Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1
Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temp...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2015-07, Vol.112 (27), p.8320-8325 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Milenkovic, Ljiljana Lucien E. Weiss Joshua Yoon Theodore L. Roth YouRong S. Su Steffen J. Sahl Matthew P. Scott W. E. Moerner |
| description | Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation. |
| doi_str_mv | 10.1073/pnas.1510094112 |
| format | Article |
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Weiss ; Joshua Yoon ; Theodore L. Roth ; YouRong S. Su ; Steffen J. Sahl ; Matthew P. Scott ; W. E. Moerner</creator><creatorcontrib>Milenkovic, Ljiljana ; Lucien E. Weiss ; Joshua Yoon ; Theodore L. Roth ; YouRong S. Su ; Steffen J. Sahl ; Matthew P. Scott ; W. E. Moerner</creatorcontrib><description>Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.1510094112</identifier><identifier>PMID: 26100903</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Algorithms ; Animals ; Binding sites ; Biological Sciences ; Cell Tracking - methods ; Cells, Cultured ; cilia ; Cilia - metabolism ; Embryo, Mammalian - cytology ; Fibroblasts - metabolism ; Hedgehog Proteins - genetics ; Hedgehog Proteins - metabolism ; Hedgehog signaling ; image analysis ; Kinetics ; Ligands ; Membranes ; Mice, Knockout ; Mice, Transgenic ; Microscopy, Confocal ; Patched Receptors ; Patched-1 Receptor ; primary cilia ; Protein Binding ; Proteins ; Receptors, Cell Surface - genetics ; Receptors, Cell Surface - metabolism ; Receptors, G-Protein-Coupled - genetics ; Receptors, G-Protein-Coupled - metabolism ; Scientific imaging ; Signal Transduction ; single-molecule microscopy ; single-particle tracking ; Smoothened ; Smoothened Receptor</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2015-07, Vol.112 (27), p.8320-8325</ispartof><rights>Volumes 1–89 and 106–112, copyright as a collective work only; author(s) retains copyright to individual articles</rights><rights>Copyright National Academy of Sciences Jul 7, 2015</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c590t-b83da6839e8a6166f585408b3242399946e4b75e14d82ca39648fe5daff897603</citedby><cites>FETCH-LOGICAL-c590t-b83da6839e8a6166f585408b3242399946e4b75e14d82ca39648fe5daff897603</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/112/27.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/26463704$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/26463704$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,723,776,780,799,881,27903,27904,53769,53771,57995,58228</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26100903$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Milenkovic, Ljiljana</creatorcontrib><creatorcontrib>Lucien E. 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Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.</description><subject>Algorithms</subject><subject>Animals</subject><subject>Binding sites</subject><subject>Biological Sciences</subject><subject>Cell Tracking - methods</subject><subject>Cells, Cultured</subject><subject>cilia</subject><subject>Cilia - metabolism</subject><subject>Embryo, Mammalian - cytology</subject><subject>Fibroblasts - metabolism</subject><subject>Hedgehog Proteins - genetics</subject><subject>Hedgehog Proteins - metabolism</subject><subject>Hedgehog signaling</subject><subject>image analysis</subject><subject>Kinetics</subject><subject>Ligands</subject><subject>Membranes</subject><subject>Mice, Knockout</subject><subject>Mice, Transgenic</subject><subject>Microscopy, Confocal</subject><subject>Patched Receptors</subject><subject>Patched-1 Receptor</subject><subject>primary cilia</subject><subject>Protein Binding</subject><subject>Proteins</subject><subject>Receptors, Cell Surface - genetics</subject><subject>Receptors, Cell Surface - metabolism</subject><subject>Receptors, G-Protein-Coupled - genetics</subject><subject>Receptors, G-Protein-Coupled - metabolism</subject><subject>Scientific imaging</subject><subject>Signal Transduction</subject><subject>single-molecule microscopy</subject><subject>single-particle tracking</subject><subject>Smoothened</subject><subject>Smoothened Receptor</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFks1v1DAQxSMEokvhzAmw1AuXtP6OfamEqkKRKoG09Gw5ySTxKokXOylaiT8eh122hQsna8a_9_xsT5a9Jvic4IJdbEcbz4kgGGtOCH2SrQjWJJdc46fZCmNa5IpTfpK9iHGDEyUUfp6dULkoMFtlP9dubHvIB99DNfeA3GDb1EK-QTdQt9D5Fm3t1P2wO7QNfgI3ovXg_dTBCDVK1TYkTdihyvXOogD3YPuISjfWi08qxymmdjv3dkqKcoe-2qnqoCYvs2dNYuHVYT3N7j5ef7u6yW-_fPp89eE2r4TGU14qVlupmAZlJZGyEUpwrEpGOWVaay6Bl4UAwmtFK8u05KoBUdumUbqQmJ1ml3vf7VwOUFcpUbC9OQQ33jrz987oOtP6e8NFekKlk8H7g0Hw32eIkxlcrKDv7Qh-joYoUijKCBf_R6UWpOCKLujZP-jGz2FML7FQKXdBfoe_2FNV8DEGaI65CTbLEJhlCMzDECTF28fXPfJ_fj0B7w7AojzaEWpoYRSjy6Fv9sQmTj48cuCSFZg_ODTWG9sGF83dmuKUF9N0RnL4BblKyz8</recordid><startdate>20150707</startdate><enddate>20150707</enddate><creator>Milenkovic, Ljiljana</creator><creator>Lucien E. 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Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>26100903</pmid><doi>10.1073/pnas.1510094112</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Algorithms Animals Binding sites Biological Sciences Cell Tracking - methods Cells, Cultured cilia Cilia - metabolism Embryo, Mammalian - cytology Fibroblasts - metabolism Hedgehog Proteins - genetics Hedgehog Proteins - metabolism Hedgehog signaling image analysis Kinetics Ligands Membranes Mice, Knockout Mice, Transgenic Microscopy, Confocal Patched Receptors Patched-1 Receptor primary cilia Protein Binding Proteins Receptors, Cell Surface - genetics Receptors, Cell Surface - metabolism Receptors, G-Protein-Coupled - genetics Receptors, G-Protein-Coupled - metabolism Scientific imaging Signal Transduction single-molecule microscopy single-particle tracking Smoothened Smoothened Receptor |
| title | Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1 |
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