Abstract
Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, we show that Hh relay and direct patterning of the 3-4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3-4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions.
Highlights
Hedgehog (Hh) morphogens are dually lipidated 19 kDa proteins that are firmly anchored to the cell membrane of producing cells
Additional targeted deletion of N-palmitate restores wing patterning, demonstrating that one role of palmitoylated membrane anchors is to prevent the release of un- or incompletely processed Hh clusters in vivo. These genetic data are supported by the nano-structure of Hh clusters as revealed by immunoelectron microscopy (IEM) and provide new insights into how Hh relay from the producing cell membrane or between membranes could be achieved
Visualization of Hh multimer nano-architecture by IEM
Summary
Hedgehog (Hh) morphogens are dually lipidated 19 kDa proteins that are firmly anchored to the cell membrane of producing cells. The N-palmitate plays two indirect roles for Hh biofunction: first, it ensures reliable membrane-proximal positioning of inhibitory N-terminal peptides as a prerequisite for their efficient proteolytic processing, and second, by its continued association with the cell membrane, it ensures that only fully processed (=activated) Hh clusters are released This model predicts that inhibition of N-palmitoylation will result in release of inactive soluble proteins with masked Ptc-binding sites (Jakobs et al, 2014; Jakobs et al, 2016; Ohlig et al, 2011; Ohlig et al, 2012). These genetic data are supported by the nano-structure of Hh clusters as revealed by immunoelectron microscopy (IEM) and provide new insights into how Hh relay from the producing cell membrane or between membranes could be achieved
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