The Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-lasting questions about this pathway are how dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells, and how the resistance-nodulation-division transporter Dispatched (Disp) regulates this process. Here we show that Disp inactivation in Shh expressing cells impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. We also show reduced cholesterol export from Disp-deficient cells, that these cells contain increased cholesterol amounts in the plasma membrane, and that Shh shedding from Disp-deficient cells is restored by pharmacological membrane cholesterol extraction and by overexpressed transgenic Disp or structurally related Patched (Ptc, a putative cholesterol transporter). These data suggest that Disp can regulate Shh function via controlled cell surface shedding and that membrane cholesterol-related molecular mechanisms shared by Disp and Ptc exercise such sheddase control.

Hedgehog (Hh) morphogens are involved in embryonic development and stem cell biology and, if misregulated, can contribute to cancer. One important post-translational modification with profound impact on Hh biofunction is its C-terminal cholesteroylation during biosynthesis. The current hypothesis is that the cholesterol moiety is a decisive factor in Hh association with the outer plasma membrane leaflet of producing cells, cell-surface Hh multimerization, and its transport and signaling. Yet, it is not decided whether the cholesterol moiety is directly involved in all of these processes, because their functional interdependency raises the alternative possibility that the cholesterol initiates early processes directly and that these processes can then steer later stages of Hh signaling independent of the lipid. We generated variants of the C-terminal Hh peptide and observed that these cholesteroylated peptides variably impaired several post-translational processes in producing cells and Hh biofunction in Drosophila melanogaster eye and wing development. We also found that substantial Hh amounts separated from cholesteroylated peptide tags in vitro and in vivo and that tagged and untagged Hh variants lacking their C-cholesterol moieties remained bioactive. Our approach thus confirms that Hh cholesteroylation is essential during the early steps of Hh production and maturation but also suggests that it is dispensable for Hh signal reception at receiving cells.

The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We previously showed that Disp enhances proteolytic Shh solubilization from its lipidated terminal peptide anchors. This process, called shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as soluble sinks for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is required and sufficient for Disp-mediated transfer because mCherry linked to cholesteroylated peptides associates with HDL in a Disp-dependent manner, but an N-palmitoylated Shh variant that lacks C-cholesterol does not. Disp-mediated Shh transfer to HDL is finalized by proteolytic processing of the palmitoylated N-terminal membrane anchor. The resulting mono-lipidated Shh variant may help meet the demands for Hh activity regulation in different cell types and developing tissues.

Morphogens determine cellular differentiation in many developing tissues in a concentration dependent manner. As a central model for gradient formation during animal development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in the Drosophila wing disc. Although heparan sulfate (HS) expression in the disc is essential for this process, it is not known whether HS regulates Hh signaling and spread in a direct or in an indirect manner. To answer this question, we systematically screened two composite Hh binding areas for HS in vitro and expressed mutated proteins in the Drosophila wing disc. We found that selectively impaired HS binding of the second site reduced Hh signaling close to the source and caused striking wing mispatterning phenotypes more distant from the source. These observations suggest that HS constrains Hh to the wing disc epithelium in a direct manner, and that interfering with this constriction converts Hh into freely diffusing forms with altered signaling ranges and impaired gradient robustness.

Vertebrate and invertebrate Hedgehog (Hh) morphogens signal over short and long distances to direct cell fate decisions during development and to maintain tissue homeostasis after birth. One of the most important questions in Hh biology is how such Hh signaling to distant target cells is achieved, because all Hh proteins are secreted as dually lipidated proteins that firmly tether to the outer plasma membrane leaflet of their producing cells. There, Hhs multimerize into light microscopically visible storage platforms that recruit factors required for their regulated release. One such recruited release factor is the soluble glycoprotein Scube2 (Signal sequence, cubulin domain, epidermal-growth-factor-like protein 2), and maximal Scube2 function requires concomitant activity of the resistance-nodulation-division (RND) transporter Dispatched (Disp) at the plasma membrane of Hh-producing cells. Although recently published cryo-electron microscopy-derived structures suggest possible direct modes of Scube2/Disp-regulated Hh release, the mechanism of Disp-mediated Hh deployment is still not fully understood. In this review, we discuss suggested direct modes of Disp-dependent Hh deployment and relate them to the structural similarities between Disp and the related RND transporters Patched (Ptc) and Niemann-Pick type C protein 1. We then discuss open questions and perspectives that derive from these structural similarities, with particular focus on new findings that suggest shared small molecule transporter functions of Disp to deplete the plasma membrane of cholesterol and to modulate Hh release in an indirect manner.

Two posttranslational lipid modifications present on all Hedgehog (Hh) morphogensan N-terminal palmitate and a C-terminal cholesterol-are established and essential regulators of Hh biofunction. Yet, for several decades, the question of exactly how both lipids contribute to Hh signaling remained obscure. Recently, cryogenic electron microscopy revealed different modes by which one or both lipids may contribute directly to Hh binding and signaling to its receptor Patched1 (Ptc). Some of these modes demand that the established release factor Dispatched1 (Disp) extracts dual-lipidated Hh from the cell surface, and that another known upstream signaling modulator called Scube2 chaperones the dual-lipidated morphogen to Ptc. By mechanistically and biochemically aligning this concept with established in vivo and recent in vitro findings, this reflection identifies remaining questions in lipidated Hh transport and evaluates additional mechanisms of Disp-and Scube2-regulated release of a second bioactive Hh fraction that has one or both lipids removed.

The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We previously showed that Disp enhances proteolytic Shh solubilization from its lipidated terminal peptide anchors. This process, called shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as soluble sinks for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is required and sufficient for Disp-mediated transfer because mCherry linked to cholesteroylated peptides associates with HDL in a Disp-dependent manner, but an N-palmitoylated Shh variant that lacks C-cholesterol does not. Disp-mediated Shh transfer to HDL is finalized by proteolytic processing of the palmitoylated N-terminal membrane anchor. The resulting mono-lipidated Shh variant may help meet the demands for Hh activity regulation in different cell types and developing tissues.

The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We previously showed that Disp enhances proteolytic Shh solubilization from its lipidated terminal peptide anchors. This process, called shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as soluble sinks for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is required and sufficient for Disp-mediated transfer because mCherry linked to cholesteroylated peptides associates with HDL in a Disp-dependent manner, but an N-palmitoylated Shh variant that lacks C-cholesterol does not. Disp-mediated Shh transfer to HDL is finalized by proteolytic processing of the palmitoylated N-terminal membrane anchor. The resulting mono-lipidated Shh variant may help meet the demands for Hh activity regulation in different cell types and developing tissues.