title: Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1.
abstract: 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.
authors.affiliation: Department of Developmental Biology, Genetics and Bioengineering, Stanford University, Stanford, CA 94305;
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Milenkovic
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305;
authors.forename: Lucien E
authors.initials: LE
authors.lastname: Weiss
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305; Department of Applied Physics, Stanford University, Stanford, CA 94305.
authors.forename: Joshua
authors.initials: J
authors.lastname: Yoon
authors.affiliation: Department of Developmental Biology, Genetics and Bioengineering, Stanford University, Stanford, CA 94305;
authors.forename: Theodore L
authors.initials: TL
authors.lastname: Roth
authors.affiliation: Department of Developmental Biology, Genetics and Bioengineering, Stanford University, Stanford, CA 94305;
authors.forename: YouRong S
authors.initials: YS
authors.lastname: Su
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305;
authors.forename: Steffen J
authors.initials: SJ
authors.lastname: Sahl
authors.affiliation: Department of Developmental Biology, Genetics and Bioengineering, Stanford University, Stanford, CA 94305;
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Scott
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305; wmoerner@stanford.edu.
authors.forename: W E
authors.initials: WE
authors.lastname: Moerner
mesh_terms: D000465:Algorithms; D000818:Animals; D058948:Cell Tracking / Q000379:methods; D002478:Cells, Cultured; D002923:Cilia / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D018345:Mice, Knockout; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000072086:Smoothened Receptor
chemical_list: D053823:Hedgehog Proteins; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; C000606240:Ptch1 protein, mouse; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C491022:Smo protein, mouse; D000072086:Smoothened Receptor
keywords: Hedgehog signaling; Smoothened; primary cilia; single-molecule microscopy; single-particle tracking