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1.  Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states 
eLife  2014;3:e02950.
The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation.
eLife digest
In animals, cells use a process called Notch signaling to communicate with neighboring cells. During this process, a protein known as a DSL ligand from one cell binds to a protein called a Notch receptor on a neighboring cell. This triggers a series of events in the neighboring cell that change how the genes in this cell are expressed. Notch signaling is involved in many processes including the early growth of embryos, the formation of organs and limbs, and the maintenance of stem cells throughout adult life.
Enzymes called Fringe enzymes can control Notch signaling by blocking or promoting the formation of the DSL ligand-Notch receptor pairs. It is also possible for a DSL ligand and a Notch receptor from the same cell to interact. This is thought to be important because it prevents an individual cell from sending and receiving Notch signals at the same time.
There are several different DSL ligands, Notch receptors and Fringe enzymes, so it is difficult to determine which configurations of receptors, ligands and Fringe enzymes can enable Notch signals to be sent or received. To address this problem, LeBon et al. investigated how Fringe enzymes acted on several different DSL-Notch receptor pairs in mammalian cells, and also in fruit flies. They focused in particular on the interactions that occurred within the same cell, as the role of Fringe enzymes in this type of interaction has not been examined previously.
The experiments revealed that Fringe proteins modify specific same-cell interactions in a way that enables a cell to receive one type of Notch signal from a neighboring cell and send a different type of Notch signal to another cell at the same time. More generally, these results show how an unconventional, ‘bottom-up’ approach can reveal the design principles of cell signaling systems, and suggest that it should now be possible to use these principles to try to understand which cell types send signals to which other cell types in many different contexts.
PMCID: PMC4174579  PMID: 25255098
cell signaling; developmental patterning; notch pathway; systems biology; D. melanogaster; other
3.  The Protein O-glucosyltransferase Rumi Modifies Eyes Shut to Promote Rhabdomere Separation in Drosophila 
PLoS Genetics  2014;10(11):e1004795.
The protein O-glucosyltransferase Rumi/POGLUT1 regulates Drosophila Notch signaling by adding O-glucose residues to the Notch extracellular domain. Rumi has other predicted targets including Crumbs (Crb) and Eyes shut (Eys), both of which are involved in photoreceptor development. However, whether Rumi is required for the function of Crb and Eys remains unknown. Here we report that in the absence of Rumi or its enzymatic activity, several rhabdomeres in each ommatidium fail to separate from one another in a Notch-independent manner. Mass spectral analysis indicates the presence of O-glucose on Crb and Eys. However, mutating all O-glucosylation sites in a crb knock-in allele does not cause rhabdomere attachment, ruling out Crb as a biologically-relevant Rumi target in this process. In contrast, eys and rumi exhibit a dosage-sensitive genetic interaction. In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells. The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a ∼50% decrease in the total level of Eys. Moreover, removing one copy of an endoplasmic reticulum chaperone enhances the rhabdomere attachment in rumi mutant animals. Altogether, our data suggest that O-glucosylation of Eys by Rumi ensures rhabdomere separation by promoting proper Eys folding and stability in a critical time window during the mid-pupal stage. Human EYS, which is mutated in patients with autosomal recessive retinitis pigmentosa, also harbors multiple Rumi target sites. Therefore, the role of O-glucose in regulating Eys may be conserved.
Author Summary
Glycosylation (addition of sugars to proteins and other organic molecules) is important for protein function and animal development. Each form of glycosylation is usually present on multiple proteins. Therefore, a major challenge in understanding the role of sugars in animal development is to identify which protein(s) modified by a specific sugar require the sugar modification for proper functionality. We have previously shown that an enzyme called Rumi adds glucose molecules to an important cell surface receptor called Notch, and that glucose plays a key role in the function of Notch both in fruit flies and in mammals. Using fruit flies, we have now identified a new Rumi target called “Eyes shut”, a secreted protein with a critical role in the optical isolation of neighboring photoreceptors in the fly eye. Our data suggest that glucose molecules on Eyes shut promote its folding and stability in a critical time window during eye development. Mutations in human Eyes shut result in a devastating form of retinal degeneration and loss of vision. Since human Eyes shut is also predicted to harbor glucose molecules, our work provides a framework to explore the role of sugar modifications in the biology of a human disease protein.
PMCID: PMC4238978  PMID: 25412384
4.  Negative Regulation of Notch Signaling by Xylose 
PLoS Genetics  2013;9(6):e1003547.
The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14–20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16–20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16–20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16–20 of Notch.
Author Summary
In multi-cellular organisms, neighboring cells need to communicate with each other to ensure proper cell fate decisions and differentiation. Signaling through the Notch receptors is the primary means by which local cell-cell communication is accomplished in animals. Given the broad usage of Notch signaling in animals and the host of human disease caused by Notch pathway misregulation, sophisticated mechanisms are required to adjust the strength of Notch signaling in each context. We have previously shown that addition of glucose residues to the Notch receptor promotes Notch signaling. Since these glucose residues on Notch can be extended by addition of xylose residues, we sought to determine whether xylose also plays a role in the regulation of Notch signaling. In contrast to glucose, we determine that xylose residues decrease Notch signaling in certain contexts by controlling Notch surface levels. Moreover, the xylose residues reside in a specific domain of Notch, unlike the glucose residues which are distributed throughout the Notch extracellular domain. Our data provide an example of signaling pathway regulation by altering the distribution of the short or elongated forms of a saccharide on a receptor protein, and offer a potential avenue for modulating Notch signaling as both a therapeutic modality and a tool in regenerative medicine.
PMCID: PMC3675014  PMID: 23754965
5.  Drosophila IAP1-Mediated Ubiquitylation Controls Activation of the Initiator Caspase DRONC Independent of Protein Degradation 
PLoS Genetics  2011;7(9):e1002261.
Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition (“undead” cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in “undead” cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs.
Author Summary
The Drosophila inhibitor of apoptosis 1 (DIAP1) readily promotes ubiquitylation of the CASPASE-9–like initiator caspase DRONC in vitro and in vivo. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by effector caspase inhibition—producing so-called “undead” cells—it has been proposed that DIAP1-mediated ubiquitylation would target full-length DRONC for proteasomal degradation, ensuring survival of normal cells. However, this has never been tested rigorously in vivo. By examining loss and gain of diap1 function, we show that DIAP1-mediated ubiquitylation does not trigger degradation of full-length DRONC. Our analysis demonstrates that DIAP1-mediated ubiquitylation controls DRONC processing and activation in a non-proteolytic manner. Interestingly, once DRONC is processed and activated, it has reduced protein stability. We also demonstrate that “undead” cells induce transcription of dronc, explaining increased protein levels of DRONC in these cells. This study re-defines the mechanism by which IAP-mediated ubiquitylation regulates caspase activity.
PMCID: PMC3164697  PMID: 21909282
6.  Dual roles of Drosophila p53 in cell death and cell differentiation 
Cell death and differentiation  2009;17(6):912-921.
The mammalian p53-family consists of p53, p63 and p73. While p53 accounts for tumor suppression through cell cycle arrest and apoptosis, the functions of p63 and p73 are more diverse and also include control of cell differentiation. The Drosophila genome contains only one p53 homolog, Dp53. Previous work has established that Dp53 induces apoptosis, but not cell cycle arrest. Here, by using the developing eye as a model, we show that Dp53-induced apoptosis is primarily dependent on the pro-apoptotic gene hid, but not reaper, and occurs through the canonical apoptosis pathway. Importantly, similar to p63 and p73, expression of Dp53 also inhibits cellular differentiation of photoreceptor neurons and cone cells in the eye independently of its apoptotic function. Intriguingly, expression of the human cell cycle inhibitor p21 or its Drosophila homolog dacapo can suppress both Dp53-induced cell death and differentiation defects in Drosophila eyes. These findings provide new insights into the pathways activated by Dp53 and reveal that Dp53 incorporates functions of multiple p53-family members.
PMCID: PMC3014827  PMID: 19960025
7.  Genetic control of programmed cell death (apoptosis) in Drosophila 
Fly  2009;3(1):78-90.
Programmed cell death, or apoptosis, is a highly conserved cellular process that has been intensively investigated in nematodes, flies and mammals. The genetic conservation, the low redundancy, the feasibility for high-throughput genetic screens and the identification of temporally and spatially regulated apoptotic responses make Drosophila melanogaster a great model for the study of apoptosis. Here, we review the key players of the cell death pathway in Drosophila and discuss their roles in apoptotic and non-apoptotic processes.
PMCID: PMC2702463  PMID: 19182545
programmed cell death; apoptosis; reaper; hid; grim; IAPs; caspases; non-apoptotic processes; compensatory proliferation
8.  Inactivation of Effector Caspases through Nondegradative Polyubiquitylation 
Molecular cell  2008;32(4):540-553.
Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase’s proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1’s E3 activity or drICE’s ubiquitin-acceptor lysines, abrogates DIAP1’s ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an “inactive” conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 “activation” and nondegradative polyubiquitylation.
PMCID: PMC2713662  PMID: 19026784
9.  Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila 
Development (Cambridge, England)  2005;132(24):5343-5352.
Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. We show that a subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. We provide evidence that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation.
PMCID: PMC2760325  PMID: 16280349
Mis-specification; Cell death; Transformation; Bicoid; Oskar; Hid; Drosophila
10.  The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously 
Development (Cambridge, England)  2007;135(1):43-52.
Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.
PMCID: PMC2277323  PMID: 18045837
Uba1; E1; Ubiquitin-activating enzyme; Apoptosis; Proliferation; Drosophila; Autonomous control; Non autonomous control

Results 1-10 (10)