Armel Gallet

Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog

The mechanisms involved in transduction of the Hedgehog (Hh) signal are of considerable interest to developmental and cancer biologists. Stabilization of the integral membrane protein Smoothened (Smo) at the plasma membrane is a crucial step in Hh signalling but the molecular events immediately downstream of Smo remain to be elucidated. We have shown previously that the transcriptional mediator Cubitus interruptus (Ci) is associated in a protein complex with at least two other proteins, the kinesin-like Costal2 (Cos2) and the serine–threonine kinase Fused (Fu). This protein complex governs the access of Ci to the nucleus. Here we show that, consequent on the stabilization of Smo, Cos2 and Fu are destabilized. Moreover, we find that the Cos2–Fu–Ci protein complex is associated with Smo in membrane fractions both in vitro and in vivo. We also show that Cos2 binding on Smo is necessary for the Hh-dependent dissociation of Ci from this complex. We propose that the association of the Cos2 protein complex with Smo at the plasma membrane controls the stability of the complex and allows Ci activation, eliciting its nuclear translocation.

Cholesterol modification of hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to hedgehog.

Hedgehog family members are secreted proteins involved in numerous patterning mechanisms. Different posttranslational modifications have been shown to modulate Hedgehog biological activity. We investigated the role of these modifications in regulating subcellular localization of Hedgehog in the Drosophila embryonic epithelium. We demonstrate that cholesterol modification of Hedgehog is responsible for its assembly in large punctate structures and apical sorting through the activity of the sterol-sensing domain-containing Dispatched protein. We further show that movement of these specialized structures through the cellular field is contingent upon the activity of proteoglycans synthesized by the heparan sulfate polymerase Tout-Velu. Finally, we show that the Hedgehog large punctate structures are necessary only for a subset of Hedgehog target genes across the parasegmental boundary, suggesting that presentation of Hedgehog from different membrane compartments is responsible for Hedgehog functional diversity in epithelial cells.

Cellular Trafficking of the Glypican Dally-like Is Required for Full-Strength Hedgehog Signaling and Wingless Transcytosis

Hedgehog (Hh) and Wingless (Wg) morphogens specify cell fate in a concentration-dependent manner in the Drosophila wing imaginal disc. Proteoglycans, components of the extracellular matrix, are involved in Hh and Wg stability, spreading, and reception. In this study, we demonstrate that the glycosyl-phosphatidyl-inositol (GPI) anchor of the glypican Dally-like (Dlp) is required for its apical internalization and its subsequent targeting to the basolateral compartment of the epithelium. Dlp endocytosis from the apical surface of Hh-receiving cells catalyzes the internalization of Hh bound to its receptor Patched (Ptc). The cointernalization of Dlp with the Hh/Ptc complex is dynamin dependent and necessary for full-strength Hh signaling. We also demonstrate that Wg is secreted apically in the disc epithelium and that apicobasal trafficking of Dlp allows Wg transcytosis to favor Wg spreading along the basolateral compartment. Thus, Dlp endocytosis is a common regulatory mechanism of both Hh and Wg morphogen action.

Cholesterol modification is necessary for controlled planar long-range activity of Hedgehog in Drosophila epithelia

The Hedgehog morphogen is a major developmental regulator that acts at short and long range to direct cell fate decisions in invertebrate and vertebrate tissues. Hedgehog is the only known metazoan protein to possess a covalently linked cholesterol moiety. Although the role of the cholesterol group of Hedgehog remains unclear, it has been suggested to be dispensable for the its long-range activity in Drosophila. Here, we provide data in three different epithelia - ventral and dorsal embryonic ectoderm, and larval imaginal disc tissue - showing that cholesterol modification is in fact necessary for the controlled long-range activity of Drosophila Hedgehog. We provide an explanation for the discrepancy between our results and previous reports by showing that unmodified Hh can act at long range, albeit in an uncontrolled manner, only when expressed in squamous cells. Our data show that cholesterol modification controls long-range Hh activity at multiple levels. First, cholesterol increases the affinity of Hh for the plasma membrane, and consequently enhances its apparent intrinsic activity, both in vitro and in vivo. In addition, multimerisation of active Hh requires the presence of cholesterol. These multimers are correlated with the assembly of Hh into apically located, large punctate structures present in active Hh gradients in vivo. By comparing the activity of cholesterol-modified Hh in columnar epithelial cells and peripodial squamous cells, we show that epithelial cells provide the machinery necessary for the controlled planar movement of Hh, thereby preventing the unrestricted spreading of the protein within the three-dimensional space of the epithelium. We conclude that, as in vertebrates, cholesterol modification is essential for controlled long-range Hh signalling in Drosophila.

The long-range activity of Hedgehog is regulated in the apical extracellular space by the glypican Dally and the hydrolase Notum.

Cell fate determination during developmental patterning is often controlled by concentration gradients of morphogens. In the epithelial field, morphogens like the Hedgehog (Hh) peptides diffuse both apically and basolaterally; however, whether both pools of Hh are sensed at the cellular level is unclear. Here, we show that interfering with the amount of apical Hh causes a dramatic change in the long-range activation of low-threshold Hh target genes, without similar effect on short-range, high-threshold targets. We provide genetic evidence that the glypican Dally upregulates apical Hh levels, and that the release of Dally by the hydrolase Notum promotes apical Hh long-range activity. Our data suggest that several pools of Hh are perceived in epithelial tissues. Thus, we propose that the overall gradient of Hh is a composite of pools secreted by different routes (apical and basolateral), and that a cellular summation of these components is required for appropriate developmental patterning.

The C-terminal domain of Armadillo binds to hypophosphorylated Teashirt to modulate Wingless signalling in Drosophila

Wnt signalling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts to stabilize Armadillo inside cells where it binds to at least two DNA‐binding factors which regulate specific target genes. One Armadillo‐binding protein in Drosophila is the zinc finger protein Teashirt. Here we show that Wingless signalling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C‐terminal end of Armadillo. Finally, we present evidence that the serine/threonine kinase Shaggy is associated with Teashirt in a complex. We discuss these results with respect to current models of Armadillo/β‐catenin action for the transmission of the Wingless/Wnt pathway.

Phosphorylation of the atypical kinesin Costal2 by the kinase Fused induces the partial disassembly of the Smoothened-Fused-Costal2-Cubitus interruptus complex in Hedgehog signalling

The Hedgehog (Hh) family of secreted proteins is involved both in developmental and tumorigenic processes. Although many members of this important pathway are known, the mechanism of Hh signal transduction is still poorly understood. In this study, we analyse the regulation of the kinesin-like protein Costal2 (Cos2) by Hh. We show that a residue on Cos2, serine 572 (Ser572), is necessary for normal transduction of the Hh signal from the transmembrane protein Smoothened (Smo) to the transcriptional mediator Cubitus interruptus (Ci). This residue is located in the serine/threonine kinase Fused (Fu)-binding domain and is phosphorylated as a consequence of Fu activation. Although Ser572 does not overlap with known Smo- or Ci-binding domains, the expression of a Cos2 variant mimicking constitutive phosphorylation and the use of a specific antibody to phosphorylated Ser572 showed a reduction in the association of phosphorylated Cos2 with Smo and Ci, both in vitro and in vivo. Moreover, Cos2 proteins with an Ala or Asp substitution of Ser572 were impaired in their regulation of Ci activity. We propose that, after activation of Smo, the Fu kinase induces a conformational change in Cos2 that allows the disassembly of the Smo-Fu-Cos2-Ci complex and consequent activation of Hh target genes. This study provides new insight into the mechanistic regulation of the protein complex that mediates Hh signalling and a unique antibody tool for directly monitoring Hh receptor activity in all activated cells.

Hedgehog morphogen: from secretion to reception

A major challenge of developmental biology is to understand how cells coordinate developmental behaviors with their neighbors. To achieve this, cells often employ signaling molecules that emanate from a local source and act at a distance on target cells. The Hedgehog morphogen is an essential signaling molecule required for numerous processes during animal development. Emphasizing the importance of this molecule for both growth control and patterning, Hedgehog signaling activity is often deregulated during cancer formation and progression. The secretion and spread of Hedgehog are not passive processes, but require accessory molecules involved in Hedgehog processing, release, spread and reception. In this review, I focus on the factors that are required to control the spread and activity of Hedgehog, highlighting recent data that have shed light on these processes.

Distinct Phosphorylations on Kinesin Costal-2 Mediate Differential Hedgehog Signaling Strength

The graded Hedgehog (Hh) signal is transduced by the transmembrane Smoothened (Smo) proteins in both vertebrates and invertebrates. In Drosophila, associations between Smo and the Fused (Fu)/Costal-2 (Cos2)/Cubitus Interruptus (Ci) cytoplasmic complex lead to pathway activation, but it remains unclear how the cytoplasmic complex responds to and transduces different levels of Hh signaling. We show here that, within the Hh gradient field, low- and high-magnitude Smo activations control differentially the phosphorylation of Cos2 on two distinct serines. We also provide evidence that these phosphorylations depend on the Fu kinase activity and lead to a shift of Cos2 distribution from the cytoplasm to the plasma membrane. Moreover, the distinct Cos2 phosphorylation states mediate differential Hh signaling magnitude, suggesting that phosphorylation and relocation of Cos2 to the plasma membrane facilitate high-level Hh signaling through the control of Ci nuclear translocation and transcriptional activity.

Dally and Notum regulate the switch between low and high level Hedgehog pathway signalling.

During development, secreted morphogens, such as Hedgehog (Hh), control cell fate and proliferation. Precise sensing of morphogen levels and dynamic cellular responses are required for morphogen-directed morphogenesis, yet the molecular mechanisms responsible are poorly understood. Several recent studies have suggested the involvement of a multi-protein Hh reception complex, and have hinted at an understated complexity in Hh sensing at the cell surface. We show here that the expression of the proteoglycan Dally in Hh-receiving cells in Drosophila is necessary for high but not low level pathway activity, independent of its requirement in Hh-producing cells. We demonstrate that Dally is necessary to sequester Hh at the cell surface and to promote Hh internalisation with its receptor. This internalisation depends on both the activity of the hydrolase Notum and the glycosyl-phosphatidyl-inositol (GPI) moiety of Dally, and indicates a departure from the role of the second glypican Dally-like in Hh signalling. Our data suggest that hydrolysis of the Dally-GPI by Notum provides a switch from low to high level signalling by promoting internalisation of the Hh-Patched ligand-receptor complex.