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

Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and...

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Autores principales: Geneva, Ivayla I., Tan, Han Yen, Calvert, Peter D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305262/
https://www.ncbi.nlm.nih.gov/pubmed/27974638
http://dx.doi.org/10.1091/mbc.E16-07-0549
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author Geneva, Ivayla I.
Tan, Han Yen
Calvert, Peter D.
author_facet Geneva, Ivayla I.
Tan, Han Yen
Calvert, Peter D.
author_sort Geneva, Ivayla I.
collection PubMed
description Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and apical membranes inherent to confocal microscopes. Theory shows that cilia will appear more than threefold brighter than the surrounding apical membrane when the densities of fluorescently labeled proteins are the same, thus providing a benchmark for ciliary enrichment. Using this benchmark, we examined the ciliary enrichment signals of two G protein–coupled receptors (GPCRs)—the somatostatin receptor 3 and rhodopsin. Remarkably, we found that the C-terminal VxPx motif, required for efficient enrichment of rhodopsin within rod photoreceptor sensory cilia, inhibited enrichment of the somatostatin receptor in primary cilia. Similarly, VxPx inhibited primary cilium enrichment of a chimera of rhodopsin and somatostatin receptor 3, where the dual Ax(S/A)xQ ciliary targeting motifs within the third intracellular loop of the somatostatin receptor replaced the third intracellular loop of rhodopsin. Rhodopsin was depleted from primary cilia but gained access, without being enriched, with the dual Ax(S/A)xQ motifs. Ciliary enrichment of these GPCRs thus operates via distinct mechanisms in different cells.
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spelling pubmed-53052622017-04-30 Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways Geneva, Ivayla I. Tan, Han Yen Calvert, Peter D. Mol Biol Cell Articles Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and apical membranes inherent to confocal microscopes. Theory shows that cilia will appear more than threefold brighter than the surrounding apical membrane when the densities of fluorescently labeled proteins are the same, thus providing a benchmark for ciliary enrichment. Using this benchmark, we examined the ciliary enrichment signals of two G protein–coupled receptors (GPCRs)—the somatostatin receptor 3 and rhodopsin. Remarkably, we found that the C-terminal VxPx motif, required for efficient enrichment of rhodopsin within rod photoreceptor sensory cilia, inhibited enrichment of the somatostatin receptor in primary cilia. Similarly, VxPx inhibited primary cilium enrichment of a chimera of rhodopsin and somatostatin receptor 3, where the dual Ax(S/A)xQ ciliary targeting motifs within the third intracellular loop of the somatostatin receptor replaced the third intracellular loop of rhodopsin. Rhodopsin was depleted from primary cilia but gained access, without being enriched, with the dual Ax(S/A)xQ motifs. Ciliary enrichment of these GPCRs thus operates via distinct mechanisms in different cells. The American Society for Cell Biology 2017-02-15 /pmc/articles/PMC5305262/ /pubmed/27974638 http://dx.doi.org/10.1091/mbc.E16-07-0549 Text en © 2017 Geneva et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.
spellingShingle Articles
Geneva, Ivayla I.
Tan, Han Yen
Calvert, Peter D.
Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways
title Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways
title_full Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways
title_fullStr Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways
title_full_unstemmed Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways
title_short Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways
title_sort untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305262/
https://www.ncbi.nlm.nih.gov/pubmed/27974638
http://dx.doi.org/10.1091/mbc.E16-07-0549
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