scholarly journals Ykt6-dependent endosomal recycling is required for Wnt secretion in the Drosophila wing epithelium

Development ◽  
2020 ◽  
Vol 147 (15) ◽  
pp. dev185421 ◽  
Author(s):  
Karen Linnemannstöns ◽  
Leonie Witte ◽  
Pradhipa Karuna M ◽  
Jeanette Clarissa Kittel ◽  
Adi Danieli ◽  
...  
Keyword(s):  
Imaginal Disc ◽  
Regulatory Control ◽  
Secreting Cell ◽  
Recycling Endosomes ◽  
Signalling Molecules ◽  
Drosophila Wing ◽  
Endosomal Recycling ◽  
Biochemical Analyses

ABSTRACTMorphogens are important signalling molecules for tissue development and their secretion requires tight regulation. In the wing imaginal disc of flies, the morphogen Wnt/Wingless is apically presented by the secreting cell and re-internalized before final long-range secretion. Why Wnt molecules undergo these trafficking steps and the nature of the regulatory control within the endosomal compartment remain unclear. Here, we have investigated how Wnts are sorted at the level of endosomes by the versatile v-SNARE Ykt6. Using in vivo genetics, proximity-dependent proteomics and in vitro biochemical analyses, we show that most Ykt6 is present in the cytosol, but can be recruited to de-acidified compartments and recycle Wnts to the plasma membrane via Rab4-positive recycling endosomes. Thus, we propose a molecular mechanism by which producing cells integrate and leverage endocytosis and recycling via Ykt6 to coordinate extracellular Wnt levels.

scholarly journals Heterodimer Formation of the Homodimeric ABC Transporter OpuA

2021 ◽  
Vol 22 (11) ◽  
pp. 5912
Author(s):  
Patricia Alvarez-Sieiro ◽  
Hendrik R. Sikkema ◽  
Bert Poolman
Keyword(s):  
Abc Transporter ◽  
Conformational Dynamics ◽  
Host Organism ◽  
Affinity Tag ◽  
Practical Applications ◽  
Fundamental Research ◽  
Protein Multimerization ◽  
Biochemical Analyses

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

scholarly journals Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

2011 ◽  
Vol 80 (1) ◽  
pp. 3-13 ◽  
Author(s):  
Chen Li ◽  
Kurniyati ◽  
Bo Hu ◽  
Jiang Bian ◽  
Jianlan Sun ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biofilm Formation ◽  
Adult Population ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Multiple Organs ◽  
Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

scholarly journals Stable Transfection of the BovineNRAMP1 Gene into Murine RAW264.7 Cells: Effect onBrucella abortus Survival

2001 ◽  
Vol 69 (5) ◽  
pp. 3110-3119 ◽  
Author(s):  
Robert Barthel ◽  
Jianwei Feng ◽  
Jorge A. Piedrahita ◽  
David N. McMurray ◽  
Joe W. Templeton ◽  
...  
Keyword(s):  
Cell Lines ◽  
In Vitro Model ◽  
Regulatory Control ◽  
Natural Resistance ◽  
Ifn Γ ◽  
Flanking Region ◽  
Vitro Model ◽  
Raw264.7 Macrophage

ABSTRACT Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. BovineNRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovine NRAMP1 as stable transgenes under the regulatory control of the bovineNRAMP1 promoter in the murine RAW264.7 macrophage cell line (Bcg s ) to analyze the regulation of the NRAMP1 gene and its role in macrophage function. We demonstrated that the 5′-flanking region of bovineNRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expression of a transgene in murine macrophages. A polymorphism within a microsatellite in the 3′ untranslated region critically affects the expression of bovineNRAMP1 and the control of in vitro replication ofBrucella abortus but not Salmonella enterica serovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-γ)- and IFN-γ–lipopolysaccharide (LPS)-treated transfected cell lines, yet the resistant transfected cell lines produced significantly less NO than other cell lines, following stimulation with LPS at 24 and 48 h.

scholarly journals Evolution of a cis-acting SNP that controls Type VI Secretion in Vibrio cholerae

2022 ◽  
Author(s):  
Siu Lung Ng ◽  
Sophia A. Kammann ◽  
Gabi Steinbach ◽  
Tobias Hoffmann ◽  
Peter J. Yunker ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Phenotypic Diversity ◽  
Constitutive Expression ◽  
Regulatory Elements ◽  
Regulatory Control ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Intergenic Regions

Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes to new niches. Regulatory architecture is often inferred from transcription factor identification and genome analysis using purely computational approaches. However, there are few examples of phenotypic divergence that arise from the rewiring of bacterial regulatory circuity by mutations in intergenic regions, because locating regulatory elements within regions of DNA that do not code for protein requires genomic and experimental data. We identify a single cis-acting single nucleotide polymorphism (SNP) dramatically alters control of the type VI secretion system (T6), a common weapon for inter-bacterial competition. Tight T6 regulatory control is necessary for adaptation of the waterborne pathogen Vibrio cholerae to in vivo conditions within the human gut, which we show can be altered by this single non-coding SNP that results in constitutive expression in vitro. Our results support a model of pathogen evolution through cis-regulatory mutation and preexisting, active transcription factors, thus conferring different fitness advantages to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches.

Development and differentiation in vitro of Drosophila imaginal disc cells from dissociated early embryos

Development ◽  
1975 ◽  
Vol 33 (2) ◽  
pp. 487-498
Author(s):  
Andreas Dübendorfer ◽  
Glen Shields ◽  
James H. Sang
Keyword(s):  
Imaginal Disc ◽  
Larval Instar ◽  
Cell Types ◽  
The Third ◽  
Early Embryos ◽  
Disc Cells ◽  
Development And Differentiation ◽  
Pattern Specification

Embryos of Drosophila melanogaster, 6–8 h after oviposition, were dissociated and the cells cultured in vitro. Besides larval cell types, imaginal disc cells, assembled and growing in bloated monolayered vesicles, were obtained. The cells of these vesicles become competent to differentiate adult structures when treated with α-ecdysone or ecdysterone in vitro. Recognizable patterns of the adult fly are not formed though. If metamorphosis of imaginal cell vesicles from in vitro-cultures is induced in vivo by transplantation into host larvae of various ages within the third larval instar, recognizable patterns can differentiate provided the host larva does not metamorphose prior to 2 days after transplantation. The frequency of specific patterns in the implants can be increased by providing 9 days of culture in vivo (adult host flies) before metamorphosis. Passage through the third larval instar is not essential for these cells to produce identifiable patterns since culture in adult flies alone can achieve this. The quality of the differentiated pattern is not correlated with the extent of cell proliferation in the cultured tissues. The problem of pattern specification in vitro and in vivo is discussed.

scholarly journals The C-terminal region of the plasmid partitioning protein TubY is a tetramer that can bind membranes and DNA

2020 ◽  
Vol 295 (51) ◽  
pp. 17770-17780
Author(s):  
Ikuko Hayashi
Keyword(s):  
Lipid Membranes ◽  
Binding Protein ◽  
Coiled Coil ◽  
Type Iii ◽  
Daughter Cells ◽  
Lysine Residues ◽  
Stable Maintenance ◽  
Biochemical Analyses

Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY. Although the TubZ filament is sufficient to transport the TubR-centromere complex in vitro, TubY is still necessary for the stable maintenance of the plasmid. TubY contains an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coiled-coil followed by a cluster of lysine residues. This study determined the crystal structure of the C-terminal domain of TubY from the Bacillus cereus pXO1-like plasmid and showed that it forms a tetrameric parallel four-helix bundle that differs from the typical MerR family proteins with a dimeric anti-parallel coiled-coil. Biochemical analyses revealed that the C-terminal tail with the conserved lysine cluster helps TubY to stably associate with the TubR-centromere complex as well as to nonspecifically bind DNA. Furthermore, this C-terminal tail forms an amphipathic helix in the presence of lipids but must oligomerize to localize the protein to the membrane in vivo. Taken together, these data suggest that TubY is a component of the nucleoprotein complex within the partitioning machinery, and that lipid membranes act as mediators of type III systems.

scholarly journals Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo

2007 ◽  
Vol 178 (3) ◽  
pp. 465-476 ◽  
Author(s):  
Insa Geffers ◽  
Katrin Serth ◽  
Gavin Chapman ◽  
Robert Jaekel ◽  
Karin Schuster-Gossler ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Mouse Embryos ◽  
Functional Equivalence ◽  
Presomitic Mesoderm ◽  
Drosophila Wing ◽  
Wing Discs ◽  
Golgi Network ◽  
Notch Ligands

The Notch ligands Dll1 and Dll3 are coexpressed in the presomitic mesoderm of mouse embryos. Despite their coexpression, mutations in Dll1 and Dll3 cause strikingly different defects. To determine if there is any functional equivalence, we replaced Dll1 with Dll3 in mice. Dll3 does not compensate for Dll1; DLL1 activates Notch in Drosophila wing discs, but DLL3 does not. We do not observe evidence for antagonism between DLL1 and DLL3, or repression of Notch activity in mice or Drosophila. In vitro analyses show that differences in various domains of DLL1 and DLL3 individually contribute to their biochemical nonequivalence. In contrast to endogenous DLL1 located on the surface of presomitic mesoderm cells, we find endogenous DLL3 predominantly in the Golgi apparatus. Our data demonstrate distinct in vivo functions for DLL1 and DLL3. They suggest that DLL3 does not antagonize DLL1 in the presomitic mesoderm and warrant further analyses of potential physiological functions of DLL3 in the Golgi network.

The Role of Insulin in the Regulation of PEPCK and Gluconeogenesis In Vivo

2009 ◽  
Vol 05 (01) ◽  
pp. 34 ◽  
Author(s):  
Christopher J Ramnanan ◽  
Dale S Edgerton ◽  
Alan D Cherrington ◽  
◽  
◽  
...  
Keyword(s):  
Messenger Rna ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Hepatic Glucose Production ◽  
Glycogen Synthesis ◽  
Glucose Production ◽  
Regulatory Control ◽  
Glycolytic Flux ◽  
Large Animals

The regulation of gluconeogenesis by insulin is complex and can involve insulin-mediated events in the liver, as well as in several non-hepatic tissues. Given the complexity of this regulation, it is no surprise that there is considerable debate regarding insulin’s ability to regulate the rate of gluconeogenic formation of glucose-6-phosphate (GNG flux to G6P)in vivo. Conventional ‘textbook’ teaching (based onin vitrostudies of rat liver) depicts that insulin can inhibit this pathway by suppressing the transcription of the enzyme phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is widely considered to be a ‘rate-limiting’ enzyme with high control strength. Additionally, recent data in rodents have led to the conclusion that hyperinsulinemia in the brain can inhibit GNG flux to G6P, likely through transcriptional regulation of PEPCK. Recent data from the authors’ lab have confirmed that the molecular regulation of PEPCK messenger RNA (mRNA) and protein by insulin is conserved in large animals. Acute physiological hyperinsulinemia does not alter gluconeogenic formation of G6P, however, despite substantial reductions in PEPCK protein. This indicates that PEPCK has poor regulatory control over the pathwayin vivo. A physiological rise in insulin suppresses hepatic glucose production by inhibiting glycogenolysis and promoting glycogen synthesis, stimulating glycolytic flux, and redirecting gluconeogenically derived carbon to glycogen. This review documents the relevant ways in which insulin can regulate GNG flux to G6Pin vivo.

Hosting the preimplantation embryo: potentials and limitations of different approaches for analysing embryo - endometrium interactions in cattle

2013 ◽  
Vol 25 (1) ◽  
pp. 62 ◽  
Author(s):  
Susanne E. Ulbrich ◽  
Eckhard Wolf ◽  
Stefan Bauersachs
Keyword(s):  
Embryonic Development ◽  
In Vitro Models ◽  
Sexual Cycle ◽  
Factors Affecting ◽  
Signalling Molecules ◽  
Comprehensive Information ◽  
New Findings ◽  
Suitable Environment

Ongoing detailed investigations into embryo–maternal communication before implantation reveal that during early embryonic development a plethora of events are taking place. During the sexual cycle, remodelling and differentiation processes in the endometrium are controlled by ovarian hormones, mainly progesterone, to provide a suitable environment for establishment of pregnancy. In addition, embryonic signalling molecules initiate further sequences of events; of these molecules, prostaglandins are discussed herein as specifically important. Inadequate receptivity may impede preimplantation development and implantation, leading to embryonic losses. Because there are multiple factors affecting fertility, receptivity is difficult to comprehend. This review addresses different models and methods that are currently used and discusses their respective potentials and limitations in distinguishing key messages out of molecular twitter. Transcriptome, proteome and metabolome analyses generate comprehensive information and provide starting points for hypotheses, which need to be substantiated using further confirmatory methods. Appropriate in vivo and in vitro models are needed to disentangle the effects of participating factors in the embryo–maternal dialogue and to help distinguish associations from causalities. One interesting model is the study of somatic cell nuclear transfer embryos in normal recipient heifers. A multidisciplinary approach is needed to properly assess the importance of the uterine milieu for embryonic development and to use the large number of new findings to solve long-standing issues regarding fertility.

Wound healing and regenerative response of fragments of the Drosophila wing imaginal disc cultured in vitro

1979 ◽  
Vol 25 (12) ◽  
pp. 913-924 ◽  
Author(s):  
Margery J. Fain ◽  
Howard A. Schneiderman
Keyword(s):  
Wound Healing ◽  
Imaginal Disc ◽  
Wing Imaginal Disc ◽  
Drosophila Wing ◽  
Regenerative Response
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