Kazuya Hori

Notch signaling at a glance

Summary Cell–cell interactions define a quintessential aspect of multicellular development. Metazoan morphogenesis depends on a handful of fundamental, conserved cellular interaction mechanisms, one of which is defined by the Notch signaling pathway. Signals transmitted through the Notch surface receptor have a unique developmental role: Notch signaling links the fate of one cell with that of a cellular neighbor through physical interactions between the Notch receptor and the membrane-bound ligands that are expressed in an apposing cell. The developmental outcome of Notch signals is strictly dependent on the cellular context and can influence differentiation, proliferation and apoptotic cell fates. The Notch pathway is conserved across species (Artavanis-Tsakonas et al., 1999; Bray, 2006; Kopan and Ilagan, 2009). In humans, Notch malfunction has been associated with a diverse range of diseases linked to changes in cell fate and cell proliferation including cancer (Louvi and Artavanis-Tsakonas, 2012). In this Cell Science at a Glance article and the accompanying poster we summarize the molecular biology of Notch signaling, its role in development and its relevance to disease.

Drosophila Deltex mediates Suppressor of Hairless-independent and late-endosomal activation of Notch signaling

Notch (N) signaling is an evolutionarily conserved mechanism that regulates many cell-fate decisions. deltex (dx) encodes an E3-ubiquitin ligase that binds to the intracellular domain of N and positively regulates N signaling. However, the precise mechanism of Dx action is unknown. Here, we found that Dx was required and sufficient to activate the expression of gene targets of the canonical Su(H)-dependent N signaling pathway. Although Dx required N and a cis-acting element that overlaps with the Su(H)-binding site, Dx activated a target enhancer of N signaling, the dorsoventral compartment boundary enhancer of vestigial (vgBE), in a manner that was independent of the Delta (Dl)/Serrate (Ser) ligands- or Su(H). Dx caused N to be moved from the apical cell surface into the late-endosome, where it accumulated stably and co-localized with Dx. Consistent with this, the dx gene was required for the presence of N in the endocytic vesicles. Finally, blocking the N transportation from the plasma membrane to the late-endosome by a dominant-negative form of Rab5 inhibited the Dx-mediated activation of N signaling, suggesting that the accumulation of N in the late-endosome was required for the Dx-mediated Su(H)-independent N signaling.

Drosophila HOPS and AP-3 complex genes are required for a Deltex-regulated activation of notch in the endosomal trafficking pathway.

DSL ligands promote proteolysis of the Notch receptor, to release active Notch intracellular domain (N(ICD)). Conversely, the E3 ubiquitin ligase Deltex can activate ligand-independent Notch proteolysis and signaling. Here we show that Deltex effects require endocytic trafficking by HOPS and AP-3 complexes. Our data suggest that Deltex shunts Notch into an endocytic pathway with two possible endpoints. If Notch transits into the lysosome lumen, it is degraded. However, if HOPS and AP-3 deliver Notch to the limiting membrane of the lysosome, degradation of the Notch extracellular domain allows subsequent Presenilin-mediated release of N(ICD). This model accounts for positive and negative regulatory effects of Deltex in vivo. Indeed, we uncover HOPS/AP-3 contributions to Notch signaling during Drosophila midline formation and neurogenesis. We discuss ways in which these endocytic pathways may modulate ligand-dependent and -independent events, as a mechanism that can potentiate Notch signaling or dampen noise in the signaling network.

Synergy between the ESCRT-III complex and Deltex defines a ligand-independent Notch signal

The ESCRT-III complex component Shrub plays a pivotal rate-limiting step in late endosomal ligand-independent Notch activation.

The first deltex null mutant indicates tissue-specific deltex-dependent Notch signaling in Drosophila

Notch (N) is a single-pass transmembrane receptor. The N signaling pathway is an evolutionarily conserved mechanism that controls various cell-specification processes. Drosophila Deltex (Dx), a RING-domain E3 ubiquitin ligase, binds to the N intracellular domain, promotes N’s endocytic trafficking to late endosomes, and was proposed to activate Suppressor of Hairless [Su(H)]-independent N signaling. However, it has been difficult to evaluate the importance of dx, because no null mutant of a dx family gene has been available in any organism. Here, we report the first null mutant allele of Drosophiladx. We found that dx was involved only in the subsets of N signaling, but was not essential for it in any developmental context. A strong genetic interaction between dx and Su(H) suggested that dx might function in Su(H)-dependent N signaling. Our epistatic analyses suggested that dx functions downstream of the ligands and upstream of activated Su(H). We also uncovered a novel dx activity that suppressed N signaling downstream of N.

Expression and characterization of the Drosophila X11‐like/Mint protein during neural development

The X11‐like (X11L) protein was originally isolated as a protein bound to the cytoplasmic domain of the β‐amyloid precursor protein (APP), which is associated with Alzheimers disease. In mammals, X11L is believed to play an important role in the regulation of APP metabolism. Here we isolated and characterized the Drosophila X11L (dX11L) protein, also may be referred to this protein as Drosophila Mint (dMint), Lin 10 (dLin10) or X11 (dX11), is thought to be expressed in neuronal tissues from late embryonic through to the adult stages of the fly. The phosphotyrosine interaction domain of dX11L interacts with the cytoplasmic domain of the Drosophila amyloid precursor protein‐like (APPL) similar to the way human X11L (hX11L) interacts with APP. Overexpression of dX11L on post‐mitotic neurons had a lethal effect on flies and, when it was localized to the eye imaginal disc, disruption of compound eye morphology due to enhanced apoptosis of neuronal cells was observed. Overexpression of hX11L and the PDZ domain of dX11L resulted in identical eye phenotypes. The PDZ domain is highly conserved between Drosophila and human, and appears to be responsible for this phenotype. Our findings suggest that the X11L family may be involved with the regulation of apoptosis during neural cell development and that aberrant X11L function could be contribute in this way to the neuronal degeneration observed in Alzheimers disease.

Genetic circuitry of Survival motor neuron, the gene underlying spinal muscular atrophy

Significance Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is a devastating neurodegenerative disease caused by reduced levels of Survival Motor Neuron (SMN) gene activity. Despite well-characterized aspects of the involvement of SMN in small nuclear ribonucleoprotein biogenesis, the gene circuitry affecting SMN activity remains obscure. Here, we use Drosophila as a model system to integrate results from large-scale genetic and proteomic studies and bioinformatic analyses to define a unique SMN interactome to provide a basis for a better understanding of SMA. Such efforts not only help dissect Smn biology but also may point to potential clinically relevant targets. The clinical severity of the neurodegenerative disorder spinal muscular atrophy (SMA) is dependent on the levels of functional Survival Motor Neuron (SMN) protein. Consequently, current strategies for developing treatments for SMA generally focus on augmenting SMN levels. To identify additional potential therapeutic avenues and achieve a greater understanding of SMN, we applied in vivo, in vitro, and in silico approaches to identify genetic and biochemical interactors of the Drosophila SMN homolog. We identified more than 300 candidate genes that alter an Smn-dependent phenotype in vivo. Integrating the results from our genetic screens, large-scale protein interaction studies, and bioinformatic analysis, we define a unique interactome for SMN that provides a knowledge base for a better understanding of SMA.

An extracellular region of Serrate is essential for ligand-induced cis-inhibition of Notch signaling

Cell-to-cell communication via the Notch pathway is mediated between the membrane-bound Notch receptor and either of its canonical membrane-bound ligands Delta or Serrate. Notch ligands mediate receptor transactivation between cells and also mediate receptor cis-inhibition when Notch and ligand are co-expressed on the same cell. We demonstrate in Drosophila that removal of any of the EGF-like repeats (ELRs) 4, 5 or 6 results in a Serrate molecule capable of transactivating Notch but exhibiting little or no Notch cis-inhibition capacity. These forms of Serrate require Epsin (Liquid facets) to transduce a signal, suggesting that ELR 4-6-deficient ligands still require endocytosis for Notch activation. We also demonstrate that ELRs 4-6 are responsible for the dominant-negative effects of Serrate ligand forms that lack the intracellular domain and are therefore incapable of endocytosis in the ligand-expressing cell. We find that ELRs 4-6 of Serrate are conserved across species but do not appear to be conserved in Delta homologs.

Regulation of ligand-independent Notch signal through intracellular trafficking.

Notch signaling is an evolutionarily conserved mechanism that defines a key cell fate control mechanism in metazoans. Notch signaling relies on the surface interaction between the Notch receptor and membrane bound ligands in an apposing cell. In our recent study,22 we uncover a non-canonical receptor activation path that relies on a ligand-independent, intracellular activation of the receptor as it travels through the endosomal compartments. We found that Notch receptor, targeted for degradation lysosomal degradation through multivesicular bodies (MVBs) is “diverted” toward activation upon mono-ubiquitination through a synergy between the ubiquitin ligase Deltex, the non-visual β-arrestin Kurtz and the ESCRT-III component Shrub. This activation path is not universal but appears to depend on the cellular context.

Genetic regions that interact with loss- and gain-of-function phenotypes of deltex implicate novel genes in Drosophila Notch signaling

The Notch signaling pathway is an evolutionarily conserved mechanism that regulates many cell fate decisions. The deltex (dx) gene encodes an E3-ubiquitin ligase that binds to the intracellular domain of the Notch protein and regulates Notch signaling in a positive manner. However, it is still not clear how Dx does this. We generated a transgenic line, GMR-dx, which overexpresses dx in the developing Drosophila eye disc. The GMR-dx line showed a rough-eye phenotype, specific transformation of a photoreceptor cell (R3 to R4), and a rotation defect in the ommatidia. This phenotype was suppressed in combination with a dx loss-of-function mutant, indicating that it was due to a dx gain-of-function. We previously reported that overexpression of Dx results in the stabilization of Notch in late endosomes. Here, we found that three motifs in Dx, a region that binds to Notch, a proline-rich motif and a RING-H2 finger, were required for this stabilization, although the relative activity of these variants in this assay did not always correspond to the severity of the rough-eye phenotype. In an attempt to identify novel genes of the Notch pathway, we tested a large collection of chromosomal deficiencies for the ability to modify the eye phenotypes of the GMR-dx line. Twelve genomic segments that enhanced the rough-eye phenotype of GMR-dx were identified. To evaluate the specificity of these interactions, we then determined whether the deletions also interacted with the wing phenotypes associated with a loss-of-function mutation of dx, dx24. Analyses based on whole-genome information allowed us to conclude that we have identified two novel loci that probably include uncharacterized genes involved in Dx-mediated Notch signaling.