Nanoscale organization of hedgehog is essential for long-range signaling.
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.
Vyas
Neha
N
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bellary Road, Bangalore 560 065, India.
Goswami
Debanjan
D
Manonmani
A
A
Sharma
Pranav
P
Ranganath
H A
HA
VijayRaghavan
K
K
Shashidhara
L S
LS
Sowdhamini
R
R
Mayor
Satyajit
S
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
D053823:Hedgehog Proteins; D019812:Heparan Sulfate Proteoglycans; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins