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1.
J Biol Res (Thessalon) ; 25: 15, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30116723

ABSTRACT

BACKGROUND: The insect-trapping leaves of Dionaea muscipula provide a model for studying the secretory pathway of an inducible plant secretory system. The leaf glands were induced with bovine serum albumin to secrete proteases that were characterized via zymogram activity gels over a 6-day period. The accompanying morphological changes of the endoplasmic reticulum (ER) and Golgi were analyzed using 3D electron tomography of glands preserved by high-pressure freezing/freeze substitution methods. RESULTS: Secretion of multiple cysteine and aspartic proteases occurred biphasically. The majority of the Golgi was organized in clusters consisting of 3-6 stacks surrounded by a cage-like system of ER cisternae. In these clusters, all Golgi stacks were oriented with their cis-most C1 cisterna facing an ER export site. The C1 Golgi cisternae varied in size and shape consistent with the hypothesis that they form de novo. Following induction, the number of ER-bound polysomes doubled, but no increase in COPII vesicles was observed. Golgi changes included a reduction in the number of cisternae per stack and a doubling of cisternal volume without increased surface area. Polysaccharide molecules that form the sticky slime cause swelling of the trans and trans Golgi network (TGN) cisternae. Peeling of the trans-most cisternae gives rise to free TGN cisternae. One day after gland stimulation, the free TGNs were frequently associated with loose groups of oriented actin-like filaments which were not seen in any other samples. CONCLUSIONS: These findings suggest that the secretory apparatus of resting gland cells is "overbuilt" to enable the cells to rapidly up-regulate lytic enzyme production and secretion in response to prey trapping.

2.
PLoS Genet ; 10(6): e1004343, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24945799

ABSTRACT

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.


Subject(s)
Drosophila Proteins/metabolism , Ecdysone/biosynthesis , Insect Hormones/biosynthesis , Nuclear Proteins/metabolism , POU Domain Factors/metabolism , Repressor Proteins/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/biosynthesis , Animals , Binding Sites , Biological Transport/genetics , Cholesterol/metabolism , DNA-Binding Proteins , Drosophila Proteins/biosynthesis , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Ecdysone/genetics , Ecdysone/metabolism , Gene Expression Regulation , Gene Expression Regulation, Developmental , Insect Hormones/metabolism , Membrane Proteins/biosynthesis , POU Domain Factors/biosynthesis , POU Domain Factors/genetics , RNA Interference , RNA, Small Interfering , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , TOR Serine-Threonine Kinases/biosynthesis , Transcription, Genetic
3.
Development ; 140(23): 4730-9, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24173800

ABSTRACT

Steroid hormones trigger the onset of sexual maturation in animals by initiating genetic response programs that are determined by steroid pulse frequency, amplitude and duration. Although steroid pulses coordinate growth and timing of maturation during development, the mechanisms generating these pulses are not known. Here we show that the ecdysone steroid pulse that drives the juvenile-adult transition in Drosophila is determined by feedback circuits in the prothoracic gland (PG), the major steroid-producing tissue of insect larvae. These circuits coordinate the activation and repression of hormone synthesis, the two key parameters determining pulse shape (amplitude and duration). We show that ecdysone has a positive-feedback effect on the PG, rapidly amplifying its own synthesis to trigger pupariation as the onset of maturation. During the prepupal stage, a negative-feedback signal ensures the decline in ecdysone levels required to produce a temporal steroid pulse that drives developmental progression to adulthood. The feedback circuits rely on a developmental switch in the expression of Broad isoforms that transcriptionally activate or silence components in the ecdysone biosynthetic pathway. Remarkably, our study shows that the same well-defined genetic program that stimulates a systemic downstream response to ecdysone is also utilized upstream to set the duration and amplitude of the ecdysone pulse. Activation of this switch-like mechanism ensures a rapid, self-limiting PG response that functions in producing steroid oscillations that can guide the decision to terminate growth and promote maturation.


Subject(s)
Drosophila melanogaster/embryology , Ecdysone/metabolism , Insect Hormones/metabolism , Sexual Maturation/genetics , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Ecdysone/biosynthesis , Gene Expression Regulation, Developmental , Larva/growth & development , Larva/metabolism , Metamorphosis, Biological/genetics , Promoter Regions, Genetic , Signal Transduction
4.
PLoS One ; 7(5): e36548, 2012.
Article in English | MEDLINE | ID: mdl-22563507

ABSTRACT

Animals use TGF-ß superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-ß/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, "BMP-specific" Smads can be phosphorylated in response to TGF-ß/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, we find that Baboon, the Drosophila TGF-ß/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, we find that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. We propose that the dual activity of TGF-ß/Activin receptors is an ancient feature, and we discuss several ways this activity can modulate TGF-ß signaling output.


Subject(s)
Activin Receptors/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Smad2 Protein/metabolism , Transcription Factors/metabolism , Activin Receptors/genetics , Animals , Blotting, Western , Cell Line , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Female , Larva/growth & development , Larva/metabolism , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Receptor Cross-Talk , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction , Smad Proteins, Receptor-Regulated , Smad2 Protein/genetics , Transcription Factors/genetics , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Wings, Animal/growth & development , Wings, Animal/metabolism
5.
J Neurosci ; 27(29): 7740-50, 2007 Jul 18.
Article in English | MEDLINE | ID: mdl-17634368

ABSTRACT

Recently, several evolutionary conserved signaling pathways that play prominent roles in regulating early neurodevelopment have been found to regulate synaptic remodeling in the adult. To test whether adult neuronal expression of bone morphogenic protein (BMP) signaling components also plays a postnatal role in regulating neuronal plasticity, we modulated BMP signaling in mice both in vivo and in vitro by genetic removal of the BMP inhibitor chordin or by perfusing recombinant BMP signaling pathway components onto acute hippocampal slices. Chordin null mice exhibited a significant increase in presynaptic transmitter release from hippocampal neurons, resulting in enhanced paired-pulse facilitation and long-term potentiation. These mice also showed a decreased acquisition time in a water maze test along with less exploratory activity during Y-maze and open-field tests. Perfusion of BMP ligands onto hippocampal slices replicated the presynaptic phenotype of chordin null slices, but bath application of Noggin, another antagonist of BMP signaling pathway, significantly decrease the frequency of miniature EPSCs. These results demonstrate that the BMP signaling pathway contributes to synaptic plasticity and learning likely through a presynaptic mechanism.


Subject(s)
Glycoproteins/metabolism , Hippocampus/cytology , Intercellular Signaling Peptides and Proteins/metabolism , Learning/physiology , Neuronal Plasticity/physiology , Presynaptic Terminals/metabolism , Spatial Behavior/physiology , Animals , Animals, Newborn , Bone Morphogenetic Proteins/metabolism , Cells, Cultured , Dose-Response Relationship, Radiation , Electric Stimulation/methods , Embryo, Mammalian , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Excitatory Postsynaptic Potentials/radiation effects , Exploratory Behavior/physiology , Glycoproteins/deficiency , Hippocampus/physiology , In Vitro Techniques , Intercellular Signaling Peptides and Proteins/deficiency , Male , Maze Learning/physiology , Mice , Mice, Knockout , Microscopy, Electron, Transmission/methods , Neuronal Plasticity/genetics , Neurons/cytology , Neurons/drug effects , Neurons/radiation effects , Patch-Clamp Techniques , Presynaptic Terminals/ultrastructure , Time Factors
6.
Plant Cell ; 18(10): 2567-81, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17012602

ABSTRACT

We have investigated the transport of storage proteins, their processing proteases, and the Vacuolar Sorting Receptor-1/Epidermal Growth Factor Receptor-Like Protein1 (VSR-1/ATELP1) receptor during the formation of protein storage vacuoles in Arabidopsis thaliana embryos by means of high-pressure freezing/freeze substitution, electron tomography, immunolabeling techniques, and subcellular fractionation. The storage proteins and their processing proteases are segregated from each other within the Golgi cisternae and packaged into separate vesicles. The storage protein-containing vesicles but not the processing enzyme-containing vesicles carry the VSR-1/ATELP1 receptor. Both types of secretory vesicles appear to fuse into a type of prevacuolar multivesicular body (MVB). We have also determined that the proteolytic processing of the 2S albumins starts in the MVBs. We hypothesize that the compartmentalized processing of storage proteins in the MVBs may allow for the sequential activation of processing proteases as the MVB lumen gradually acidifies.


Subject(s)
Arabidopsis/embryology , Plant Proteins/metabolism , Protein Processing, Post-Translational , Seeds/metabolism , Amino Acid Sequence , Golgi Apparatus/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Microscopy, Electron , Molecular Sequence Data , Plant Proteins/chemistry , Subcellular Fractions/metabolism
7.
Nutr Clin Care ; 7(2): 46-55, 2004.
Article in English | MEDLINE | ID: mdl-15481738

ABSTRACT

We examine the physiologic changes involved in the onset of cardiovascular disease (CVD) as well as multiple dietary and lifestyle factors that either promote or prevent CVD. Dietary fats (saturated, monounsaturated, n-3 and n-6 polyunsaturated, and trans fats), antioxidants, and carbohydrates, as well as alcohol consumption, exercise, smoking, and infections, are evaluated. Epidemiologic studies and clinical trials are discussed in light of the underlying mechanisms.


Subject(s)
Cardiovascular Diseases/prevention & control , Diet/methods , Life Style , Alcohol Drinking , Antioxidants/administration & dosage , Cardiovascular Diseases/etiology , Dietary Carbohydrates/administration & dosage , Dietary Fats/administration & dosage , Humans , Motor Activity/physiology , Risk Factors , Smoking/adverse effects
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