In vivo evidence that Patched and Smoothened constitute distinct binding and transducing components of a Hedgehog receptor complex

Development ◽  
1998 ◽  
Vol 125 (24) ◽  
pp. 4943-4948 ◽  
Author(s):  
Y. Chen ◽  
G. Struhl
Keyword(s):  
Null Allele ◽  
Transmembrane Protein ◽  
Thin Strip ◽  
Receptor Complex ◽  
Wild Type ◽  
Posterior Compartment ◽  
Transmembrane Receptors ◽  
Anterior Compartment ◽  
Do So

During Drosophila development, cells belonging to the posterior compartment of each segment organize growth and patterning by secreting Hedgehog (Hh), a protein which induces a thin strip of adjacent cells in the anterior compartment to express the morphogens Decapentaplegic (Dpp) and Wingless (Wg). Hedgehog is bound and transduced by a receptor complex that includes Smoothened (Smo), a member of the Frizzled (Fz) family of seven-pass transmembrane receptors, as well as the multiple-pass transmembrane protein Patched (Ptc). Ptc is required for the binding of Hh to the complex as well as for the Hh-dependent activation of Smo within the complex. Here, we identify a likely null allele of the smo gene and use it to determine whether Hh is bound by Ptc alone, or by Smo in concert with Ptc. We find that cells devoid of Smo can sequester Hh, but that their ability to do so depends, as in wild-type cells, on the expression of high levels of Ptc protein. These results suggest that Ptc normally binds Hh without any help from Smo and hence favor a mechanism of signal transduction in which Hh binds specifically to Ptc and induces a conformational change leading to the release of latent Smo activity.

scholarly journals N-3-Hydroxy Dodecanoyl-DL-homoserine Lactone (OH-dDHL) Triggers Apoptosis of Bone Marrow-Derived Macrophages through the ER- and Mitochondria-Mediated Pathways

2021 ◽  
Vol 22 (14) ◽  
pp. 7565
Author(s):  
Kyungho Woo ◽  
Dong Ho Kim ◽  
Man Hwan Oh ◽  
Ho Sung Park ◽  
Chul Hee Choi
Keyword(s):  
Bone Marrow ◽  
Quorum Sensing ◽  
Deletion Mutant ◽  
Transmembrane Protein ◽  
Cell Communication ◽  
Homoserine Lactone ◽  
Host Responses ◽  
Wild Type ◽  
Mutant Strains

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N-3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI-deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI-deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.

Hedgehog is required for activation of engrailed during regeneration of fragmented Drosophila imaginal discs

Development ◽  
1999 ◽  
Vol 126 (8) ◽  
pp. 1591-1599 ◽  
Author(s):  
M.C. Gibson ◽  
G. Schubiger
Keyword(s):  
Normal Growth ◽  
Imaginal Discs ◽  
Ideal Model ◽  
Posterior Compartment ◽  
Anterior Compartment ◽  
In Vivo Culture ◽  
Pattern Regulation ◽  
Regulative Capacity ◽  
Anterior Posterior

Surgically fragmented Drosophila appendage primordia (imaginal discs) engage in wound healing and pattern regulation during short periods of in vivo culture. Prothoracic leg disc fragments possess exceptional regulative capacity, highlighted by the ability of anterior cells to convert to posterior identity and establish a novel posterior compartment. This anterior/posterior conversion violates developmental lineage restrictions essential for normal growth and patterning of the disc, and thus provides an ideal model for understanding how cells change fate during epimorphic pattern regulation. Here we present evidence that the secreted signal encoded by hedgehog directs anterior/posterior conversion by activating the posterior-specific transcription factor engrailed in regulating anterior cells. In the absence of hedgehog activity, prothoracic leg disc fragments fail to undergo anterior/posterior conversion, but can still regenerate missing anterior pattern elements. We suggest that hedgehog-independent regeneration within the anterior compartment (termed integration) is mediated by the positional cues encoded by wingless and decapentaplegic. Taken together, our results provide a novel mechanistic interpretation of imaginal disc pattern regulation and permit speculation that similar mechanisms could govern appendage regeneration in other organisms.

scholarly journals Ankylosis homologue mediates cellular efflux of ATP, not pyrophosphate

2021 ◽  
Author(s):  
Flora Szeri ◽  
Fatemeh Niaziorimi ◽  
Sylvia Donnelly ◽  
Nishat Fariha ◽  
Mariia Tertyshnaia ◽  
...  
Keyword(s):  
Culture Medium ◽  
Bone Matrix ◽  
Atp Release ◽  
Hek293 Cells ◽  
Wild Type ◽  
Plasma Membrane Protein ◽  
Mineralized Bone Matrix ◽  
Rule Out ◽  
Do So

AbstractThe plasma membrane protein Ankylosis Homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints, but we recently showed that ANKH releases large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. This ATP is converted extracellularly into PPi and AMP by the ectoenzyme Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANK. We now addressed this question by determining the effect of a complete absence of ENPP1 on ANKH-dependent extracellular PPi concentrations. Introduction of ANKH in ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi seen in ENPP1-proficient cells.Ank-activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1-/- mice contained < 2.5% of the PPi found in bones of wild type mice, showing that Enpp1-activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60-70 % of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification, therefore do so by extruding NTPs rather than PPi itself.

scholarly journals BACTERIOPHAGE T4 ENDONUCLEASES II AND IV, OPPOSITELY AFFECTED BY dCMP HYDROXYMETHYLASE ACTIVITY, HAVE DIFFERENT ROLES IN THE DEGRADATION AND IN THE RNA POLYMERASE-DEPENDENT REPLICATION OF T4 CYTOSINE-CONTAINING DNA

Genetics ◽  
1986 ◽  
Vol 114 (3) ◽  
pp. 669-685
Author(s):  
Karin Carlson ◽  
Aud Ȗvervatin
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Bacteriophage T4 ◽  
The Other ◽  
Wild Type ◽  
E Coli ◽  
Phage Dna ◽  
Dna Template ◽  
Do So

ABSTRACT Bacteriophage T4 mutants defective in gene 56 (dCTPase) synthesize DNA where cytosine (Cyt) partially or completely replaces hydroxymethylcytosine (HmCyt). This Cyt-DNA is degraded in vivo by T4 endonucleases II and IV, and by the exonuclease coded or controlled by genes 46 and 47.—Our results demonstrate that T4 endonuclease II is the principal enzyme initiating degradation of T4 Cyt-DNA. The activity of endonuclease IV, but not that of endonuclease II, was stimulated in the presence of a wild-type dCMP hydroxymethylase, also when no HmCyt was incorporated into phage DNA, suggesting the possibility of direct endonuclease IV-dCMP hydroxymethylase interactions. Endonuclease II activity, on the other hand, was almost completely inhibited in the presence of very small amounts of HmCyt (3-9% of total Cyt + HmCyt) in the DNA. Possible mechanisms for this inhibition are discussed.—The E. coli RNA polymerase modified by the products of T4 genes 33 and 55 was capable of initiating DNA synthesis on a Cyt-DNA template, although it probably cannot do so on an HmCyt template. In the presence of an active endonuclease IV, Cyt-DNA synthesis was arrested 10-30 min after infection, probably due to damage to the template. Cyt-DNA synthesis dependent on the unmodified (33  -  55  -) RNA polymerase was less sensitive to endonuclease IV action.

scholarly journals Effect of abx, bx and pbx mutations on expression of homeotic genes in Drosophila larvae.

Genetics ◽  
1990 ◽  
Vol 124 (4) ◽  
pp. 899-908 ◽  
Author(s):  
J W Little ◽  
C A Byrd ◽  
D L Brower
Keyword(s):  
Ectopic Expression ◽  
Wing Disc ◽  
Homeotic Genes ◽  
Wild Type ◽  
Posterior Compartment ◽  
Altered Expression ◽  
Anterior Compartment ◽  
Regulatory Domains ◽  
Wild Type Phenotype ◽  
Drosophila Larvae

Abstract We have examined the patterns of expression of the homeotic gene Ubx in imaginal discs of Drosophila larvae carrying mutations in the abx, bx and pbx regulatory domains. In haltere discs, all five bx insertion mutations examined led to a general reduction in Ubx expression in the anterior compartment; for a given allele, the strength of the adult cuticle phenotype correlated with the degree of Ubx reduction. Deletions mapping near or overlapping the sites of bx insertions, including three abx alleles and the bx34e-prv(bx-prv) allele, showed greatly reduced Ubx expression in parts of the anterior compartment of the haltere disc; however, anterior patches of strong Ubx expression often remained, in highly variable patterns. As expected, the pbx1 mutation led to reduced Ubx expression in the posterior compartment of the haltere disc; surprisingly, pbx1 also led to altered expression of the en protein near the compartment border in the central region of the disc. In the metathoracic leg, all the bx alleles caused extreme reduction in Ubx expression in the anterior regions, with no allele-specific differences. In contrast, abx and bx-prv alleles resulted in patchy anterior reductions in third leg discs. In the larval central nervous system, abx but not bx alleles affected Ubx expression; the bx-prv deletion gave a wild-type phenotype, but it could not fully complement abx mutations. In the posterior wing disc, the bx-prv allele, and to a much lesser extent the bx34e chromosome from which it arose, led to ectopic expression of Ubx. Unlike other grain-of-function mutations in the BX-C, this phenotype appeared to be partially recessive to wild type. Finally, we asked whether the ppx transformation, which results from early lack of Ubx+ function in the mesothorax and is seen in abx animals, is due to ectopic Scr expression. Some mesothoracic leg and wing discs from abx2 larvae displayed ectopic expression of Scr, which was variable in extent but always confined to the posterior compartment.

Abstract 73: A Novel Role of E2F1 in Stress-induced Cardiac Fibrosis

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Dauren Biyashev ◽  
Chan Boriboun ◽  
Gangjian Qin
Keyword(s):  
Cardiovascular System ◽  
Cardiac Fibrosis ◽  
Cardiovascular Health ◽  
Transmembrane Protein ◽  
Ang Ii ◽  
Tgf Beta ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts

Rationale: E2F1 transcription factor is best known as a cell cycle regulator. Recent reports indicate the importance of E2F1 in cardiovascular system, though its exact role is not clear. TGF-beta/Smad2,3 signaling pathway, on the other hand, has been long implicated in the regulation of cardiovascular health and numerous disease, including cardiac fibrosis. Interaction between these two major pathways has been reported in the cancer settings. Objective: To identify the possible interactions between E2F1 and TGF-beta/Smad2,3 signaling pathways in cardiovascular system and determine the functional outcome of these interactions in cardiac health. Methods and Results: E2F1-/- mice developed significantly higher degree of cardiac fibrosis than wild type mice in the Angiotensin II - induced cardiac fibrosis model. The levels of phosphorylated Smad2 and Smad3 were significantly higher in the hearts of E2F1-/- mice, as well as in mouse embryonic fibroblasts derived from E2F1-/- animals. Associated expression of collagen I was significantly increased in mouse embryonic fibroblasts derived from E2F1-/- animals, and treatment with TGF-beta resulted in higher collagen deposition compared to wild type fibroblasts. Treating animals with SB 431542, chemical inhibitor of Smad2,3 signaling, obliterated the difference in the degree of cardiac fibrosis between wild type and E2F1 knockout animals in the Ang II model. We discovered that levels of syndecan-4, heparan sulfate proteoglycan transmembrane protein implicated in fibrosis and known to interact with TGF-beta are significantly increased in both E2F1-/- fibroblasts and hearts. siRNA-mediated knockdown of syndecan-4 using siRNA resulted in decreased Smad2,3 phosphorylation in E2F1-/- MEFs. Similarly, down regulation of syndecan-4 in-vivo using morpholino lead to decreased cardiac fibrosis in E2F1-/- mice in Ang II model. Conclusions: E2F1 suppresses activation of TGF-beta/Smad 2,3 pathway. The E2F1-dependent suppression of cardiac fibrosis through TGF-beta/Smad 2,3 pathway is at least partially regulated by syndecan-4.

scholarly journals VPS35 Protects Against TMEM230-Mutation-Induced Progressive Locomotor Deficits in Drosophila

2021 ◽  
Author(s):  
Chao Ma ◽  
Xiaobo Wang ◽  
Wanli W Smith ◽  
Zhaohui Liu
Keyword(s):  
Protein Trafficking ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Transmembrane Protein ◽  
Critical Role ◽  
In Vivo Studies ◽  
Wild Type ◽  
Dopaminergic Neurodegeneration ◽  
Locomotor Deficits

Abstract BackgroundRecently, four Parkinson’s disease (PD)-linked mutations (Y92C, R141L, 184PGext*5 and 184Wext*5) in transmembrane protein 230 (TMEM230) were identified in PD patients, and these mutations have implications in protein trafficking and neurodegeneration. However, there is a lack of in vivo studies on the roles of PD-related variants of TMEM230 in PD pathogenesis.MethodsIn this study, we generated human wild-type (WT) and mutant TMEM230 (Y92C, R141L, 184PGext*5 and 184Wext*5) transgenic Drosophila using isoform Ⅱ cDNA. ResultsWe found that the expression of TMEM230 184PGext*5 in pan-neurons or dopaminergic neurons in Drosophila induced PD-like phenotypes, which included impaired locomotor ability, a shortened lifespan, reduced TH levels, and increased phosphorylated JNK and cleaved caspase-3 levels. Moreover, rotenone, a common pesticide, enhanced TMEM230-184PGext*5-induced PD-like phenotypes. In contrast, the overexpression of wild-type (WT) VPS35 rescued TMEM230-184PGext*5-induced PD-like phenotypes, while the knockdown of VPS35 by RNA interference (RNAi) or the expression of mutant VPS35 D620N worsened PD-like phenotypes. ConclusionThese results indicate that VPS35, as a downstream effector of TMEM230, plays a critical role in TMEM230-linked JNK/caspase-3 signalling pathways and that mutations in TMEM230 and VPS35 disrupt these pathways, resulting in dopaminergic neurodegeneration and PD-like phenotypes. These findings provide novel insight into the molecular mechanisms of mutant TME230- and VPS35-induced abnormalities underlying the pathogenesis of PD.

scholarly journals The fixABCX Genes in Rhodospirillum rubrum Encode a Putative Membrane Complex Participating in Electron Transfer to Nitrogenase

2004 ◽  
Vol 186 (7) ◽  
pp. 2052-2060 ◽  
Author(s):  
Tomas Edgren ◽  
Stefan Nordlund
Keyword(s):  
Electron Transfer ◽  
Electron Transport ◽  
Western Blotting ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Transmembrane Protein ◽  
Wild Type ◽  
Bisphosphate Carboxylase

ABSTRACT In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxgenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.

scholarly journals Dosage requirements of Ultrabithorax and bithoraxoid in the determination of segment identity in Drosophila melanogaster.

Genetics ◽  
1990 ◽  
Vol 124 (2) ◽  
pp. 357-366 ◽  
Author(s):  
S M Smolik-Utlaut
Keyword(s):  
Drosophila Embryo ◽  
Bithorax Complex ◽  
Wild Type ◽  
Posterior Compartment ◽  
Anterior Compartment ◽  
First Instar ◽  
Multiple Copies ◽  
Cellular Identity ◽  
Segmental Identity

Abstract The wild-type Ultrabithorax (Ubx) and bithoraxoid (bxd) functions are primarily responsible for establishing the identity of parasegment 6 (PS6) in the Drosophila embryo and thus the identity of the posterior compartment of the third thoracic segment (pT3) and the anterior compartment of the first abdominal segment (aA1) in the adult. The experiments described were designed to test the ability of an increased dosage of Ubx+ and bxd+ to affect the transformation of PS5 toward PS6. The results are consistent with the ideas that (1) multiple copies of Ubx+ and bxd+ cause some cells within PS5 to take on the characteristics of PS6 cells but do not cause an overall parasegmental transformation of PS5 toward PS6, (2) cellular identity depends not only on the activity of Ubx+ but on its concentration as well, and (3) that an interaction between Ubx+ and the wild-type Antennapedia (Antp) gene establishes segmental identity in pT2. In the first instar larvae carrying eight copies of Ubx+ and bxd+ the fine hairs of the T3 setal belt are transformed toward the hook-like structures of the A1 setal belt. Other structures within this segment are unaffected. In the adult, the haltere is reduced in size. The transformation of pT2 cells (wing) toward pT3 cells (haltere) is seen in adults carrying eight doses of wild type Ubx and bxd by decreasing the amount of the bithorax complex (BX-C) regulator Polycomb (Pc). However, the transformation of the T3 setal belt is not enhanced in the larvae of these animals. The interaction between the genes of the Antennapedia complex (ANT-C) and the Ubx+ and bxd+ functions in pT2 is dosage sensitive only when the animals carry one copy of Pc. In these animals, the transformation of wing toward haltere is significantly enhanced.

Analysis of cubitus interruptus regulation in Drosophila embryos and imaginal disks

Development ◽  
1995 ◽  
Vol 121 (6) ◽  
pp. 1625-1635 ◽  
Author(s):  
C. Schwartz ◽  
J. Locke ◽  
C. Nishida ◽  
T.B. Kornberg
Keyword(s):  
Zinc Finger Protein ◽  
Regulatory Region ◽  
Regulatory Sequences ◽  
Posterior Compartment ◽  
Cubitus Interruptus ◽  
Anterior Compartment ◽  
C Elegans ◽  
Imaginal Disks

The cubitus interruptus (ci) gene of Drosophila is expressed in all anterior compartment cells in both embryos and imaginal disks where it encodes a putative zinc-finger protein related to the vertebrate Gli and C. elegans Tra-1 proteins. Using ci/lacZ fusions, we located regulatory sequences responsible for the normal pattern of ci expression, and obtained evidence that separate elements regulate its expression in embryos and imaginal disks. Mutants that delete a portion of this regulatory region express ci ectopically in the posterior compartments of their wing imaginal disks and have wings with malformed posterior compartments. Similar deletions of ci/lacZ fusion constructs also result in ectopic posterior compartment expression. Evidence that the engrailed protein normally represses ci in posterior compartments includes the expansion of ci expression into posterior compartment cells that lack engrailed function, diminution of ci expression upon overexpression of engrailed protein in anterior compartment cells, and the ability of engrailed protein to bind to the ci regulatory region in vivo and in vitro. We suggest that engrailed protein directly represses ci expression in posterior compartment cells.

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