title: Simultaneous measurement of smoothened entry into and exit from the primary cilium.
abstract: Ciliary accumulation of signaling proteins must result from a rate of ciliary entry that exceeds ciliary exit, but approaches for distinguishing ciliary entry vs. exit are lacking. Using a photoconvertible fluorescent protein tag, we establish an assay that allows a separate but simultaneous examination of ciliary entry and exit of the Hedgehog signaling protein Smoothened in individual cells. We show that KAAD-cyclopamine selectively blocks entry, whereas ciliobrevin interferes initially with exit and eventually with both entry and exit of ciliary Smoothened. Our study provides an approach to understanding regulation of ciliary entry vs. exit of Hedgehog signaling components as well as other ciliary proteins.
authors.affiliation: Departments of Biochemistry and Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States of America; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Jynho
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Anatomy and Regenerative Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, United States of America.
authors.forename: Elaine Y C
authors.initials: EY
authors.lastname: Hsia
authors.affiliation: Departments of Biochemistry and Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States of America; Division of Hematology-Oncology, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern, Dallas, TX, United States of America.
authors.forename: James
authors.initials: J
authors.lastname: Kim
authors.affiliation: Departments of Biochemistry and Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States of America; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Navdar
authors.initials: N
authors.lastname: Sever
authors.affiliation: Departments of Biochemistry and Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States of America; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Beachy
authors.affiliation: Departments of Biochemistry and Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States of America; Department of Anatomy and Regenerative Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, United States of America.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Zheng
mesh_terms: D000818:Animals; D002923:Cilia / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D041681:NIH 3T3 Cells; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015398:Signal Transduction; D000072086:Smoothened Receptor
chemical_list: D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; C491022:Smo protein, mouse; D000072086:Smoothened Receptor