Insect cuticle, an in vivo model of protein trafficking

1999 ◽  
Vol 112 (13) ◽  
pp. 2113-2124 ◽  
Author(s):  
G. Csikos ◽  
K. Molnar ◽  
N.H. Borhegyi ◽  
G.C. Talian ◽  
M. Sass
Keyword(s):  
Protein Trafficking ◽  
Developmental Stages ◽  
Fat Body ◽  
Epidermal Cells ◽  
In Vivo Model ◽  
Larval Stages ◽  
Lepidopteran Larvae ◽  
Cuticle Proteins ◽  
Pupal Cuticle

In the course of this study more than 20 proteins have been isolated from the larval cuticle of Manduca sexta. Synthesis, secretion, transport and accumulation of four particular proteins, representative members of four characteristic groups, were followed during metamorphosis by immunoblot and immuncytochemical methods and are described in detail in this paper. We established that only some of the proteins of the soft cuticle of Lepidopteran larvae are synthesized in epidermal cells at the beginning of the larval stages and are digested during the moulting period (MsCP29). Other proteins (MsCP30/11) are secreted into the cuticle by the epidermal cells in different forms during various developmental stages. Some proteins are secreted apically during the feeding period, but before ecdysis they are then taken up by epidermal cells and transported in a basolateral direction back into the hemolymph and saved in an immunologically intact form by the fat body cells (MsCP12.3). Some cuticle proteins have a non-epidermal origin. They are transported from the hemolymph into the cuticle. Before and during ecdysis these molecules reappear in the hemolymph and are detectable again in the pupal cuticle (MsCP78). Our data prove that the cuticle is not a non-living part of the insect body: it is not only an inert, protective armor, but maintains a continuous and dynamic metabolic connection with the other organs of the organism.

scholarly journals Proteomics of protein trafficking by in vivo tissue-specific labeling

2020 ◽  
Author(s):  
Ilia A. Droujinine ◽  
Dan Wang ◽  
Yanhui Hu ◽  
Namrata D. Udeshi ◽  
Luye Mu ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Large Scale ◽  
Fat Body ◽  
Secreted Proteins ◽  
Intracellular Protein ◽  
Quantitative Mass Spectrometry ◽  
Subcellular Compartment ◽  
Human Orthologs ◽  
Identification And Characterization

AbstractSecreted interorgan communication factors encode key regulators of homeostasis. However, long-standing questions surround their origins/destinations, mechanisms of interactions, and the number of proteins involved. Progress has been hindered by the lack of methodologies for these factors’ large-scale identification and characterization, as conventional approaches cannot identify low-abundance factors and the origins and destinations of secreted proteins. We established an in vivo platform to investigate secreted protein trafficking between organs proteome-wide, whereby engineered promiscuous biotin ligase BirA*G3 (a relative of TurboID) biotinylates all proteins in a subcellular compartment of one tissue, and biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Using this platform, we identified 51 putative muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, of which 60-70% have human orthologs. We demonstrate, in particular, that conserved fat body-derived novel interorgan communication factors CG31326, CG2145, and CG4332 promote muscle activity. Our results indicate that the communication network of secreted proteins is vast, and we identified systemic functions for a number of these factors. This approach is widely applicable to studies in interorgan, local and intracellular protein trafficking networks, non-conventional secretion, and to mammalian systems, under healthy or diseased states.One Sentence SummaryWe developed an in vivo platform to investigate protein trafficking between organs proteome-wide, provide a resource for interorgan communication factors, and determined conserved adipokines that affect muscles.

scholarly journals Screening for insulin-independent pathways that modulate glucose homeostasis identifies androgen receptor antagonists

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sri Teja Mullapudi ◽  
Christian SM Helker ◽  
Giulia LM Boezio ◽  
Hans-Martin Maischein ◽  
Anna M Sokol ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Glucose Homeostasis ◽  
Rapid Screening ◽  
In Vivo Model ◽  
Glucose Levels ◽  
Larval Stages ◽  
Insulin Dependent ◽  
Screening Platform ◽  
Core Characteristics

Pathways modulating glucose homeostasis independently of insulin would open new avenues to combat insulin resistance and diabetes. Here, we report the establishment, characterization, and use of a vertebrate ‘insulin-free’ model to identify insulin-independent modulators of glucose metabolism. insulin knockout zebrafish recapitulate core characteristics of diabetes and survive only up to larval stages. Utilizing a highly efficient endoderm transplant technique, we generated viable chimeric adults that provide the large numbers of insulin mutant larvae required for our screening platform. Using glucose as a disease-relevant readout, we screened 2233 molecules and identified three that consistently reduced glucose levels in insulin mutants. Most significantly, we uncovered an insulin-independent beneficial role for androgen receptor antagonism in hyperglycemia, mostly by reducing fasting glucose levels. Our study proposes therapeutic roles for androgen signaling in diabetes and, more broadly, offers a novel in vivo model for rapid screening and decoupling of insulin-dependent and -independent mechanisms.

scholarly journals Blood Vessel Imaging at Pre-Larval Stages of Zebrafish Embryonic Development

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 886
Author(s):  
Alexander S. Machikhin ◽  
Mikhail V. Volkov ◽  
Alexander B. Burlakov ◽  
Demid D. Khokhlov ◽  
Andrey V. Potemkin
Keyword(s):  
Embryonic Development ◽  
Blood Vessel ◽  
Developmental Stages ◽  
Cardiac Activity ◽  
Spatiotemporal Analysis ◽  
Invasive Technique ◽  
Cardiovascular Research ◽  
Larval Stages ◽  
Vessel Imaging

The zebrafish (Danio rerio) is an increasingly popular animal model biological system. In cardiovascular research, it has been used to model specific cardiac phenomena as well as to identify novel therapies for human cardiovascular disease. While the zebrafish cardiovascular system functioning is well examined at larval stages, the mechanisms by which vessel activity is initiated remain a subject of intense investigation. In this research, we report on an in vivo stain-free blood vessel imaging technique at pre-larval stages of zebrafish embryonic development. We have developed the algorithm for the enhancement, alignment and spatiotemporal analysis of bright-field microscopy images of zebrafish embryos. It enables the detection, mapping and quantitative characterization of cardiac activity across the whole specimen. To validate the proposed approach, we have analyzed multiple data cubes, calculated vessel images and evaluated blood flow velocity and heart rate dynamics in the absence of any anesthesia. This non-invasive technique may shed light on the mechanism of vessel activity initiation and stabilization as well as the cardiovascular system’s susceptibility to environmental stressors at early developmental stages.

Prepupal differentiation in Drosophila: distinct cell types elaborate a shared structure, the pupal cuticle, but accumulate transcripts in unique patterns

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 649-656 ◽  
Author(s):  
K. Fechtel ◽  
D.K. Fristrom ◽  
J.W. Fristrom
Keyword(s):  
Cell Types ◽  
Imaginal Discs ◽  
Specific Expression ◽  
Distinct Cell ◽  
Cuticle Proteins ◽  
Shared Structure ◽  
Pupal Cuticle

The components of the pupal cuticle are the main differentiation products synthesized by both the larval and adult epidermis during the prepupal period of Drosophila development. The pupal cuticle is formed in vitro by imaginal discs in response to a 6 h pulse of 20-hydroxyecdysone (20-HE). We previously described the isolation and initial characterization of four ecdysone-dependent genes (EDGs) whose expression in imaginal discs occurs only in response to a pulse of 20-HE. In this report, we demonstrate that the pattern of temporal and tissue-specific expression of these EDGs in vivo is like that expected for genes that encode pupal cuticle proteins. Transcripts of these genes are detected in prepupae only in the epidermis and only when cuticle components are synthesized and secreted. Nonetheless, their temporal and spatial patterns of accumulation differ. EDG-84A-1 transcripts accumulate only in prepupae and only in imaginal cells. EDG-78E and EDG-64CD transcripts accumulate at the same time in both larval and imaginal cells. EDG42-A transcripts appear first in prepupae in imaginal cells and then, after a 2–4 h lag, in larval cells. It is evident that some genes are not restricted in their expression to only larval or imaginal epidermis.

scholarly journals Evaluation of Cosmetic Properties of Natural Ingredients in the Trás-os-Montes area: a PhD Project

2021 ◽  
Author(s):  
Sara Gonçalves ◽  
Isabel Gaivão
Keyword(s):  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Human Lymphocytes ◽  
Ecological Impact ◽  
Model Organism ◽  
The Body ◽  
In Vivo Model ◽  
Dna Integrity ◽  
The European Union

The term cosmetics refers to a product applied to the body for the purpose of beautifying, cleansing or improving appearance and enhancing attractive features. The natural cosmetics market has grown since the consumer took consciousness of the concept of natural-based ingredients. A great number of cosmetics have noxious and chemically-potent substances and have an ecological impact on the environment. A study performed by the Danish Council THINK Chemicalsfound that in total 65 chemicals of concern were found in 39 products. This means consumers are exposed to these chemicals, perhaps in a daily basis. They also found that three products contained illegal ingredients in the European Union. Thus, the use of natural and organic cosmetics becomes increasingly important. This requires a strong investigation into the benefits that fruits and plants can bring to health. The PhD project will focus on four natural ingredients common in the Trás-os-Montes area: almond (Prunus dulcis), elderberry (Sambucus nigra), olive (Olea europaea) and grapes (Vitis vinifera). The general purpose of this PhD project is to evaluate the cosmetic properties of the natural ingredients towards the DNA integrity promotion. Additionally, it is intended to evaluate genoprotection, longevity and prolificacy of the natural ingredients in Drosophila melanogaster. The short life cycle, the distinct developmental stages, the availability of various tools and reagents, known genome sequence and the physiological similarity of Drosophila with humans make them an excellent in vivo model organism to rapidly test toxicity in whole organism and elucidate the molecular mechanisms underlying the toxicity. The natural product with the best result will be used to evaluate genoprotection in human lymphocytes. These are used as a surrogate tissue, as they are easily obtained, in large numbers, do not require cell culture, are diploids and are almost all in the same phase of the cell cycle. This project is in an initial phase and lacks results, which will be available along this year.

scholarly journals Iron sequestration by transferrin 1 mediates nutritional immunity in Drosophila melanogaster

2020 ◽  
Vol 117 (13) ◽  
pp. 7317-7325 ◽  
Author(s):  
Igor Iatsenko ◽  
Alice Marra ◽  
Jean-Philippe Boquete ◽  
Jasquelin Peña ◽  
Bruno Lemaitre
Keyword(s):  
Drosophila Melanogaster ◽  
Defense Mechanism ◽  
Fat Body ◽  
Immune Defense ◽  
In Vivo Model ◽  
Nutritional Immunity ◽  
Iron Sequestration

Iron sequestration is a recognized innate immune mechanism against invading pathogens mediated by iron-binding proteins called transferrins. Despite many studies on antimicrobial activity of transferrins in vitro, their specific in vivo functions are poorly understood. Here we use Drosophila melanogaster as an in vivo model to investigate the role of transferrins in host defense. We find that systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires Drosophila nuclear factor κB (NF-κB) immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. Next, we show that the iron transporter Tsf1 is induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, we discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, our study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. Our in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy.

scholarly journals The pupal cuticle of Drosophila: biphasic synthesis of pupal cuticle proteins in vivo and in vitro in response to 20-hydroxyecdysone.

1985 ◽  
Vol 101 (1) ◽  
pp. 189-200 ◽  
Author(s):  
J Doctor ◽  
D Fristrom ◽  
J W Fristrom
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Ultrastructural Changes ◽  
Low Molecular Weight ◽  
Before And After ◽  
Cuticle Protein ◽  
Cuticle Proteins ◽  
Pupal Cuticle

We investigated the synthesis and localization of Drosophila pupal cuticle proteins by immunochemical techniques using both a complex antiserum and monoclonal antibodies. A set of low molecular weight (15,000-25,000) pupal cuticle proteins are synthesized by the imaginal disk epithelium before pupation. After pupation, synthesis of the low molecular weight proteins ceases and a set of unrelated high molecular weight proteins (40,000-82,000) are synthesized and incorporated into the pupal cuticle. Ultrastructural changes in the cuticle deposited before and after pupation correlate with the switch in cuticle protein synthesis. A similar biphasic accumulation of low and high molecular weight pupal cuticle proteins is also seen in imaginal discs cultured in vitro. The low molecular weight pupal cuticle proteins accumulate in response to a pulse of the insect steroid hormone 20-hydroxyecdysone and begin to appear 6 h after the withdrawal of the hormone from the culture medium. The high molecular weight pupal cuticle proteins accumulate later in culture; a second pulse of hormone appears to be necessary for the accumulation of two of these proteins.

Transcriptional repression of TRH promoter function by T3: analysis by in vivo gene transfer

2000 ◽  
Vol 78 (3) ◽  
pp. 155-163 ◽  
Author(s):  
Hajer Guissouma ◽  
Nathalie Becker ◽  
Isabelle Seugnet ◽  
Barbara A Demeneix
Keyword(s):  
Thyroid Hormone ◽  
Gene Transfer ◽  
Transcriptional Repression ◽  
Developmental Stages ◽  
Hormone Secretion ◽  
In Vivo Model ◽  
Viral Gene ◽  
Newborn Mice ◽  
Binding Forms

We consider how an integrated in vivo model can be used to study the specific transcriptional effects of specific receptors in neuroendocrine systems. Our example is the role of thyroid receptor (TR) isoforms in mediating negative feedback effects of T3 on TRH (thyrotropin releasing hormone) expression. The in vivo transfection method employed polyethylenimine (PEI) to introduce genes directly into specifc regions of the brains of mice, rats, and Xenopus tadpoles. In the mouse model, the technique has served to examine TR effects on TRH transcription and on the pituitary-thyroid axis end point: thyroid hormone secretion. When a TRH-luciferase construct is introduced into the hypothalami of newborn mice TRH-luciferase transcription is regulated physiologically, being significantly increased in hypothyroidism and decreased in T3-treated animals. When various T3-binding forms of TRβ or TRα are expressed in the hypothalamus, all TRβ isoforms give T3-dependent regulation of TRH transcription, whereas TRα isoforms block T3-dependent transcription. Moreover, TR transcriptional effects are correlated with physiological consequences on circulating T4. Thus, somatic gene transfer shows TR subtypes to have distinct, physiologically relevant effects on TRH transcription. The approach is an appealing alternative to germinal transgenesis for studying specific neuroendocrine regulations at defined developmental stages in different species.Key words: thyroid hormone, TRH, mouse central nervous system, non viral gene transfer, polyethylenimine.

scholarly journals Proteomics of protein trafficking by in vivo tissue-specific labeling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ilia A. Droujinine ◽  
Amanda S. Meyer ◽  
Dan Wang ◽  
Namrata D. Udeshi ◽  
Yanhui Hu ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Serum Proteins ◽  
Fat Body ◽  
Model Systems ◽  
Secreted Proteins ◽  
Subcellular Compartment ◽  
Secreted Factors ◽  
Cells Of Origin ◽  
Human Ortholog

AbstractConventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.

scholarly journals Immune-related genes of the larval Holotrichia parallela in response to entomopathogenic nematodes Heterorhabditis beicherriana LF

2020 ◽  
Author(s):  
Ertao Li ◽  
Jianhui Qin ◽  
Honglin Feng ◽  
Jinqiao Li ◽  
Xiaofeng Li ◽  
...  
Keyword(s):  
Entomopathogenic Nematodes ◽  
Target Genes ◽  
Developmental Stages ◽  
Fat Body ◽  
Holotrichia Parallela ◽  
Peptidoglycan Recognition Protein ◽  
Recognition Protein ◽  
Transcriptome Resource ◽  
Immune Related Genes

Abstract Background Entomopathogenic nematodes (EPNs) emerge as compatible alternatives to conventional insecticides in controlling Holotrichia parallela larvae (Coleoptera: Scarabaeidae). However, the immune responses of H. parallela against EPNs infection remain unclear. Results In present research, RNA-Seq was firstly performed. A total of 89427 and 85741 unigenes were achieved from the midgut of H. parallela larvae treated with Heterorhabditis beicherriana LF for 24 and 72 h, respectively; 2545 and 3156 unigenes were differentially regulated, respectively. Among those differentially expressed genes (DEGs), 74 were identified potentially related to the immune response. Notably, some immune-related genes, such as peptidoglycan recognition protein SC1 (PGRP-SC1), pro-phenoloxidase activating enzyme-I (PPAE-I) and glutathione s-transferase (GST), were induced at both treatment points. Bioinformatics analysis showed that PGRP-SC1, PPAE-I and GST were all involved in anti-parasitic immune process. Quantitative real-time PCR (qRT-PCR) results showed that the three immune-related genes were expressed in all developmental stages; PGRP-SC1 and PPAE-I had higher expressions in midgut and fat body, respectively, while GST exhibited high expression in both of them. Moreover, in vivo silencing of them resulted in increased susceptibility of H. parallela larvae to H. beicherriana LF. Conclusion These results suggest that PGRP-SC1, PPAE-I and GST could be used as target genes to disturb the immune system of H. parallela. This study provides the first comprehensive transcriptome resource of H. parallela exposure to nematode challenge that will help to support further comparative studies on host-EPN interactions.

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