

References 35 publications

International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature

International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature

The route of the visual receptor rhodopsin along the cilium

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

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