Extracellular carriers control lipid-dependent secretion, delivery, and activity of WNT morphogens.

WNT morphogens trigger signaling pathways fundamental for embryogenesis, regeneration, and cancer. WNTs are modified with palmitoleate, which is critical for binding Frizzled (FZD) receptors and activating signaling. However, it is unknown how WNTs are released and spread from cells, given their strong lipid-dependent membrane attachment.

Structural basis for catalyzed assembly of the Sonic hedgehog-Patched1 signaling complex.

The dually lipidated Sonic hedgehog (SHH) morphogen signals through the tumor suppressor membrane protein Patched1 (PTCH1) to activate the Hedgehog pathway, which is fundamental in development and cancer. SHH engagement with PTCH1 requires the GAS1 coreceptor, but the mechanism is unknown. We demonstrate a unique role for GAS1, catalyzing SHH-PTCH1 complex assembly in vertebrate cells by direct SHH transfer from the extracellular SCUBE2 carrier to PTCH1.

Hedgehog pathway modulation by glypican 3-conjugated heparan sulfate.

Glypicans are a family of cell surface heparan sulfate proteoglycans that play critical roles in multiple cell signaling pathways. Glypicans consist of a globular core, an unstructured stalk modified with sulfated glycosaminoglycan chains, and a glycosylphosphatidylinositol anchor. Though these structural features are conserved, their individual contribution to glypican function remains obscure.

Structural insights into proteolytic activation of the human Dispatched1 transporter for Hedgehog morphogen release.

The membrane protein Dispatched (Disp), which belongs to the RND family of small molecule transporters, is essential for Hedgehog (Hh) signaling, by catalyzing the extracellular release of palmitate- and cholesterol-modified Hh ligands from producing cells. Disp function requires Furin-mediated proteolytic cleavage of its extracellular domain, but how this activates Disp remains obscure. Here, we employ cryo-electron microscopy to determine atomic structures of human Disp1 (hDisp1), before and after cleavage, and in complex with lipid-modified Sonic hedgehog (Shh) ligand.

Hedgehog Pathway Activation Requires Coreceptor-Catalyzed, Lipid-Dependent Relay of the Sonic Hedgehog Ligand.

Hedgehog signaling governs critical processes in embryogenesis, adult stem cell maintenance, and tumorigenesis. The activating ligand, Sonic hedgehog (SHH), is highly hydrophobic because of dual palmitate and cholesterol modification, and thus, its release from cells requires the secreted SCUBE proteins. We demonstrate that the soluble SCUBE-SHH complex, although highly potent in cellular assays, cannot directly signal through the SHH receptor, Patched1 (PTCH1).

Distinct Cation Gradients Power Cholesterol Transport at Different Key Points in the Hedgehog Signaling Pathway.

Cholesterol plays two critical roles in Hedgehog signaling, a fundamental pathway in animal development and cancer: it covalently modifies the Sonic hedgehog (SHH) ligand, restricting its release from producing cells, and directly activates Smoothened in responding cells. In both contexts, a membrane protein related to bacterial RND transporters regulates cholesterol: Dispatched1 controls release of cholesterylated SHH, and Patched1 antagonizes Smoothened activation by cholesterol. The mechanism and driving force for eukaryotic RND proteins, including Dispatched1 and Patched1, are unknown.

Morphogenesis of neurons and glia within an epithelium.

To sense the outside world, some neurons protrude across epithelia, the cellular barriers that line every surface of our bodies. To study the morphogenesis of such neurons, we examined the amphid, in which dendrites protrude through a glial channel at the nose. During development, amphid dendrites extend by attaching to the nose via DYF-7, a type of protein typically found in epithelial apical ECM.

Sending and Receiving Hedgehog Signals.

Communication between cells pervades the development and physiology of metazoans. In animals, this process is carried out by a relatively small number of signaling pathways, each consisting of a chain of biochemical events through which extracellular stimuli control the behavior of target cells. One such signaling system is the Hedgehog pathway, which is crucial in embryogenesis and is implicated in many birth defects and cancers.

SNAP-25 contains non-acylated thiol pairs that can form intrachain disulfide bonds: possible sites for redox modulation of neurotransmission.

Intrachain disulfide bond formation among the cysteine thiols of SNAP-25, a component of the SNARE protein complex required for neurotransmitter release, has been hypothesized to link oxidative stress and inhibition of synaptic transmission. However, neither the availability in vivo of SNAP-25 thiols, which are known targets of S-palmitoylation, nor the tendency of these thiols to form intrachain disulfide bonds is known. We have examined, in rat brain extracts, both the availability of closely spaced, or vicinal, thiol pairs in SNAP-25 and the propensity of these dithiols toward disulfide bond formation using a method improved by us recently that exploits the high chemoselectivity of phenylarsine oxide (PAO) for vicinal thiols.