Multiple Signaling Mechanisms of the UNC-6/netrin Receptors UNC-5 and UNC-40/DCC in Vivo

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1071-1080 ◽  
Author(s):  
David C Merz ◽  
Hong Zheng ◽  
Marie T Killeen ◽  
Aldis Krizus ◽  
Joseph G Culotti
Keyword(s):  
Dominant Negative ◽  
Genetic Interactions ◽  
Receptor Subtypes ◽  
Negative Effects ◽  
Receptor Complexes ◽  
Guidance Cue ◽  
Distal Tip Cells ◽  
Multiple Signaling ◽  
Tip Cells

Abstract Cell and growth cone migrations along the dorsoventral axis of Caenorhabditis elegans are mediated by the UNC-5 and UNC-40 receptor subtypes for the secreted UNC-6 guidance cue. To characterize UNC-6 receptor function in vivo, we have examined genetic interactions between unc-5 and unc-40 in the migrations of the hermaphrodite distal tip cells. We report that cell migration defects as severe as those associated with a null mutation in unc-6 are produced only by null mutations in both unc-5 and unc-40, indicating that either receptor retains some partial function in the absence of the other. We show that hypomorphic unc-5 alleles exhibit two distinct types of interallelic genetic interactions. In an unc-40 wild-type genetic background, some pairs of hypomorphic unc-5 alleles exhibit a partial allelic complementation. In an unc-40 null background, however, we observed that unc-5 hypomorphs exhibit dominant negative effects. We propose that the UNC-5 and UNC-40 netrin receptors can function to mediate chemorepulsion in DTC migrations either independently or together, and the observed genetic interactions suggest that this flexibility in modes of signaling results from the formation of a variety of oligomeric receptor complexes.

Lack of dominant-negative effects of a truncated γc on retroviral-mediated gene correction of immunodeficient mice

Blood ◽  
2001 ◽  
Vol 97 (6) ◽  
pp. 1618-1624 ◽  
Author(s):  
Makoto Otsu ◽  
Kazuo Sugamura ◽  
Fabio Candotti
Keyword(s):  
Gene Therapy ◽  
Murine Model ◽  
Severe Combined Immunodeficiency ◽  
Dominant Negative ◽  
Interleukin 4 ◽  
Gene Correction ◽  
Negative Effects ◽  
Gamma Chain ◽  
Immunodeficient Mice

A recent clinical trial of gene therapy for X-linked severe combined immunodeficiency (XSCID) has shown that retroviral-mediated gene correction of bone marrow stem cells can lead to the development of normal immune function. These exciting results have been preceded by successful immune reconstitution in several XSCID mouse models, all carrying null mutations of the common gamma chain (γc). One question not formally addressed by these previous studies is that of possible dominant-negative effects of the endogenous mutant γc protein on the activity of the wild-type transferred gene product. The present work was therefore undertaken to study whether corrective gene transfer was applicable to an XSCID murine model with preserved expression of a truncated γc molecule (Δγc+-XSCID). Gene correction of Δγc+-XSCID mice resulted in the reconstitution of lymphoid development, and preferential repopulation of lymphoid organs by gene-corrected cells demonstrated the selective advantage of γc-expressing cells in vivo. Newly developed B cells showed normalization of lipopolysaccharide-mediated proliferation and interleukin-4 (IL-4)–induced immunoglobulin G1 isotype switching. Splenic T cells and thymocytes of treated animals proliferated normally to mitogens and responded to the addition of IL-2, IL-4, and IL-7, indicating functional reconstitution of γc-sharing receptors. Repopulated thymi showed a clear increase of CD4−/CD8− and CD8+fractions, both dramatically reduced in untreated Δγc+-XSCID mice. These improvements were associated with the restoration of Bcl-2 expression levels and enhanced cell survival. These data indicate that residual expression of the endogenous truncated γc did not lead to dominant-negative effects in this murine model and suggest that patient selection may not be strictly necessary for gene therapy of XSCID.

scholarly journals Novel Transcription Coactivator Complex Containing Activating Signal Cointegrator 1

2002 ◽  
Vol 22 (14) ◽  
pp. 5203-5211 ◽  
Author(s):  
Dong-Ju Jung ◽  
Hee-Sook Sung ◽  
Young-Wha Goo ◽  
Hyun Mi Lee ◽  
Ok Ku Park ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Serum Response Factor ◽  
Ectopic Expression ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Mutant Form ◽  
Negative Effects ◽  
Direct Binding ◽  
Serum Response

ABSTRACT Human activating signal cointegrator 1 (hASC-1) was originally isolated as a transcriptional coactivator of nuclear receptors. Here we report that ASC-1 exists as a steady-state complex associated with three polypeptides, P200, P100, and P50, in HeLa nuclei; stimulates transactivation by serum response factor (SRF), activating protein 1 (AP-1), and nuclear factor κB (NF-κB) through direct binding to SRF, c-Jun, p50, and p65; and relieves the previously described transrepression between nuclear receptors and either AP-1 or NF-κB. Interestingly, ectopic expression of Caenorhabditis elegans ASC-1 (ceASC-1), an ASC-1 homologue that binds P200 and P100, like hASC-1, while weakly interacting only with p65, in HeLa cells appears to replace endogenous hASC-1 from the hASC-1 complex and exerts potent dominant-negative effects on AP-1, NF-κB, and SRF transactivation. In addition, neutralization of endogenous P50 by single-cell microinjection of a P50 antibody inhibits AP-1 transactivation; the inhibition is relieved by coexpression of wild-type P50, but not of P50ΔKH, a mutant form that does not interact with P200. Overall, these results suggest that the endogenous hASC-1 complex appears to play an essential role in AP-1, SRF, and NF-κB transactivation and to mediate the transrepression between nuclear receptors and either AP-1 or NF-κB in vivo.

scholarly journals SRP54 mutations induce Congenital Neutropenia via dominant-negative effects on XBP1 splicing

Blood ◽  
2020 ◽  
Author(s):  
Christoph Schürch ◽  
Thorsten Schaefer ◽  
Joëlle Seraina Müller ◽  
Pauline Hanns ◽  
Marlon Arnone ◽  
...  
Keyword(s):  
De Novo ◽  
Dominant Negative ◽  
Severe Neutropenia ◽  
In Vivo Model ◽  
Factor X ◽  
Negative Effects ◽  
Congenital Neutropenia ◽  
Developmental Defects ◽  
Xbp1 Mrna

Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN), displaying symptoms overlapping with Shwachman-Diamond-Syndrome (SDS).1 Here, we investigate srp54 KO zebrafish as the first in vivo model of SRP54 deficiency. srp54-/- zebrafish are embryonically lethal and display, next to severe neutropenia, multi-systemic developmental defects. In contrast, srp54+/- zebrafish are viable, fertile and only show mild neutropenia. Interestingly, injection of human SRP54 mRNAs carrying mutations observed in patients (T115A, T117Δ and G226E) aggravated neutropenia and induced pancreatic defects in srp54+/- fish, mimicking the corresponding human clinical phenotypes. These data suggest that the variable phenotypes observed in patients may be due to mutation-specific dominant negative effects on the functionality of the residual wildtype SRP54 protein. Consistently, overexpression of mutated SRP54 also induced neutropenia in wildtype fish and impaired granulocytic maturation of human promyelocytic HL-60 cells as well as of healthy cord-blood derived CD34+ HSPCs. Mechanistically, srp54 mutant fish and human cells show impaired unconventional splicing of the transcription factor X-box binding protein 1 (Xbp1). Vice-versa, xbp1 morphants recapitulate phenotypes observed in srp54 deficiency and, importantly, injection of spliced, but not unspliced xbp1 mRNA rescues neutropenia in srp54+/- zebrafish. Together, these data indicate that SRP54 is critical for the development of various tissues, with neutrophils reacting most sensitively to SRP54 loss. The heterogenic phenotypes observed in patients, ranging from mild CN to SDS-like disease, may be due to different dominant negative effects of mutated SRP54 proteins on downstream XBP1 splicing, which represents a potential therapeutic target.

Ectopic expression of a Drosophila InsP3R channel mutant has dominant-negative effects in vivo

Cell Calcium ◽  
2006 ◽  
Vol 39 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Sonal Srikanth ◽  
Santanu Banerjee ◽  
Gaiti Hasan
Keyword(s):  
Ectopic Expression ◽  
Dominant Negative ◽  
Negative Effects

Regulation of the UNC-5 netrin receptor initiates the first reorientation of migrating distal tip cells in Caenorhabditis elegans

Development ◽  
2000 ◽  
Vol 127 (3) ◽  
pp. 585-594 ◽  
Author(s):  
M. Su ◽  
D.C. Merz ◽  
M.T. Killeen ◽  
Y. Zhou ◽  
H. Zheng ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Developmental Stage ◽  
Phase 1 ◽  
Critical Role ◽  
The Body ◽  
Dependent Manner ◽  
C Elegans ◽  
Guidance Cue ◽  
Distal Tip Cells ◽  
Tip Cells

Cell migrations play a critical role in animal development and organogenesis. Here, we describe a mechanism by which the migration behaviour of a particular cell type is regulated temporally and coordinated with over-all development of the organism. The hermaphrodite distal tip cells (DTCs) of Caenorhabditis elegans migrate along the body wall in three sequential phases distinguished by the orientation of their movements, which alternate between the anteroposterior and dorsoventral axes. The ventral-to-dorsal second migration phase requires the UNC-6 netrin guidance cue and its receptors UNC-5 and UNC-40, as well as additional, UNC-6-independent guidance systems. We provide evidence that the transcriptional upregulation of unc-5 in the DTCs is coincident with the initiation of the second migration phase, and that premature UNC-5 expression in these cells induces precocious turning in an UNC-6-dependent manner. The DAF-12 steroid hormone receptor, which regulates developmental stage transitions in C. elegans, is required for initiating the first DTC turn and for coincident unc-5 upregulation. We also present evidence for the existence of a mechanism that opposes or inhibits UNC-5 function during the longitudinal first migration phase and for a mechanism that facilitates UNC-5 function during turning. The facilitating mechanism presumably does not involve transcriptional regulation of unc-5 but may represent an inhibition of the phase 1 mechanism that opposes or inhibits UNC-5. These results, therefore, reveal the existence of two mechanisms that regulate the UNC-5 receptor that are critical for responsiveness to the UNC-6 netrin guidance cue and for linking the directional guidance of migrating distal tip cells to developmental stage advancements.

scholarly journals In vivo Modeling Implicates APOL1 in Nephropathy: Evidence for Dominant Negative Effects and Epistasis under Anemic Stress

PLoS Genetics ◽  
2015 ◽  
Vol 11 (7) ◽  
pp. e1005349 ◽  
Author(s):  
Blair R. Anderson ◽  
David N. Howell ◽  
Karen Soldano ◽  
Melanie E. Garrett ◽  
Nicholas Katsanis ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Negative Effects

scholarly journals Correction: In vivo Modeling Implicates APOL1 in Nephropathy: Evidence for Dominant Negative Effects and Epistasis under Anemic Stress

PLoS Genetics ◽  
2015 ◽  
Vol 11 (9) ◽  
pp. e1005459 ◽  
Author(s):  
Blair R. Anderson ◽  
David N. Howell ◽  
Karen Soldano ◽  
Melanie E. Garrett ◽  
Nicholas Katsanis ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Negative Effects

scholarly journals Probing the domain structure of FtsZ by random truncation and insertion of GFP

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 4033-4043 ◽  
Author(s):  
Masaki Osawa ◽  
Harold P. Erickson
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Wild Type ◽  
Negative Effects ◽  
Terminal Domain ◽  
Z Ring ◽  
Green Fluorescent

Random transposon-mediated mutagenesis has been used to create truncations and insertions of green fluorescent protein (GFP), and Venus-yellow fluorescent protein (YFP), in Escherichia coli FtsZ. Sixteen unique insertions were obtained, and one of them, in the poorly conserved C-terminal spacer, was functional for cell division with the Venus-YFP insert. The insertion of enhanced GFP (eGFP) at this same site was not functional; Venus-YFP was found to be superior to eGFP in other respects too. Testing the constructs for dominant negative effects led to the following general conclusion. The N-terminal domain, aa 1–195, is an independently folding domain that can poison Z-ring function when expressed without a functional C-terminal domain. The effects were weak, requiring expression of the mutant at 3–5 times the level of wild-type FtsZ. The C-terminal domain, aa 195–383, was also independently folding, but had no activity in vivo. The differential activity of the N- and C-terminal domains suggests that FtsZ protofilament assembly is directional, with subunits adding primarily at the bottom of the protofilament. Directional assembly could occur by either a treadmilling or a dynamic instability mechanism.

scholarly journals Disrupted choline clearance and sustained acetylcholine release in vivo by a common choline transporter coding variant associated with poor attentional control in humans

2021 ◽  
Author(s):  
Eryn Donovan ◽  
Cassandra Avila ◽  
Vinay Parikh ◽  
Cristina Fenollar-Ferrer ◽  
Randy D. Blakely ◽  
...  
Keyword(s):  
Frontal Cortex ◽  
Conformational Changes ◽  
Cognitive Disorders ◽  
Cholinergic Neurons ◽  
Dominant Negative ◽  
Ach Release ◽  
Negative Effects ◽  
Choline Transport ◽  
Choline Transporter

Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional capacity as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, using in vivo cortical choline biosensing, CHT-mediated choline transport, and ACh release. CHT-mediated clearance of choline in mice expressing one or two Val89 alleles was reduced by over 7-fold relative to wild type (WT) mice, suggesting dominant-negative effects. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10 min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. Consistent with this hypothesis, structural modeling revealed that Val89 may attenuate choline transport by changing the ability of choline to induce conformational changes of CHT that support normal transport rates. Our findings suggest that diminished, sustained cholinergic signaling capacity in the frontal cortex underlies perturbed attentional performance in individuals expressing CHT Val89. Our work supports the utility of the CHT Val89 mouse model as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction.

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