The intracellular deletions of Delta and Serrate define dominant negative forms of the Drosophila Notch ligands

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2465-2474 ◽  
Author(s):  
X. Sun ◽  
S. Artavanis-Tsakonas
Keyword(s):  
Notch Pathway ◽  
Notch Signalling ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Wild Type ◽  
Mutant Forms ◽  
Intracellular Domains ◽  
Notch Ligands ◽  
Enhancer Of Split

We examined the function of the intracellular domains of the two known Drosophila Notch ligands, Delta and Serrate, by expressing wild-type and mutant forms in the developing Drosophila eye under the sevenless promoter. The expression of intracellularly truncated forms of either Delta (sev-DlTM) or Serrate (sev-SerTM) leads to extra photoreceptor phenotypes, similar to the eye phenotypes associated with loss-of-function mutations of either Notch or Delta. Consistent with the notion that the truncated ligands reduce. Notch signalling activity, the eye phenotypes of sev-DlTM and sev-SerTM are enhanced by loss-of-function mutations in the Notch pathway elements, Notch, Delta, mastermind, deltex and groucho, but are suppressed by a duplication of Delta or mutations in Hairless, a negative regulator of the pathway. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split m delta, a target of Notch signalling, is down-regulated by the truncated ligands highly expressed in neighbouring cells. We conclude that the truncated ligands act as antagonists of Notch signalling.

Secreted forms of DELTA and SERRATE define antagonists of Notch signaling in Drosophila

Development ◽  
1997 ◽  
Vol 124 (17) ◽  
pp. 3439-3448 ◽  
Author(s):  
X. Sun ◽  
S. Artavanis-Tsakonas
Keyword(s):  
Notch Signaling ◽  
Molecular Level ◽  
Notch Pathway ◽  
Dominant Negative ◽  
Genetic Interactions ◽  
Wing Margin ◽  
Loss Of Function ◽  
Dominant Negative Mutations ◽  
Notch Ligands ◽  
Enhancer Of Split

We examined the function of secreted forms of the two known Drosophila Notch ligands, DELTA and SERRATE, by expressing them under various promoters in the Drosophila developing eye and wing. The phenotypes associated with the expression of secreted Delta (DlS) or secreted Serrate (SerS) forms mimic loss-of-function mutations in the Notch pathway. Both genetic interactions between DlS or SerS transgenics and duplications or loss-of-function mutations of Delta or Serrate indicate that DlS and SerS behave as dominant negative mutations. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split mdelta, a target of Notch signaling, is down-regulated by SERS. The antagonistic nature of the two mutant secreted ligand forms in the eye is consistent with their behavior in the wing, where they are capable of down-regulating wing margin specific genes opposite to the effects of the endogenous ligands. This analysis uncovers secreted molecular antagonists of Notch signaling and provides evidence of qualitative differences in the actions of the two ligands DLS and SERS.

The Drosophila gene Bearded encodes a novel small protein and shares 3′ UTR sequence motifs with multiple Enhancer of split complex genes

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 4039-4051 ◽  
Author(s):  
M.W. Leviten ◽  
E.C. Lai ◽  
J.W. Posakony
Keyword(s):  
Sequence Similarity ◽  
Notch Pathway ◽  
Alpha Helix ◽  
Sequence Motifs ◽  
Wild Type ◽  
Gain Of Function ◽  
Small Protein ◽  
Drosophila Gene ◽  
Amphipathic Alpha Helix ◽  
Enhancer Of Split

Gain-of-function alleles of the Drosophila gene Bearded (Brd) cause sensory organ multiplication and loss phenotypes indistinguishable at the cellular level from those caused by loss-of-function mutations in the genes of the Notch pathway (Leviten, M. W. and Posakony, J. W. (1996). Dev. Biol. 176, 264–283). We have carried out a molecular analysis of the structure and expression of both wild-type and mutant Brd transcription units. We find that the Brd transcript is truncated and accumulates to substantially higher levels in the gain-of-function mutants, due to the insertion of a transposable element of the blood family in the Brd 3′ untranslated region (UTR). The wild-type Brd 3′ UTR includes three copies of a 9-nucleotide sequence (CAGCTTTAA) that we refer to as the ‘Brd box’. Moreover, the 3′ UTRs of Brd and of the m4 transcription unit of the Enhancer of split gene complex [E(spl)-C] exhibit an unusually high degree of sequence identity that includes not only Brd box sequences but also a second motif we refer to as the ‘GY box’ (GTCTTCC). We find that both the Brd box and the GY box are also present in the 3′ UTRs of several basic helix-loop-helix repressor-encoding genes of the E(spl)-C, often in multiple copies, suggesting that a novel mode of post-transcriptional regulation applies to Brd and many E(spl)-C genes. The fact that the more abundant Brd mutant mRNA lacks the GY box and two of the Brd boxes present in wild-type Brd mRNA suggests that either or both of these elements may confer instability on transcripts that contain them. Finally, we find that Brd encodes a novel small protein of only 81 amino acids that is predicted to include a basic amphipathic alpha-helix. The deduced Brd protein shows sequence similarity to the E(spl)m4 protein, which is likewise expected to include a basic amphipathic alpha-helix, suggesting that the two proteins have related biochemical functions.

Interactions among Delta, Serrate and Fringe modulate Notch activity during Drosophila wing development

Development ◽  
1998 ◽  
Vol 125 (15) ◽  
pp. 2951-2962 ◽  
Author(s):  
T. Klein ◽  
A.M. Arias
Keyword(s):  
Notch Signalling ◽  
Dominant Negative ◽  
Wing Development ◽  
Notch Signalling Pathway ◽  
Notch Ligand ◽  
Drosophila Wing ◽  
Signalling Molecule ◽  
Dorsoventral Boundary ◽  
Notch Ligands ◽  
Specific Nuclear Protein

The Notch signalling pathway plays an important role during the development of the wing primordium, especially of the wing blade and margin. In these processes, the activity of Notch is controlled by the activity of the dorsal specific nuclear protein Apterous, which regulates the expression of the Notch ligand, Serrate, and the Fringe signalling molecule. The other Notch ligand, Delta, also plays a role in the development and patterning of the wing. It has been proposed that Fringe modulates the ability of Serrate and Delta to signal through Notch and thereby restricts Notch signalling to the dorsoventral boundary of the developing wing blade. Here we report the results of experiments aimed at establishing the relationships between Fringe, Serrate and Delta during wing development. We find that Serrate is not required for the initiation of wing development but rather for the expansion and early patterning of the wing primordium. We provide evidence that, at the onset of wing development, Delta is under the control of apterous and might be the Notch ligand in this process. In addition, we find that Fringe function requires Su(H). Our results suggest that Notch signalling during wing development relies on careful balances between positive and dominant negative interactions between Notch ligands, some of which are mediated by Fringe.

scholarly journals NITROGEN LIMITATION ADAPTATION functions as a negative regulator of Arabidopsis immunity

2021 ◽  
Author(s):  
Beatriz Val Torregrosa ◽  
Mireia Bundo ◽  
Tzyy Jen Chiou ◽  
Victor Flors ◽  
Blanca San Segundo
Keyword(s):  
Immune Responses ◽  
Transcriptional Activation ◽  
Fungal Pathogens ◽  
Nitrogen Limitation ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Wild Type ◽  
Pathogen Infection ◽  
Fungal Elicitors ◽  
Resistance To Infection

Background: Phosphorus is an important macronutrient required for plant growth and development. It is absorbed through the roots in the form of inorganic phosphate (Pi). To cope with Pi limitation, plants have evolved an array of adaptive mechanisms to facilitate Pi acquisition and protect them from stress caused by Pi starvation. The NITROGEN LIMITATION ADAPTION (NLA) gene plays a key role in the regulation of phosphate starvation responses (PSR), its expression being regulated by the microRNA miR827. Stress caused by Pi limiting conditions might also affect the plant response to pathogen infection. However, cross-talk between phosphate signaling pathways and immune responses remains unclear. Results: In this study, we investigated whether NLA plays a role in Arabidopsis immunity. We show that loss-of-function of NLA and MIR827 overexpression causes an increase in phosphate (Pi) content which results in resistance to infection by the fungal pathogen Plectosphaerella cucumerina. The nla mutant plants accumulated callose in their leaves, a response that is also observed in wild-type plants that have been treated with high Pi. We also show that pathogen infection and treatment with fungal elicitors is accompanied by transcriptional activation of MIR827 and down-regulation of NLA. Upon pathogen challenge, nla plants exhibited higher levels of the phytoalexin camalexin compared to wild type plants. Camalexin level also increases in wild type plants treated with high Pi. Furthermore, the nla mutant plants accumulated salicylic acid (SA) and jasmonic acid (JA) in the absence of pathogen infection whose levels further increased upon pathogen. Conclusions: This study shows that NLA acts as a negative regulator of Arabidopsis immunity. Overaccumulation of Pi in nla plants positively affects resistance to infection by fungal pathogens. This piece of information reinforces the idea of signaling convergence between Pi and immune responses for the regulation of disease resistance in Arabidopsis.

Gtpase Immunity-Associated Protein Family Member 4 Contributes to the Enhanced Expansion of HSPCs in RUNX1 Deficient Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4344-4344
Author(s):  
Amanda Scholl ◽  
Kentson Lam ◽  
Alex Muselman ◽  
Tingdong Tang ◽  
Shinobu Matsuura ◽  
...  
Keyword(s):  
Family Member ◽  
Conflicts Of Interest ◽  
Protein Family ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Wild Type ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor

Abstract RUNX1 is the transcription factor described as the master regulator of hematopoiesis. Due to its central role during blood development, numerous RUNX1 mutations have been reported in hematologic abnormalities. Mice null for Runx1 die during embryogenesis, lacking definitive HSCs. Conditional Runx1Δ/Δ mice are viable, but exhibit a variety of blood abnormalities. The most salient defect in these Runx1Δ/Δ mice is expansion of the hematopoietic stem and progenitor cell (HSPC) population, measured as an increase in number of lineage negative, Sca1 positive, cKit positive (LSK) cells. A shortened form of RUNX1 (RUNX1SF) lacking the C-terminal and part of the N-terminal domain (41-214) acts as a dominant negative regulator of RUNX1 and hence also models RUNX1 loss-of-function. A differential gene expression analysis of HSPCs derived from Runx1Δ/Δ compared to wild type mice uncovered GTPase immunity-associated protein family member 4 (GIMAP4) as one of the genes most highly upregulated. Previous studies have focused almost exclusively on the role of GIMAP4 as a pro-apoptotic protein during T-cell development. This study illuminates a novel non-apoptotic role of GIMAP4 in a formerly unstudied HSPC context. Runx1Δ/Δ mice were crossed with Gimap4-/- mice to generate a double knockout (dKO) mouse line. These dKO mice exhibited attenuated HSPC proliferation in comparison to Runx1Δ/Δ mice, suggesting that GIMAP4 functions in this HSPC expansion phenotype. BMT experiments using lethally irradiated C57 mice and RUNX1SF transduced wild type versus Gimap4-/-bone marrow confirmed this result. GIMAP4 also worked independently and coordinately with RUNX1 to influence individual progenitor populations. Common lymphoid progenitors (CLP) were affected only by GIMAP4. Gimap4-/- mice exhibited an expansion of the CLP population, consistent with its pro-apoptotic role in lymphoid populations. Conversely, both RUNX1 and GIMAP4 coordinately exerted an effect on myeloid progenitor populations. Runx1Δ/Δ mice harbored expanded granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) populations. This expansion was not observed when GIMAP4 was also ablated. This suggests a pro-proliferative role of GIMAP4 specifically in myeloid populations. These opposing roles of GIMAP4 in lymphoid versus myeloid cells suggest a more contextual, cell-specific role of this GTPase protein. Ultimately, this study provides insight into how RUNX1 and GIMAP4 may coordinate to maintain HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the Trans-Golgi Network

2000 ◽  
Vol 151 (6) ◽  
pp. 1207-1220 ◽  
Author(s):  
Mona Wilcke ◽  
Ludger Johannes ◽  
Thierry Galli ◽  
Véronique Mayau ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Intracellular Compartments ◽  
Golgi Network ◽  
Mutant Forms ◽  
In Cells

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20°C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

A subset of notch functions during Drosophila eye development require Su(H) and the E(spl) gene complex

Development ◽  
1998 ◽  
Vol 125 (15) ◽  
pp. 2893-2900 ◽  
Author(s):  
P. Ligoxygakis ◽  
S.Y. Yu ◽  
C. Delidakis ◽  
N.E. Baker
Keyword(s):  
Cell Fate ◽  
Notch Pathway ◽  
Signal Transduction Pathway ◽  
Notch Signalling ◽  
Transduction Pathway ◽  
Notch Signalling Pathway ◽  
Bhlh Genes ◽  
Drosophila Eye ◽  
Enhancer Of Split ◽  
Signalling Process

The Notch signalling pathway is involved in many processes where cell fate is decided. Previous work showed that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signalling represses atonal expression during lateral specification. In this paper we investigate which known components of the Notch pathway are involved in each signalling process. Using clonal analysis we show that a ligand of Notch, Delta, is required along with Notch for both proneural enhancement and lateral specification, while the downstream components Suppressor-of-Hairless and Enhancer-of-Split are involved only in lateral specification. Our data point to a distinct signal transduction pathway during proneural enhancement by Notch. Using misexpression experiments we also show that particular Enhancer-of-split bHLH genes can differ greatly in their contribution to lateral specification.

scholarly journals Fluorescence Resonance Energy Transfer Microscopy of the Helicobacter pylori Vacuolating Cytotoxin within Mammalian Cells

2002 ◽  
Vol 70 (7) ◽  
pp. 3824-3832 ◽  
Author(s):  
David C. Willhite ◽  
Dan Ye ◽  
Steven R. Blanke
Keyword(s):  
Helicobacter Pylori ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dominant Negative ◽  
Wild Type ◽  
Vacuolating Cytotoxin ◽  
Vacuolated Cells ◽  
Mutant Forms

ABSTRACT The Helicobacter pylori vacuolating cytotoxin (VacA) binds and enters mammalian cells to induce cellular vacuolation. To investigate the quaternary structure of VacA within the intracellular environment where toxin cytotoxicity is elaborated, we employed fluorescence resonance energy transfer (FRET) microscopy. HeLa cells coexpressing full-length and truncated forms of VacA fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) were analyzed for FRET to indicate direct associations. These studies revealed that VacA-CFP and VacA-YFP interact within vacuolated cells, supporting the belief that monomer associations at an intracellular site are important for the toxin's vacuolating activity. In addition, the two fragments of proteolytically nicked VacA, p37 and p58, interact when coexpressed within mammalian cells. Because p37 and p58 function in trans when expressed separately within mammalian cells, these data suggest that the mechanism by which these two fragments induce vacuolation requires direct association. FRET microscopy also demonstrated interactions between mutant forms of VacA, as well as wild-type VacA with mutant forms of the toxin within vacuolated cells. Finally, a dominant-negative form of the toxin directly associates with wild-type VacA in cells where vacuolation was not detectable, suggesting that the formation of complexes comprising wild-type and dominant-negative forms of toxin acts to block intracellular toxin function.

scholarly journals The Membrane Dynamics of Pexophagy Are Influenced by Sar1p in Pichia pastoris

2008 ◽  
Vol 19 (11) ◽  
pp. 4888-4899 ◽  
Author(s):  
Laura A. Schroder ◽  
Michael V. Ortiz ◽  
William A. Dunn
Keyword(s):  
Pichia Pastoris ◽  
Membrane Dynamics ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Guanosine Triphosphate ◽  
Guanosine Diphosphate ◽  
Mutant Forms ◽  
Negative Mutant

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy.

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