Functions of O-fucosyltransferase in Notch trafficking and signaling: towards the end of a controversy?

The precise role of the O-fucosyltransferase Ofut1 in Notch-receptor trafficking has remained controversial. A recent study sheds new light on the non-catalytic activity of Ofut1 and provides further evidence that Ofut1 acts as a chaperone in the endoplasmic reticulum

Indirect evidence for Delta-dependent intracellular processing of Notch in Drosophila embryos

AbstractCell–cell signalling mediated by the receptor Notch regulates the differentiation of a wide variety of cell types in invertebrate and vertebrate species [1], but the mechanism of signal transduction following receptor activation is unknown. A recent model proposes that ligand binding induces intracellular processing of Notch [2–4]; the processed intracellular form of Notch then translocates to the nucleus and interacts with DNA-bound Suppressor of Hairless (Su(H)), a transcription factor required for target gene expression [5–8]. As intracellular processing of endogenous Notch has so far escaped immunodetection [1], we devised a sensitive nuclear-activity assay to monitor indirectly the processing of an engineered Notch in vivo. First, we show that the intracellular domain of Notch, fused to the DNA-binding domain of Gal4, regulated transcription, in a Delta-independent manner. Second, we show that full-length Notch, containing the Gal4 DNA-binding domain inserted 27 amino acids carboxy terminal to the transmembrane domain, activated transcription in a Delta-dependent manner. These results provide indirect evidence for a ligand-dependent intracellular processing event in vivo, supporting the view that Su(H)-dependent Notch signalling involves intracellular cleavage, and transcriptional regulation by processed Notch

Drosophila Ric-8 regulates Gαi cortical localization to promote Gαi-dependent planar orientation of the mitotic spindle during asymmetric cell division

International audienceLocalization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pI) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of Gαi depends on Partner of Inscuteable (Pins). We establish here that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Gαi, regulates cortical localization of the subunits Gαi and Gβ13F. Ric-8, Gαi and Pins are not necessary for the control of the antero-posterior orientation of the mitotic spindle during pI cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis

Crystal structure, biochemical and biophysical characterisation of NHR1 domain of E3 Ubiquitin ligase neutralized

International audienceNotch signaling controls diverse developmental decisions of central importance to cell activity. One of the conserved positive regulators of No- tch signaling is Neuralized, the E3 Ubiquitin li-gase enzyme that regulates signaling activity by endocytosis. Neuralized has two novel repeats, NHR1 and NHR2, with a RING finger motif at the C-terminus. Both endocytosis of the Notch ligand, Delta, and inhibition of Notch signaling by Tom, a bearded family member, require the NHR1 domain. Here we describe the first crystal structure of NHR1 domain from Drosophila me- lanogaster, solved to 2.1 Å resolution by X-ray analysis. Using NMR and other biophysical tech- niques we define a minimal binding region of Tom, consisting of 12 residues, which interacts with NHR1 and show by interfacial analysis of protein monolayers that NHR1 binds PI4P. Taken together, the studies provide insight into mo-lecular interactions that are important for Notch signaling

Stem Cell Proliferation Is Kept in Check by the Chromatin Regulators Kismet/CHD7/CHD8 and Trr/MLL3/4.

Chromatin remodeling accompanies differentiation, however, its role in self-renewal is less well understood. We report that in Drosophila, the chromatin remodeler Kismet/CHD7/CHD8 limits intestinal stem cell (ISC) number and proliferation without affecting differentiation. Stem-cell-specific whole-genome profiling of Kismet revealed its enrichment at transcriptionally active regions bound by RNA polymerase II and Brahma, its recruitment to the transcription start site of activated genes and developmental enhancers and its depletion from regions bound by Polycomb, Histone H1, and heterochromatin Protein 1. We demonstrate that the Trithorax-related/MLL3/4 chromatin modifier regulates ISC proliferation, colocalizes extensively with Kismet throughout the ISC genome, and co-regulates genes in ISCs, including Cbl, a negative regulator of Epidermal Growth Factor Receptor (EGFR). Loss of kismet or trr leads to elevated levels of EGFR protein and signaling, thereby promoting ISC self-renewal. We propose that Kismet with Trr establishes a chromatin state that limits EGFR proliferative signaling, preventing tumor-like stem cell overgrowths

Overexpression of Partner of Numb Induces Asymmetric Distribution of the PI4P 5-Kinase Skittles in Mitotic Sensory Organ Precursor Cells in Drosophila

Unequal segregation of cell fate determinants at mitosis is a conserved mechanism whereby cell fate diversity can be generated during development. In Drosophila, each sensory organ precursor cell (SOP) divides asymmetrically to produce an anterior pIIb and a posterior pIIa cell. The Par6-aPKC complex localizes at the posterior pole of dividing SOPs and directs the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized at the opposite pole. The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes. Here, we have examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs. Our analysis indicates that both Sktl and PIP2 reporters are uniformly distributed in mitotic SOPs. In the course of this study, we have observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP::PonLD) results in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. Our observation that Pon overexpression alters polar protein distribution is relevant because RFP::PonLD is often used as a polarity marker in dividing progenitors

Van Gogh and Frizzled Act Redundantly in the Drosophila Sensory Organ Precursor Cell to Orient Its Asymmetric Division

Drosophila sensory organ precursor cells (SOPs) divide asymmetrically along the anterior-posterior (a-p) body axis to generate two different daughter cells. Planar Cell Polarity (PCP) regulates the a-p orientation of the SOP division. The localization of the PCP proteins Van Gogh (Vang) and Frizzled (Fz) define anterior and posterior apical membrane domains prior to SOP division. Here, we investigate the relative contributions of Vang, Fz and Dishevelled (Dsh), a membrane-associated protein acting downstream of Fz, in orienting SOP polarity. Genetic and live imaging analyses suggest that Dsh restricts the localization of a centrosome-attracting activity to the anterior cortex and that Vang is a target of Dsh in this process. Using a clone border assay, we provide evidence that the Vang and fz genes act redundantly in SOPs to orient its polarity axis in response to extrinsic local PCP cues. Additionally, we find that the activity of Vang is dispensable for the non-autonomous polarizing activity of fz. These observations indicate that both Vang and Fz act as cues for downstream effectors orienting the planar polarity axis of dividing SOPs

Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster

A specific glycosphingolipid regulates the E3 ubiquitin ligase Mindbomb1, which promotes the ligand endocytosis necessary for Notch activation