title: Nanoscale organization of hedgehog is essential for long-range signaling.
abstract: Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform--incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bellary Road, Bangalore 560 065, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Vyas
authors.forename: Debanjan
authors.initials: D
authors.lastname: Goswami
authors.forename: A
authors.initials: A
authors.lastname: Manonmani
authors.forename: Pranav
authors.initials: P
authors.lastname: Sharma
authors.forename: H A
authors.initials: HA
authors.lastname: Ranganath
authors.forename: K
authors.initials: K
authors.lastname: VijayRaghavan
authors.forename: L S
authors.initials: LS
authors.lastname: Shashidhara
authors.forename: R
authors.initials: R
authors.lastname: Sowdhamini
authors.forename: Satyajit
authors.initials: S
authors.lastname: Mayor
mesh_terms: D000818:Animals; D019521:Body Patterning; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster / Q000737:chemistry / Q000196:embryology / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D053823:Hedgehog Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D053823:Hedgehog Proteins; D019812:Heparan Sulfate Proteoglycans; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins